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Fungal Genetics and Biology 70 (2014) 42ndash67

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Fungal Genetics and Biology

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Review

Fungal model systems and the elucidation of pathogenicity determinants

httpdxdoiorg101016jfgb2014060111087-1845 2014 The Authors Published by Elsevier IncThis is an open access article under the CC BY-NC-ND license (httpcreativecommonsorglicensesby-nc-nd30)

uArr Corresponding author Fax +34 957212072E-mail address ge2penaeucoes (E Perez-Nadales)

Elena Perez-Nadales auArr Maria Filomena Almeida Nogueira b Clara Baldin cd Soacutenia Castanheira eMennat El Ghalid a Elisabeth Grund f Klaus Lengeler g Elisabetta Marchegiani h Pankaj Vinod Mehrotra bMarino Moretti i Vikram Naik i Miriam Oses-Ruiz j Therese Oskarsson g Katja Schaumlfer aLisa Wasserstrom g Axel A Brakhage cd Neil AR Gow b Regine Kahmann i Marc-Henri Lebrun hJoseacute Perez-Martin e Antonio Di Pietro a Nicholas J Talbot j Valerie Toquin k Andrea Walther gJuumlrgen Wendland g

a Department of Genetics Edificio Gregor Mendel Planta 1 Campus de Rabanales University of Cordoba 14071 Cordoba Spainb Aberdeen Fungal Group School of Medical Sciences Institute of Medical Sciences University of Aberdeen Aberdeen UKc Department of Molecular and Applied Microbiology Leibniz Institute for Natural Product Research and Infection Biology ndash Hans Knoumlll Institute (HKI)Beutembergstr 11a 07745 Jena Germanyd Department of Microbiology and Molecular Biology Institute of Microbiology Friedrich Schiller University Jena Beutenbergstr 11a 07745 Jena Germanye Instituto de Biologiacutea Funcional y Genoacutemica CSIC Universidad de Salamanca 37007 Salamanca Spainf Functional Genomics of Plant Pathogenic Fungi UMR 5240 CNRS-UCB-INSA-Bayer SAS Bayer CropScience 69263 Lyon Franceg Carlsberg Laboratory Department of Yeast Genetics Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmarkh Evolution and Genomics of Plant Pathogen Interactions UR 1290 INRA BIOGER-CPP Campus AgroParisTech 78850 Thiverval-Grignon Francei Max-Planck-Institute for Terrestrial Microbiology Department of Organismic Interactions Karl-von-Frisch-Strasse 10 D-35043 Marburg Germanyj School of Biosciences Geoffrey Pope Building University of Exeter Exeter EX4 4QD UKk Biochemistry Department Bayer SAS Bayer CropScience CRLD 69263 Lyon France

a r t i c l e i n f o a b s t r a c t

Article historyReceived 3 February 2014Accepted 25 June 2014Available online 7 July 2014

KeywordsFungal model organismPlant fungal pathogenHuman fungal pathogenGenomicsVirulence

Fungi have the capacity to cause devastating diseases of both plants and animals causing significant har-vest losses that threaten food security and human mycoses with high mortality rates As a consequencethere is a critical need to promote development of new antifungal drugs which requires a comprehensivemolecular knowledge of fungal pathogenesis In this review we critically evaluate current knowledge ofseven fungal organisms used as major research models for fungal pathogenesis These include pathogensof both animals and plants Ashbya gossypii Aspergillus fumigatus Candida albicans Fusarium oxysporumMagnaporthe oryzae Ustilago maydis and Zymoseptoria tritici We present key insights into the virulencemechanisms deployed by each species and a comparative overview of key insights obtained from geno-mic analysis We then consider current trends and future challenges associated with the study of fungalpathogenicity 2014 The Authors Published by Elsevier Inc This is an open access article under the CC BY-NC-ND license

(httpcreativecommonsorglicensesby-nc-nd30)

Contents

1 Introduction 432 Aspergillus fumigatus 44

21 Overview of the A fumigatus genome 4422 Assessing virulence of A fumigatus 4723 Current research interests 47

231 Virulence determinants 47232 Signaling involved in virulence 47233 Host perception and response 47234 Development of anti-infective strategies 49235 Contribution of fungal secondary metabolism to virulence 49

E Perez-Nadales et al Fungal Genetics and Biology 70 (2014) 42ndash67 43

24 Conclusions 49

3 Candida species 50

31 Comparative genomics in Candida species 5032 The toolbox for C albicans 5033 Current research interests 52

331 Genome functional analysis 52332 Altered host responses 52333 Yeast-to-hypha transition 52334 Importance of the cell wall in immune recognition 52

34 Conclusions 52

4 Fusarium oxysporum 52

41 Overview of the F oxysporum genome 5342 Assessing virulence of F oxysporum 5343 Current research interests 53

431 Role of MAPK cascades in virulence 53432 Lineage specific (LS) chromosomes 54433 Secreted effectors and gene-for-gene system 54434 F oxysporum as a model for fungal trans-kingdom pathogenicity 54

44 Conclusions 54

5 Ashbya gossypii 54

51 Comparative genomics in Eremothecium 5552 Current research interests 55

521 Riboflavin production 55522 Hyphal growth 55523 Septation 55524 Nuclear division and movement 55525 Genome evolution 56

53 Conclusions 56

6 Magnaporthe oryzae 56

61 Overview of the M oryzae genome 5662 Assessing virulence of M oryzae 5663 Current research interests 57

631 Mechanisms of fungal infection 57632 Plant pathogen interactions in the rice blast fungus M oryzae 57633 Signaling 57

64 Conclusions 57

7 Ustilago maydis 57

71 Comparative genomics in U maydis 5872 Assessing virulence of U maydis 5873 Current research interests 58

731 Host perception and plant response 58732 Effector function 58733 Cell cycle dimorphism and pathogenic development 59734 Signaling and MAP kinase targets involved in virulence 59735 Understanding the molecular basis of fundamental eukaryotic processes 59

74 Conclusions 59

8 Zymoseptoria tritici 59

81 Overview of the Z tritici genome 6082 Assessing virulence of Z tritici 6083 Current research interests 60

831 Signaling pathways and protein secretion 60832 Plant-pathogen interaction effectors 60833 Disease control 61834 Evolutionary genomics 61

84 Conclusions 61

9 General conclusions 61

Acknowledgments 62Appendix A Supplementary material 62

References 62

1 Introduction

A small fraction of the estimated 5 million fungal species areresponsible for devastating diseases affecting agriculture andhuman health Emerging infectious diseases (EIDs) caused by fungiare increasingly recognized as major threats to food security andanimal health (Brown et al 2012 Fischer et al 2008) Dispersaland emergence of fungal diseases are furthermore promoted by

human activity primarily through global trade which lacks suffi-cient biosecurity measures and may be exacerbated by the impactof climate change (Brasier 2008 Fisher et al 2012 Gange et al2007 Harvell et al 1999 Ratnieks and Carreck 2010 Verweijet al 2009) Fungal pathogens are characterized by a remarkablegenetic flexibility that facilitates rapid evolution and adaptationto the host or environment (Calo et al 2013) These features whencombined with Darwinian selection support the emergence of new

44 E Perez-Nadales et al Fungal Genetics and Biology 70 (2014) 42ndash67

lineages with increased virulence or altered host range (Croll andMcDonald 2012) This highly adaptive behavior is promoted byan immense fungal genome plasticity (Calo et al 2013) Besidessexual reproduction parasexualilty (Pontecorvo 1956) aneuploidy(Selmecki et al 2006) transposons telomere instability (Starneset al 2012) or horizontal gene transfer (Ma et al 2010 Richardset al 2011) amongst others enable adaptive evolution by promot-ing mitotic recombination independent chromosomal assortmentgain or loss of chromosomes and translocations Moreover fungiexhibit morphogenetic plasticity which enables them to colonizeand invade tissue as hyphae which primarily extend at their tips(Fischer et al 2008) while often forming differentiated sporesinfection structures fruiting bodies and unicellular yeast cellsthat can aid rapid dispersal (Sudbery et al 2004)

In this review we cover an array of fungal species encompass-ing diverse taxonomic groups that cover several hundred millionyears of evolution (Fig 1) We include two major fungal pathogensof humans Aspergillus fumigatus and Candida albicans a facultativepathogen of both animals and plants Fusarium oxysporum andplant pathogenic species Magnaporthe oryzae Mycospherella gra-minicola Ashbya gossypii and Ustilago maydis which display infec-tion-associated dimorphism and cellular differentiationCollectively this provides an overview of diverse mechanisms ofpathogenesis and some unifying themes while also encompassingsome of the best-studied and understood fungal pathosystems

The purpose of the review is to highlight both specific differ-ences and unifying features of some of the best-studied fungalpathogens and to highlight the significant challenges that remainin developing a deep understanding of fungal pathogenesis

2 Aspergillus fumigatus

Aspergillus fumigatus is a filamentous fungus that can be isolatedfrom compost soil where it proliferates in organic debris and plays

Fig 1 Phylogenetic tree Fungal species phylogeny generated from a concatenated aligntaxa and 9033 amino acid positions Bootstrap values for each node are reported as perccopy genes from the 21 fungal species were aligned using Clustal W (Larkin et al 2007)and Castresana 2007) All the aligned sequences were then concatenated to one file usgenerate the phylogenetic tree with 100 bootstraps for branch support and LG as the a(Keane et al 2006) The tree was visualized using TreeDyn (Chevenet et al 2006)

an essential role in carbon and nitrogen recycling Fumigatus is themost important air-borne fungal pathogen and can grow at tem-peratures up to 55 C while its spores survive temperatures ofup to 70 C (Brakhage and Langfelder 2002) The fungus propa-gates asexually with release of thousands of spores per conidialhead into the atmosphere Due to their small size (2ndash3 lm diame-ter) the spores can easily reach lung alveoli (Latge 2001)

A first description of pulmonary aspergillosis was published in1842 by physician John H Bennett (Supplementary Fig 1A) whonoted the presence of a fungus in the lungs of a post mortempatient with pneumothorax Almost 50 years elapsed howeverbefore A fumigatus was recognized as the primary cause of theinfection (Barnes 2004) Today A fumigatus conidia infect millionsof susceptible individuals causing allergies associated withasthma allergic sinusitis and bronchoalveolitis (Denning et al2013) In cavities in the lungs of tuberculosis patients A fumigatusspores germinate and develop into a fungus ball or non-invasiveaspergilloma (Riscili and Wood 2009) In addition to these formsof aspergillosis which are not life-threatening patients withaltered immune status such as leukemia patients or transplantpatients are at risk to develop invasive aspergillosis (IA) with anestimated number of more than 200000 cases per year (Brownet al 2012 Garcia-Vidal et al 2008) Mortality rates for IA reacharound 50 of the cases when patients are treated and increaseto more than 90 when the diagnosis is missed or delayed(Brown et al 2012)

21 Overview of the A fumigatus genome

A first draft of the A fumigatus clinical isolate Af293 genomewas published in 2005 (Nierman et al 2005) Eight chromosomeswere identified containing about 10000 genes (Table 2) Threeyears later the genome sequence of a second A fumigatus isolateA1163 was released (Fedorova et al 2008) Comparative analysisshowed conservation of 98 of the sequence between the two

ment of 49 conserved fungal genes using maximum likelihood The tree covers 21entages To generate the tree protein sequences of the 49 selected conserved singleand the obtained conserved sequence blocks were sampled with G-blocks (Talaveraing Galaxy (Goecks et al 2010) Finally PhyML (Guindon et al 2010) was used tomino acid substitution model as identified by ModelGenerator (Keane et al 2006)

Table 1Overview of biological features

Species Human fungal pathogens Plant fungal pathogens

Aspergillus fumigatus Candida albicans Magnaporthe oryzae Fusarium oxysporum Zymoseptoria tritici Ustilago maydis Ashbya gossypii

Taxonomy Phylum Ascomycota Phylum Ascomycota Phylum Ascomycota Phylum Ascomycota Phylum Ascomycota Phylum Basidiomycota Phylum AscomycotaClass Eurotiomycetes Subphylum

SaccharomycotinaClass Sordariomycetes Class Sordariomyctes Class Dothideomycetes Class Ustilaginomycetes Subphylum

SaccharomycotinaOrder Eurotiales Class Saccharomycetes Order Magnaporthales Order Hypocreales Order Capnodiales Order Ustilaginales Class SaccharomycetesFamilyTrichocomaceae

Order Saccharomycetales Family Magnaporthaceae Family Nectriaceae FamilyMycosphaerellaceae

Family Ustilaginaceae Order Saccharomycetales

Genus Aspergillus FamilySaccharomycetaceae

Genus Magnaporthe Genus Fusarium Genus Zymoseptoria Genus Ustilago FamilySaccharomycetaceae

Species A fumigatus Genus Candida Species M oryzae Species F oxysporum Species Z tritici Species U maydis Genus EremotheciumSpecies C albicans Species A gossypii

Predominant cell-type Multinucleateseptated filaments

Budding yeastpseudohyphae and truehypha (in which elongatedyeast cells remain attachedafter cytokinesis)

Filamentous mycelium Filamentous myceliumMicroconidia

Dimorphic fungus yeast-like cellsfilamentousmycelium

Unicellular budding yeast multinucleate septatedfilaments

Sexual cycle Yes but in naturepredominantly asexual

Parasexual cycle (mating ofdiploid cells followed bymitosis and chromosomeloss instead of meiosis

Yes but in naturepredominantly asexual

Not identified Yes occurs duringepidemics

Yes occurs only inside theplant

not identified

Mating-type system Bipolar heterothallismMAT1-1 MAT1-2

MATa and MATa Bipolar heterothallismMAT1-1 MAT1-2

MAT1 gene identified andexpressed in F oxysporum Amixed distribution ofMAT1-1 and MAT1-2 allelesin Fusarium species complex

Bipolar heterothallismMAT1-1 MAT1-2

Tetrapolar a locus (2alleles) b locus ( 20 alleles)

bipolar MATaa

Spores Uninucleate conidiaand binucleateascospores

Chlamydospore Conidia (asexual spore) andascospores (sexual spore)

Microconidiamacroconidiaclamydospores

Ascospores andpycnidiospores

Diploid spores teliospores Ascospores

Pathogenicity Animals (can causeasthma aspergillomainvasive aspergillosis)

Candidiasis (skin infections)and Candidemia (presenceof Candida albicans in blood)in immuno-compromisedindividuals

Rice and somemonocotylous plants (e gbarley wheat)

Fusarium wilt on plantcrops Emerging cause offusariosis in humans

Bread and durum wheat(septoria tritici leaf blotch)

Corn and teosinte plants Cottoncitrus fruits

Other features Toxin production Homothallic andheterothallic mating andhaploid mating alsopossible but rare

Fusarium species complexare pathogenic especially toagriculture plants

Highly resistant to UVradiation

Riboflavin overproducer

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45

Table 2Overview of fungal genome data



Genome size (Mb) 29420 1488 4103 6136 397 2050 876Chromosomes 8 8 7 15 21 23 7GC content () 500 333 516 484 517 570 518Number of Genes 9783 6354 12827 17708 10900 6788 4726Non-coding RNAs (tRNAs) 179 132 325 308 Unknown 104 192ndash293 (216)Introns Average 18 per gene 415 introns in the entire

genomeAverage 18 per gene Unknown Average 15 per

gene3093 introns in the entiregenome average 046 pergene

226 introns in theentire genome

Avg gene sizeintergenicregion

164 kb122 kb 147 kb858 bp 2 kb14 kb 13 kb P115 kb 174 kb973 bp 19 kb340 bp

Transposons 8 (Predicted DDE1transposon-related ORF ampputative transposaseinduced by exposure tohuman airway epithelialcells)

3 (Zorro3-R ZORRO1Zorro2-1 member of L1clade of transposons andencodes a potential DNA-binding zinc-finger protein)

97 of genome comprisesrepeated sequences longer than200 base pairs (bp) and withgreater than 65 similarity

28 of the genomeidentified as repetitivesequence

20 11 DNA with similarity totransposons

no -

Predicted LINE (longinterspersed nuclearelements) LINE-like reversetranscriptase

Retroelements (copia-likeand gypsy-like LINEs (longinterspersed nuclearelements) and SINEs (shortinterspersednuclearelements) DNA transposons(Tc1-mariner hAT-likeMutator-like and MITEs)

(hobS tigR retroelements)

Predicted gypsytransposon- related ORFPredicted mariner Ant1transposon-related ORF

S cerevisiae homology 60 64 369 Unknown 225 33 (20 identity cut-off) 95Mitochondrial DNA (kb) 32 41 3495 3448 44 568 235References Nierman et al (2005) Braun et al (2005) httpwwwbroadinstituteorg http

wwwbroadinstituteorgOrton et al (2011) Kamper et al 2006 Dietrich et al (2004)

Fedorova et al (2008) Mitrovich et al (2007) Dean et al (2005) Rep and Kistler (2010) Goodwin et al(2011)

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isolates with the majority of the remaining 2 being strain-specificgenes clustered in blocks from 10 to 400 kb in size including alarge number of pseudogenes and repeat elements (Fedorovaet al 2008) Comparison of the sequenced A fumigatus genomeswith those of closely related species such as Neosartorya fischeriand Aspergillus clavatus showed the presence of lineage-specificgenes often localized to the sub-telomeric regions These genomicislands were suggested to represent a gene repository that couldplay a role in adaptation to different environments such as com-post or a living host (Fedorova et al 2008) Transcriptome studiesbased on RNA-Seq and proteome studies are routinely carried outwith A fumigatus (Horn et al 2012 Kroll et al 2014) The updatedfeatures of the A fumigatus genome are available at the AspergillusGenome Database website (Table 3)

22 Assessing virulence of A fumigatus

To understand the infection process of A fumigatus and identifyfungal factors involved in pathogenesis appropriate infectionmodels are required Mouse infection models have been crucialfor the study of pathogenicity for the characterization of host-pathogen interactions and for development of therapeuticapproaches (Clemons and Stevens 2005) According to the immu-nosuppression regimen mouse infection models are divided intoneutropenic or non-neutropenic models (Liebmann et al 2004a)Because it is believed that infection starts by inhalation of conidiain mouse infection models conidia are administered by intra-nasal intra-tracheal inoculation or inhalation Mouse models how-ever have disadvantages including an open question about theirreliability as models for human diseases (Seok et al 2013) as wellas increasing public concern regarding animal welfare and the clin-ical value of animal research which is resulting in increasing reg-ulatory demands This together with the requirement for costlyfacilities and personnel is driving the development of alternativeinfection models for fungal pathogens The larvae of the greaterwax moth Galleria mellonella for example provide importantadvantages They are readily available from suppliers at convenientcost they are large enough (around 25 cm in length) to be easilyinoculated (Mylonakis 2008) and can be maintained at 37 Cwhich means that infections can be studied at the temperature atwhich they occur in humans (Mylonakis 2008) However resultsobtained with such models are of limited value because there aresignificant differences in the immune responses of invertebratesand mammals Embryonated chicken eggs have also been used asan alternative infection model for analysis of fungal pathogenicityin A fumigatus with promising results (Jacobsen et al 2010)

23 Current research interests

231 Virulence determinantsIt is debated whether A fumigatus has true virulence factors or

simply physiological characteristics such as thermo-tolerancethat enable the fungus to grow in an immuno-compromisedhuman host Several virulence determinants of A fumigatus havehowever been characterized These determinants include the sid-erophore-mediated iron uptake system (Schrettl et al 2004) andthe pksP gene which is involved in the biosynthesis of dihydroxy-naphthalene (DHN) melanin which forms the gray-green sporepigment (Heinekamp et al 2012 Horn et al 2012 Langfelderet al 1998 Thywissen et al 2011 Volling et al 2011) DHN mel-anin inhibits both apoptosis and acidification of conidia-containingphagolysosomes of macrophages (Thywissen et al 2011 Vollinget al 2011) Mechanistically it was reported that DHN melanininhibited assembly of v-ATPase on the phagosomal membrane(Heinekamp et al 2012) A fumigatus also possesses immune-eva-sion mechanisms which reduce recognition of both immune

effector cells and the complement system (Aimanianda et al2009 Behnsen et al 2008 2010 Horn et al 2012) It can beexpected that virulence is a multifactorial process and thus morevirulence-associated traits will be discovered

232 Signaling involved in virulenceSeveral genes of the cAMP signal transduction pathway are

required for pathogenicity These include genes encoding adenyl-ate cyclase (AcyA) protein kinase A (PKA) and a stimulating Gaprotein-encoding gene designated gprA (Liebmann et al 2004bZhao et al 2006) Recently the cAMP signaling pathway has alsobeen related to regulation of the DHN melanin production (Abadet al 2010 Grosse et al 2008)

The calcineurincalmodulin signaling pathway is required forformation of cell shape and pathogenicity in A fumigatus Deletionof the gene encoding the catalytic subunit of calcineurin phospha-tase (calA) resulted in a branching defect and limited growth aswell as attenuation of virulence (da Silva Ferreira et al 2007Juvvadi et al 2013) A target of calcineurin is the zinc finger tran-scription factor CrzA which is implicated in germination polariza-tion cell wall structure asexual development and virulence(Cramer et al 2008) Of the four MAPKs found in A fumigatus itwas shown that MpkA regulates the cell wall integrity pathwayand SakA the hyperosmotic glycerol pathway (Jain et al 2011May et al 2005 Rispail et al 2009)

Two GPCRs were shown to be required for virulence in A fumig-atus GprC and GprD (Gehrke et al 2010) Little is known how-ever about the downstream multi-subunit G-proteins Afumigatus encodes only three Ga subunits while other Aspergilliharbor up to four Ga proteins For gpaB most likely involved incAMP signal transduction its involvement in pathogenicity wasdemonstrated (Liebmann et al 2004b Rispail et al 2009)

The seven transmembrane domain (7TMD) protein PalH wasrecognized as a putative pH sensor and also shown to be requiredfor virulence on mice (Grice et al 2013) Three histidine kinasereceptors TcsAFos-1 TcsB and TcsC have been characterized Dele-tion of tcsA led to a strain with reduced virulence in a systemicmurine model while the only phenotype detectable for DtcsB wasreduced sensitivity to SDS (Grice et al 2013) TcsC has been shownto play a role in the high osmotic pressure response and growth inthe presence of nitrate as nitrogen source (McCormick et al 2012)Finally a number of a cell wall stress sensors have been describedincluding Wsc1 Wsc2 Wsc3 and MidA Wsc1 is involved in resis-tance against echinocandins MidA appears to be essential for ther-motolerance at elevated temperatures and to counteract the effectsof cell wall-disrupting compounds such as congo red and calcofluorwhite Wsc1 Wsc3 and MidA show also some overlapping roles inradial growth and conidiation The role of Wsc2 remains to be elu-cidated (Dichtl et al 2012) Another conserved signal cascade isthe CPC system which links environmental stresses to amino acidhomeostasis Deletion of the transcriptional activator CpcA led toreduced virulence while deletion of the sensor kinase CpcCresulted in increased sensitivity towards amino acid starvation(Abad et al 2010)

233 Host perception and responseIn healthy individuals inhaled conidia of A fumigatus are rap-

idly attacked by the immune system of the host Physical barriersof the lung and the innate immunity are of major importance fordefense Epithelial cells of the upper respiratory tract contributeto elimination of microbes through secretion of mucus and cilia-mediated active transport (Brakhage 2005) In alveoli there areepithelial cells called pneumocytes responsible for secretion of apulmonary surfactant with antimicrobial activity (Balloy andChignard 2009) A decisive role for elimination of pathogens isplayed by macrophages and neutrophils Alveolar macrophages

Table 3Overview of molecular tools



Transformation Protoplasts amp electroporation Lithium acetate spheroplastfusion amp electroporation

PEGCaCl2 amp ATMT(Agrobacterium-mediatedtransformation)

Protoplasts amp ATMT ATMT Protoplasts Electroporation ampprotoplasts

Minimalhomology forgene deletion

gt500 bp 50 bp 500 bp gt1000 bp 500 bp 500 bp 45 bp

Episomalelements

no No episomal plasmids butintegrative plasmidsinclude CIp10 (artificialintegration at RPS10 locus)pDUPPDIS (shuttle vectorfor integration at NEUT5 l)

no self-replicative ARS(active replicatingsystem) plasmid pFNit-Lam-Tlam linear

no UmARS plasmids ScARS plasmids

Promoters [c]constitutive [r]regulatable

[r] Pyomelanin promoter PTetsystem Xylose-regulatedpromoter

[c] PADH1 [r] PACT1 [r]PTEF3 [c] PEF1-a2 [r]PGAL1 [r] PPCK1 [r] PSAP2[r] PMRP1 [r] PHEX1 [r]PMET3 [r] PMAL2 [r] PTET[r] PSAT1

[r] PICL1 [r] PNiA1 [c] PMPG1 [r] Thiaminerepressed Psti35promoter [c] PgpdA

[c] Pacu-3 [c] Pgpd [r] PTet system [r] Pnar1 [r]Pcrg1 [c] Potef

PTEF1 PHIS3 [r] PMET3[r]PTHI13

Commonly usedselectionmarkers

Hygromycin B PyrithiaminePhleomycin

URA3 LEU2 HIS1 ARG4MH3 Nourseothricin FLPand Cre-Lox technology

Hygromycin BSulfonylurea GlufosinateGeneticin G418Nourseothricin

Hygromycin BPhleomycin

Hygromycin B BialaphosGeneticin G418 Carboxin

Hygromycin B PhleomycinCarboxin NourseothricinGeneticin G418 FLPtechnology

Hygromycin B AgLEU2GEN3KanMXNourseothricin

Reporter genes lacZ GFP ADH1 ACT1 TEF3 EF1-a2GAL1 PCK1 SAP2 MRP1HEX1 MET3 MAL2 TETSAT1

GFP GUS HcRed GFP ChFP GFP GFP GUS lacZ GFP

Fluorescentprotein labels

GFP mCherry mCherry GFP YFP CFP RFPVenus

GFP RFP mCherry YFP GFP ChFP GFP GFP RFP mCherry CFP YFP Codon optimized

Cytochemical dyes DAPI Calcofluor White Mitotracker

Calcoflour White AlcianBlue

Calcofluor White DAPIFM4-64 WGA

Calcofluor White DAPIFITC

DAPI Calcofluor WhiteWGA alexa Mito tracker

DAPI FM4-64 Filipin DAPI FM4-64 CalcofluorWhite Mito tracker Filipin

Arrays Affimetrix Febit Custom made DNAmicroarray Affymetrix

Custom made DNAmicroarray Affymetrix



Custom made DNAmicroarray Affymetrixrepresenting 6297 genes

Does not apply

Roche TIGR(RNA-seq mostly replacing thearray platforms)

Pathogenicitymodels

Animal models mouse andembryonated chicken eggs

Reconstituted epithelialmodels chick chorio-allantoic model

Rice barley Plant modelsTomatoplants tomato fruitsapple fruitsMammalian modelimmunodepressedmice Invertebratemodel G mellonella

Wheat Corn plants Does not apply

Non-mammalian models Celegans D melanogaster Gmellonella B mori ZebrafishMammalian modelsmurine intravenous modelmurine gastrointestinalcolonization anddissemination model

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act very fast and within 30 h can kill 90 of inhaled conidia viaphagocytosis (Balloy and Chignard 2009) Neutrophils form thehighest number of intravascular phagocytes During infectionneutrophils are recruited to alveoli and phagocytose conidia andgermlings The direct killing mechanisms are however stillunclear Neutrophils also produce neutrophil extracellular traps(NETs) in response to A fumigatus which consist of chromatincovered with granular proteins that display some antimicrobialactivity (Bruns et al 2010)

Host protein receptors are involved in triggering the immuneresponse against A fumigatus including secreted complementfactors or those exposed on the surface of phagocytes such asdectin-1 (Steele et al 2005) As long as inhaled A fumigatus conidiaare covered with a proteinaceous layer formed by hydrophobinsthe fungus is masked and immunologically inert (Aimaniandaet al 2009) Once conidia start to germinate the rodlet layer isnot present on the cell surface leading to exposure of surfacecomponents recognized by immune cells C-type lectin and Toll-like receptors on host immune cells have for instance beenproposed to bind to fungal ligands and are involved in activationof the immune response through production of cytokines andchemokines prostaglandins and reactive oxygen intermediates(Brakhage et al 2010)

234 Development of anti-infective strategiesThe current guideline-recommended therapy against invasive

aspergillosis is the triazole antibiotic voriconazole which is supe-rior to the toxic polyene amphotericin B deoxycholate (Herbrechtet al 2002 Steinbach 2013) While a 2002 clinical trial found animproved response with voriconazole (528) versus amphotericinB (316) the field is still in need of improved antifungals withentirely novel targets As recently reported (Steinbach 2013) acombination therapy of antifungals may be promising akin toother medical disciplines whereby agents with differentmechanisms are employed to provide a synergistic response Inaddition the future holds promise for novel developments suchas the generation of immunotherapies based on T cells or den-dritic cells These therapies are based on in vitro proliferation ofthese cell types upon stimulation by fungus-specific antigensleading to cell-based vaccination (Lehrnbecher et al 2013)

235 Contribution of fungal secondary metabolism to virulenceBased on the genome sequence of A fumigatus it is estimated

that the fungus encodes at least 39 secondary metabolism geneclusters which lead to biosynthesis of various low molecularweight compounds These compounds are often synthesized bynon-ribosomal peptide synthetases (NRPSs) or polyketidesynthases (PKSs) (Brakhage 2013 Inglis et al 2013) Some ofthe compounds produced by these secondary metabolism geneclusters have been identified such as siderophores DHN melaninand gliotoxin and their involvement in virulence shown (Scharfet al 2014 Schrettl et al 2004) It is likely however that othercompounds of A fumigatus will contribute to virulence

24 Conclusions

In the last 20 years considerable progress has been made indevelopment of advanced genetic tools for A fumigatus Whilemore than 400 mutant strains have been generated (Horn et al2012) only a few virulence determinants have been characterizedAt present systems biology approaches are being explored as anpotential means to describe the interaction between human hostand pathogen However the huge amount of data generated byRNA-seq and proteome analyses of both pathogen and hostcells requires development of sophisticated bioinformatic toolsComplete genome-wise gene knock-out libraries would facilitate


screening efforts such as analysis of the mode of action of anti-fungal compounds but are not yet available for A fumigatus Inparallel huge efforts are being made to gain insight into virulencemechanisms by analyzing the interaction of A fumigatus with dif-ferent cellular systems such as neutrophils macrophages epithe-lial cells and dendritic cells with increasing complexity such ascombining different cellular systems

To bridge basic research on the pathobiology of A fumigatuswith clinical requirements a so-called Translational Systems Biol-ogy approach has been started which aims to integrate differentlsquolsquoomicsrsquorsquo data levels and image-based data of host-pathogen inter-actions into network and spatio-temporal computational modelsThe main goals are to identify and validate new drugs and newdrug targets increase the efficiency of pathogen identification dur-ing an infection and identify effective therapies (Horn et al 2012)

3 Candida species

Candida albicans is normally a commensal of the human micro-flora but is also a classic opportunistic pathogen causing mucosalthrush blood stream and systemic infections termed candidaemiaand invasive candidiasis respectively Candida infections are nor-mally associated with individuals who are immunocompromisedor traumatized due to major surgery transplantation and invasivemedical treatments that disrupt the normal protective bacterialmicroflora Five species account for around 95 of all Candidainfections namely C albicans C tropicalis C glabrata C kruseiand C parapsilosis (Pfaller et al 2009) Infections are associatedwith high mortality rates which can vary between 20 and 60depending on the specific risk factors and patient group(Calderone and Clancy 2012)

C albicans belongs to the Saccharomycetaceae which includesyeasts mycelial and pleiomorphic organisms (Table 1) Many ofthis family can undergo complete sexual cycles but C albicanshas not been demonstrated to undergo meiosis leading to asco-spore formation although most of the S cerevisiae homologs formeiosis-associated genes are present in its genome

An important feature of biology of C albicans is its ability toexist in different morphological forms (Supplementary Fig 2B)under different environmental conditions The major vegetativeforms of C albicans are (i) budding yeast cells (ii) elongated co-joined yeast cells that undergo synchronous cell division calledpseudohyphae and (iii) un-constricted branching true hyphae thatgrow exclusively by apical extension (Sudbery 2011) Each of thesegrowth forms is associated with changes in cell cycle regulationexpression of morphology-specific gene sets and differences inhost immune response Therefore morphogenesis of C albicansimpacts directly on pathogenesis (Gow and Hube 2012) Mostmonomorphic mutants are attenuated in virulence (Sudberyet al 2004) C albicans also forms asexual chlamydospores whichare seldom if ever observed in human tissues

Another important cell type in C albicans is the mating compe-tent opaque cell The white-opaque phenotypic switch was firstdescribed in the 1980s (Slutsky et al 1985 1987) and later shownto be critically important in determining mating competence ofopposite mating types (Hull and Johnson 1999 Hull et al 2000Magee and Magee 2000) Several transcription factors includingWor1 Czf1 Wor2 and Efg1 play important roles in controllingwhite-opaque switching (Miller and Johnson 2002 Zordan et al2006) Diploid strains will mate if partners are in the opaque formand homozygous (aa or aa) for the MLT (mating-type-like) locus(Miller and Johnson 2002) and mating partners form elongatedconjugation tubes called shmoos that undergo chemotropism andfusion (Lockhart et al 2003) After fusion the tetraploid nucleusundergoes concerted chromosome loss to regenerate the diploid

state (Supplementary Fig 2B) Homothallic (same sex) mating ofdiploids has also been reported in strains lacking the Bar1 proteasewhich inactivates a-pheromone enabling autocrine signaling(Alby et al 2009)

Recently a screen for aneuploid isolates discovered strains thatwere fully haploid for all eight chromosomes but retained either aMLTa or a MLTa locus on chromosome 5 These strains were oftenunstable and showed reduced overall fitness but could either mateor autodiploidise to regenerate diploid variants (Hickman et al2013) The haploid strains of C albicans have a full morphogeneticrepertoire and have significant potential in development of newgenetic strategies including forward genetic screens for analysisof C albicans biology

31 Comparative genomics in Candida species

Of the pathogenic Candida species C albicans C tropicalis andC parapsilosis are diploid species belonging to the so-called lsquolsquoCTGcladersquorsquo characterized by a somatic CTG codon reassignment in theirgenomes The CTG codon encodes serine and not leucine and there-fore results in the fungus mistranslating heterologous recombinantproteins (Santos and Tuite 1995) This discovery paved the way forgeneration of codon-corrected reporter genes and genetic markersrequired for molecular genetic analyses C glabrata and C kruseiare more distant relatives of the CTG clade species They are hap-loid and more closely related to S cerevisiae than to C albicansMembers of the CTG clade have not undergone an ancient wholegenome duplication which significantly shaped the genomes ofthe S cerevisiae group of fungi

C albicans is typical of most pathogenic Candida species in hav-ing a genome size of around 6000 predicted genes (Table 2) (Butleret al 2009) The nearest phylogenetic relative to C albicans isC dubliniensis yet this species is considerably less virulent thanC albicans and many more distantly related Candida species(Jackson et al 2009) Expansion of certain virulence-associatedgene families often occurs in the sub-telomeric regions of Candidaspecies ndash such as with the EPA gene family of adhesins of C glab-rata (Kaur et al 2007) The genetic diversity of such cell wall pro-teins within a species is often further increased by expansion oftrinucleotide repeat regions within rapidly evolving genes Also anumber of gene families are clearly enriched in pathogenic speciesof Candida including those encoding classes of cell wall proteinsiron assimilation properties and major facilitator and other trans-porters (Butler et al 2009) Genetic diversity within and betweenspecies is further increased by recombination events that havetaken place between Major Repeat Sequences (MRS) and previousretrotransposons Other chromosome changes such as the forma-tion of partially aneuploid strains and strains with isochromo-somes have been shown to affect antifungal drug sensitivities(Selmecki et al 2006)

32 The toolbox for C albicans

C albicans diploidy and codon usage present significant techni-cal difficulties in performing functional genetics (see above) Con-sequently bespoke nutritional markers used in gene disruptionprotocols and reporter genes had to be developed for use in thisorganism (Table 3) Genes from other organisms naturally devoidof CTG codons or which have been codon-corrected or fullycodon-optimized genes have been used to enable heterologousexpression (Fonzi and Irwin 1993 Gerami-Nejad et al 2009)

The progenitor method of gene disruption methodologies in Calbicans has been based on the so-called lsquolsquoUra-blasterrsquorsquo method(Fonzi and Irwin 1993) The use of this system is associated withsome well recognized problems mainly related to the issue thatnull mutants are heterozygous for URA3 and the Ura3 gene product


can become limiting in vivo affecting virulence (Brand et al 2004)In order to circumvent this URA3 can be integrated into a highexpression locus and alternative genetically marked strains havebeen generated based on complementation of leu2 his1 and arg4which do not have compromised virulence Other C albicans strainshave been generated that use non-nutritional-based selectablemarkers such as nourseothricin and flipper (FLP) or Cre-lox recom-binase to rapidly excise selectable markers (Table 3) In addition

Fig 2 Pathogenicity assays (A) Laboratory rodents used as models for fungal research oachieved via cortisone or corticosteroid treatment to mimic a leukopenia Ways of infectdirectly from spore suspension and respiration in an aerosol chamber to generate pulmon(CAM) (adapted from Gow et al 2003) (C) Larvae of the greater wax moth (Galleria meTipically the infection is performed via micro-injection in the posterior pseudopod ProEmbryonated eggs used as infection models in A fumigatus and C albicans research Thchamber is then generated applying a negative pressure from the blunt end hole Afterchamber onto the chorioallantoic chamber using a sterile syringe The holes can then beshell membrane As air sac Ys yolk sac Chm chorioallantoic membrane Alc allantoicweek old tomato seedlings (cultivar Money Maker) are inoculated with F oxysporum stvermiculite and incubated in a growth chamber at 28 C Evaluation is performed usingplant (F-G) Invasive growth assay on living fruit tissue Apple fruits (F) or tomato fruits28 C for 3 days (HndashK) M oryzae assays (H) Typical oval-shaped lesions on rice leaves c(kindly donated by Dr Michael J Kershaw) (I) Invasive hyphae on rice sheath at 29 h po6 days post-inoculation (kindly donated by Dr Michael J Kershaw) (K) Drop inoculation aold maize seedlings Disease symptoms are scored according to different categories basethe leaves 4 Large tumors on leaves 5 Heavy tumors on the base of the stem andor dthird leaf in field tests (cv CYMMIT) (N) Pycnidia produced by the strain IPO94269 on wpycnidia and cirrhi (P) Inoculation of Z tritici onto the second leaf using a paintbrush in g35 dpi isolates INRA08-FS0002 (1) and isolate IPO323 (2) on cv Apache (E) (Suffert et

several systems have been developed that use regulated promotersto control expression of a single functional allele (Samaranayakeand Hanes 2011)

A wide range of in vitro ex vivo and in vivo assays have beendeveloped to assess the virulence of wild type and mutant strainsof Candida species (Table 3) In vitro assays include exposure to pri-mary cell cultures or cell lines of epithelial immune cells and themeasurement of cell damage by the release of chromium-51 or

f A fumigatus C albicans and F oxysporum Immunosuppression of the host can beion include injection in the tail vein to generate a disseminated infection inhalationary infection (B) C albicans invading the chicken embryo chorioallantoic membranellonella) used to investigate virulence of A fumigatus C albicans and F oxysporumgression of the fungal infection is associated with melanization of the larvae (D)e egg must be perforated at the blunt end and on the side where an artificial airperforation of the shell membrane the inoculum is injected into the artificial air

sealed with paraffin Art ch artificial air chamber S shell Amc amniotic cavity Smcavity Alb albumin (EndashG) F oxysporum assays (E) Tomato plant root assay Two

rains by immersing the roots in a microconidial suspension for 30 min planted ina disease index for Fusarium vascular wilt going from 1 = healthy plant to 5 = dead(G) are inoculated with F oxysporum strains and incubated in a humid chamber atultivar (cv) Co-39 generated after spray inoculation of the pathogen (strain Guy11)st-inoculation obtained from a rice leaf sheath assay (J) Spray inoculation of barleyssay on barley 6 days post-inoculation (L) U maydis strains are injected into 7-day-

d on tumor size 1 Chlorosis 2 Small swelling at ligula or stem 3 Small tumors onead plant (MndashQ) Z tritici Symptoms on wheat leaves (M) Natural infection on theheat leaves (cv Obelisk) 21 days post inoculation (dpi) (O) Close up of (N) showingreenhouse (Q) Symptoms produced by different isolates of Z tritici on flag leaves at

al 2013) Images kindly donated by Dr Frederic Suffert and Dr Thierry C Marcel


lactate dehydrogenase from the human cells or the measurementproduction of pro-inflammatory and anti-inflammatory cytokinesby using ELISA or Cytometric Bead Arrays (CBA) as indicatorsof immune activation Interactions between Candida andphagocytes can also be visualized and quantified by live imagingvideo microscopy and fluorescence-activated cell sorting (FACS)analysis

Various in vivo models including invertebrate models mini-hosts and mouse models have been developed for Candida research(Table 3 and Fig 2) Invertebrate models include Drosophila mela-nogaster Caenorhabditis elegans the larval stages of the moth Gal-leria mellonella and the silk worm Bombyx mori These modelshave often been shown to be able to recapitulate the rank virulenceorder of strains as assessed in mice

The vertebrate mini-host model Zebrafish Danio rerio whichhas both innate and adaptive immune systems has also been usedto measure Candida virulence host response to infectious agentsthe efficacy of antifungal drugs and the cell biology of phago-cytendashpathogen interactions however the main vertebrate infectionmodel is still heavily focussed on murine studies Normally yeastcells are intravenously injected via tail vein and mouse mortalityis analyzed usually over two to four weeks Fungal organ burdensare often assessed in the kidney which is one of the main targetorgans in the mouse and in other tissues and these assays can becomplemented by measurements of in vivo cytokine levels A chickembryo chorioallantoic membrane (CAM) assay has also been usedas a pathogenicity model and invasion assay for C albicans(Fig 2B)

A Candida gut infection model has also been described whichallows disease progression across the gastrointestinal mucosaand subsequent systemic dissemination of the fungus to be inves-tigated (MacCallum 2012)


331 Genome functional analysisThe recent discovery of C albicans haploid strains is likely to

lead to emergence of new libraries of mutants and forward geneticscreens that will facilitate studies of developmental processes andthe search for antifungal drugs and drug target identificationprotocols

332 Altered host responsesA second major field of endeavor has been related to taking

advantage of emerging data from genome sequencing of thehuman genome to identify polymorphisms that correlate withaltered host resistance Such methodologies have already contrib-uted significantly to our understanding of the recognition and sig-naling pathways that articulate host responses (Lionakis andNetea 2013 Plantinga et al 2012) These studies have radicallyaltered our understanding of disease conditions such as chronicmucocutaneous candidiasis orophayngeal candidiasis persistentcandidaemia and vulvo-vaginal infections by identifying specificimmune defects in relevant pathways It is likely that this field willremain highly fertile in the next years

333 Yeast-to-hypha transitionA third area that has remained a major research focus over

many years is the regulation of the yeast-to-hypha transition(Sudbery 2011) The main pathways and cell biological processesthat regulate morphogenesis have been the subject of intenseinterest and activity with recent work defining how stressadaptation induces filamentous growth and how the elementsof the cytoskeleton vesicle secretion pathway and cell cyclemachinery articulate the modification of cell shape Hyphaldevelopment of C albicans has become the most worked on system

of filamentous growth in fungi and this is likely to retain itsposition as a pre-eminent model system for fungal developmentalbiology

334 Importance of the cell wall in immune recognitionYeastndashhyphal switching is but one of the ways in which Candida

species can alter their surface shape and surface properties Amajor field of research for this organism has therefore focused onunderstanding how the specific surface chemistry of the cell wallactivates represses or modulates immune recognition andimmune function (Gow et al 2011) Surface components are alsobeing investigated in the context of development of vaccine andnew generation diagnostics therefore a focus on cell wall chemis-try and host-fungus interactions will continue to have importancein both basic and translational research

34 Conclusions

Candida research has moved forward quickly in the molecularera with the constant enrichment of the field with new methodol-ogies This has propelled research particularly in C albicans whichhas emerged as one of the most studied microbial pathogensFuture work is seeing the translation of these technologies to theso called lsquolsquonon-albicansrsquorsquo species to address relevant clinical andbiological questions Some of the biggest clinical questions are stillto be answered and the need for vaccines sensitive diagnostics andnew drugs to improve clinical outcome remain strong researchdrivers for the future

4 Fusarium oxysporum

The first description of Fusarium was made by Link in 1809(Supplementary Fig 1C) In 1940 Snyder and Hansen grouped allthe species of the genus in 9 taxa and reclassified the infragenericgroup called elegans into a single F oxysporum species designatingdifferent formae speciales (ff spp) based on their pathogenicity ondifferent plant species (Snyder and Hansen 1940) To date morethan 150 ff spp have been reported (Michielse and Rep 2009)Interestingly molecular phylogenetic studies have revealed sub-stantial genetic diversity among isolates supporting the currentview that F oxysporum represents a species complex In 1998 apioneering study established that field isolates of the f sp cubensehave polyphyletic origins suggesting that the capacity to infect agiven plant host has arisen several times during evolution(OrsquoDonnell et al 1998)

Fusarium oxysporum causes vascular wilt disease Sporespresent in the soil germinate in response to signals from theplant host and differentiate infection hyphae which adhere tothe plant roots and penetrate them directly without the needfor specialized infection structures (Supplementary Fig 2C) Rootpenetration appears to occur predominantly through naturalopenings at the intercellular junctions of cortical cells or throughwounds (Perez-Nadales and Di Pietro 2011) Once inside theroot hyphae grow inter- and intracellularly to invade the cortexand cross the endodermis until they reach the xylem vesselsThe fungus then uses the xylem as a conduit to colonize thehost Disease symptoms include wilting chlorosis necrosispremature leaf loss browning of the vascular system andstunting which eventually will lead to plant death (Michielseand Rep 2009) Small oval-shaped microconida falcate macroco-nidia and thick-walled chlamydospores are formed on the deadplant tissue and in soil F oxysporum can survive in soil forextended time periods either as chlamydospores or by growingsaprophytically on organic compounds until a new cycle ofinfection starts (Agrios 2005)


Although a sexual stage has not yet been described the genomecontains apparently functional mating type idiomorphs (MAT1-1and MAT-1-2) similar to those found in sexual Fusarium speciesThe mat1-1-1 gene encodes a protein carrying an a-box motifwhereas mat1-2-1 encodes a transcription factor with a high-mobility-group (HMG) DNA binding domain F oxysporum MATgenes are expressed and all the predicted introns are correctly pro-cessed (Arie et al 2000) These observations indicate that similarto other fungal pathogens previously thought to lack sex thepresence of a cryptic sexual cycle may remain to be discoveredin F oxysporum

The genome sequence of the tomato pathogenic isolate F oxy-sporum f sp lycopersici was published in 2010 (Ma et al 2010)Since then eleven additional F oxysporum strains have beensequenced by the Broad Institute The availability of the completegenome sequences (accesible at the Fusarium Comparative Data-base Table 3) as well as of molecular tools and well-establishedpathogenicity assays has allowed to address the genetic basesand evolutionary origins of pathogenicity and host range inF oxysporum

41 Overview of the F oxysporum genome

The genome sequencing assembly and annotation of Fusariumoxysporum f sp lycopersici was performed by the Broad Instituteas part of the Fusarium Comparative Sequencing Project Astriking feature is that 28 of the F oxysporum genome corre-sponds to repetitive sequences including many retroelementsand short interspersed elements (SINEs) as well as class II trans-posable elements (TEs) (Table 2) Comparison of the F oxysporumgenome with those of F graminearum and F verticillioides led tothe discovery of four supernumerary chromosomes that areenriched for TEs and for genes putatively related to hostndashpathogeninteractions (Ma et al 2010) These so-called lineage-specific (LS)regions contain more than 95 of all DNA transposons Only 20of the predicted genes in the LS regions could be functionallyclassified on the basis of homology to known proteins Manyencode predicted secreted effectors virulence factors transcrip-tion factors and proteins involved in signal transduction but lesshousekeeping proteins Recent data suggest that LS regions ofF oxysporum strains with different host specificities may differconsiderably in sequence

42 Assessing virulence of F oxysporum

A variety of virulence assays have been developed in F oxyspo-rum to study invasive growth and pathogenicity (Table 3) Thetomato root infection assay is performed by immersing the rootsof two week-old seedlings in a microconidial suspension in dis-tilled water followed by planting in vermiculite and maintenancein a growth chamber at 28 C (Di Pietro and Roncero 1998) Theseverity of disease symptoms has been traditionally measuredusing a disease index ranging from 1 (healthy plant) to 5 (deadplant) (Fig 2E) Alternative methods involve measurement of vas-cular browning determination of the plant weight above the coty-ledons and using a disease index on a scale from 0 (healthy plant)to 4 (very small wilted or dead plant) (Michielse et al 2009) Morerecently disease severity has been plotted as a percentage of plantsurvival against time by recording mortality each day for 30ndash45 days This method allows statistical analysis of survival ratesby the KaplanndashMeier method and comparison among groups usingthe log-rank test (Lopez-Berges et al 2012 2013) In planta quan-tification of fungal biomass is performed by extracting total geno-mic DNA from infected tomato roots andor stems at differenttimes post-infection followed by quantitative real-time PCR anal-ysis (Pareja-Jaime et al 2010) using the Fusarium-specific six1

gene (Rep et al 2004) and normalization to the tomato gadphgene Quantitative assessment of root penetration by F oxysporumgerm tubes during the initial 12ndash24 h after inoculation wasperformed using scanning electron microscopy (Perez-Nadalesand Di Pietro 2011) The ability of fungal germlings to attach toroots is determined by incubating host roots with fungal sporesfor 24ndash48 h in potato dextrose broth diluted 150 with water (DiPietro et al 2001 Prados Rosales and Di Pietro 2008)

Rapid invasive growth assays on tomato or apple fruit tissue(Fig 2E and F) are performed by injecting a microconidial suspen-sion into tomato fruits or apple slices and evaluating invasivegrowth and maceration of the surrounding fruit tissue (Di Pietroet al 2001) The in vitro cellophane penetration assay has beenshown to correlate significantly with in vivo pathogenicity ontomato plants For this assay the fungus is allowed to grow on acellophane membrane placed on a solid agar medium plate Thecellophane with the fungal colony is removed 2ndash4 days after inoc-ulation and the ability of the fungus to penetrate the membrane toreach the underlying medium is evaluated (Prados Rosales and DiPietro 2008)

A mouse infection model was established for the tomatopathogenic F oxysporum f sp lycopersici strain making this thefirst fungal isolate to serve as a dual model for the study of fungalpathogenicity in plants and mammals (Ortoneda et al 2004)Immunosuppressed mice are inoculated by injecting microconidiainto a lateral vein of the tail Mortality of the animals is recordedeach day for 15 days and survival rates are estimated as describedabove for the plant root infection assay Fungal tissue burden inkidneys and lungs at 7 days post-infection was also evaluatedusing standard plating methods (Lopez-Berges et al 20122013 Ortoneda et al 2004) and histopathological analysis(Schaumlfer et al 2014) The greater wax moth Galleria mellonellahas been used as a non-vertebrate infection model to reduce theneed for using mammals for in vivo testing (Fig 2C) F oxysporumwas able to proliferate inside the haemocoel of G mellonellalarvae and to kill and colonize the insects Importantly mostgenes required for full virulence on immune-suppressed micealso played a significant role in G mellonella infection(Navarro-Velasco et al 2011)


431 Role of MAPK cascades in virulenceThe F oxysporum MAPK Fmk1 an orthologue of the yeast

Fus3Kss1 MAPKs was found to be essential for virulence ontomato plants (Di Pietro et al 2001) Infection-related processessuch as invasive growth vegetative hyphal fusion and rootadhesion (Di Pietro et al 2001 Prados Rosales and Di Pietro2008) absolutely require Fmk1 and are negatively controlled bythe nitrogen source ammonium (Lopez-Berges et al 2010)Because this MAPK is widely conserved among fungi anddetermines pathogenicity in all plant pathogens studied so far(Rispail et al 2009) a major effort has been directed towardselucidating the upstream and downstream components of thissignaling cascade The homeodomain transcription factor Ste12was shown to function downstream of Fmk1 and to be requiredfor invasive growth the most critical of the Fmk1-regulatedfunctions for plant infection (Rispail and Di Pietro 2009) Themucin-like transmembrane protein Msb2 was recently character-ized as an upstream component of the cascade (Perez-Nadalesand Di Pietro 2011) In addition to the Fmk1 pathway orthologuesof the S cerevisiae high osmolarity Hog1 and cell integrityMpk1 MAPK signaling cascades have also been identified inF oxysporum and are currently under investigation The Rho-typeGTPase Rho1 which functions upstream of Mpk1 was found to beessential for morphogenesis and pathogenicity (Martinez-Rocha


et al 2008) Future studies will address how signaling throughdifferent MAPK cascades is orchestrated to control infectiousgrowth in F oxysporum

432 Lineage specific (LS) chromosomesSequence characteristics of the genes present on the LS genome

regions indicate a distinct evolutionary origin from the core gen-ome suggesting that they could have been acquired through hori-zontal transfer from another Fusarium species This idea wasexperimentally supported by the finding that co-incubation oftwo strains of F oxysporum can result in transfer of small LS chro-mosomes from a tomato pathogenic to a non-pathogenic strainconverting the latter in a pathogen This led to the hypothesis thathorizontal chromosome transfer in F oxysporum can generate newpathogenic lineages (Ma et al 2010) The genetic and cellularmechanisms underlying these processes are currently the subjectof intensive study

433 Secreted effectors and gene-for-gene systemHost specificity between different races of F oxysporum f sp

lycopersici and tomato cultivars is determined by a set of pathogengenes encoding small secreted cysteine-rich effector proteinstermed SIX (Secreted In Xylem) (Rep et al 2004) One of these pro-teins Avr1 triggers a resistance response in tomato plants carryingthe matching resistance (R) gene I-1 Intriguingly Avr1 also func-tions as a virulence effector by suppressing disease resistance con-ferred by two other R genes I-2 and I-3 (Houterman et al 2008)Most six genes are located on the same LS chromosome (chromo-some 14) also called the pathogenicity chromosome and are asso-ciated with chromosomal sub-regions enriched for DNAtransposons (Ma et al 2010) Expression of most six genes is spe-cifically induced in planta and requires the transcription factorSge1 which is located on a core chromosome (Michielse and Rep2009)

434 F oxysporum as a model for fungal trans-kingdom pathogenicityF oxysporum f sp lycopersici isolate FGSC 9935 was the first

fungal strain reported to cause disease both on plant (tomato)and mammalian hosts (immunodepressed mice) (Ortonedaet al 2004) Since then a number of fungal genes have beenidentified that are either required for pathogenicity on tomatobut not on mice such as those encoding the Fmk1 MAPK or thesmall G protein Rho1 (Di Pietro et al 2001 Martinez-Rochaet al 2008) or for virulence on mice but not on plants such asthe pH response factor PacC (Caracuel et al 2003) or the secretedPathogenesis Related 1 (PR-1)ndashlike protein Fpr1 (Prados-Rosaleset al 2012) Recently HapX a transcription factor that governsiron homeostasis was characterized as the first virulencedeterminant required for both plant and animal infection in thesame fungal strain (Lopez-Berges et al 2012) Similarly thevelvet protein complex a conserved regulator of fungal develop-ment and secondary metabolism contributes to infection ofplants and mammals in part by promoting the biosynthesis ofthe depsipeptide mycotoxin beauvericin (Lopez-Berges et al2013) Most of the evidence obtained so far suggests that fungalpathogenicity on plants and animals may have fundamentallydistinct evolutionary origins

44 Conclusions

The establishment of F oxysporum as a plant and animal infec-tion model the use of molecular genetic approaches in this speciesand the genomic characterization of different ff spp has advancedour understanding on several key aspects related to fungal patho-genicity as well as our knowledge of the evolutionary origins andkey mechanisms underlying the parasitic lifestyle of fungi In the

future F oxysporum is likely to continue providing valuable newinsights into the molecular bases of both host specificity and path-ogenicity on evolutionary distant hosts

5 Ashbya gossypii

The filamentous ascomycete Ashbya gossypii belongs tothe genus Eremothecium and causes stigmatomycosis describedby Ashby and Nowell in 1926 (Supplementary Fig 1D)Stigmatomycosis or lsquoyeast spot diseasersquo was also linked to otherEremothecium species eg Eremothecium coryli (syn Nematosporacoryli) and is a fungal disease that occurs in a number of cropsespecially cotton and pistachio but also soybean pecan or citrusfruits These fungi are wound parasites and are transmitted byhemipteran insects (Dietrich et al 2013) A gossypii was alsoidentified as a natural overproducer of riboflavinvitamin B2 Inthe 1970s chemical synthesis of riboflavin was replaced bybiotechnological means using strains of A gossypii Candidafamata or Bacillus subtilis (Stahmann et al 2000) Ashbya belongsto the Saccharomycete family of yeasts This close relationship toyeast like fungi the small genome size (9 Mb) and its moleculargenetic tractability has turned Ashbya into an interesting modelsystem for fungal growth development and genome evolution(Dietrich et al 2004 Steiner et al 1995 Wendland andWalther 2005)

Needle-shaped Ashbya spores often connected by whip-likefilaments contain a single haploid nucleus each Spore germina-tion generates a ball-shaped germ cell (Wendland and Walther2005) (Supplementary Fig 2D) Polarity establishment requiringthe Cdc42-GTPase module results in germ tube emergence andthe establishment of polarized hyphal growth (Wendland andPhilippsen 2001) Once polar growth is established filamentousgrowth is maintained (Supplemental Fig 2D) Ashbya filamentsare septate and multi-nucleate Nuclear divisions in a commoncytoplasm are asynchronous based on localized G1-cyclintranscript accumulation (Gladfelter et al 2006) In juvenileAshbya mycelia additional axes of polarity are generated bylateral branches Ashbya mycelia mature upon compartmentali-zation of germlings by septation Hyphal maturation whichoccurs approximately 20 h after the isotropic-to-polar switch thatformed the first germ tube results in a dramatic increase ingrowth speed (10-fold) to about 200 lmh (Kohli et al 2008)In mature mycelia lateral branching is largely repressed andinstead new axes of polarity are established by apical dichoto-mous tip-branching events This generates the characteristicY-shaped hyphae of matured mycelia (Wendland and Walther2005)

At the end of the growth phase riboflavin overproduction is ini-tiated and a developmental programme results in the formation ofsporangia from septate hyphal segments Sporangia will usuallyharbor two bundles of 4 needle shaped uninucleate spores Thegenome of A gossypii strain ATCC 10895 contains four copies of aMATa mating type locus that harbors conserved a1 and a2 tran-scriptional regulators (Dietrich et al 2013 Wendland andWalther 2005) Three of the mating-type loci are close to the telo-meric ends on chromosomes 4 5 and 6 respectively which is rem-iniscent of the position of the silent mating-type cassettes HMRand HML on chromosome III in S cerevisiae The putative activemating-type locus on chromosome 6 shows conserved synteny toactive MAT-loci in Ascomycetes (Wendland and Walther 2005)Generally in sexually reproducing Ascomycetes mating partnersof two opposite mating-types (in S cerevisiae a or a) are attractedby a pheromone signal relay that leads to cell fusion karyogamyand formation of diploid nuclei that can undergo meiosis andspore formation Although the pheromone response pathway is


conserved in Ashbya deletion of key elements of this pathway egthe pheromone receptor genes STE2 and STE3 or the downstreamtranscription factor STE12 does not abolish sporulation(Wendland et al 2011)

51 Comparative genomics in Eremothecium

Several genomes within the genus Eremothecium have beenentirely sequenced and annotated The first was that of A gossypiiwhile the genome of E cymbalariae represents that of the typestrain of the genus that was isolated by Borzi in 1888 (Dietrichet al 2004 Wendland and Walther 2011) Recently an A gossypiiisolate from Florida was shown to bear 99 sequence identity withthe known A gossypii genome In addition Ashbya aceri representsa new species which shows 90 identity with A gossypii and itsgenome organization differs from A gossypii only by eight translo-cation events (Dietrich et al 2013)

A comparison of the A gossypii ATCC10895 and E cymbalariaegenomes revealed the basis for genome evolution in the Eremothe-cium genus The E cymbalariae genome contains 97 Mb (excludingrDNA-repeats) on eight chromosomes while that of A gossypii isonly 88 Mb distributed on only seven chromsomes (Table 2) Thisgenome reduction is largely due to shorter intergenic regions asthe number of genes is almost identical In Ashbya chromosomenumber was reduced by breakage of one chromosome at the cen-tromere and fusion of the chromosome arms to telomeres of otherchromosomes (Gordon et al 2011)

E cymbalariae hosts one (potentially non-functional) TY3-Gypsytransposon whereas in A gossypii no transposable element or LTR-remnants were found Furthermore the Ashbya genome shows avery high GC content of 52 compared to 40 in E cymbalariaeand in other Saccharomyces complex species Such a high GC con-tent may be the result of evolutionary recombination events thatdrive GC-content (Wendland et al 2011)


521 Riboflavin productionRiboflavinvitamin B2 is essential for basic cellular metabolism

since it is a precursor for many flavin-based coenzymes includingflavin mononucleotide and flavin adenine dinucleotide Whilemany microorganisms plants and fungi can synthesize riboflavinde novo from various carbon sources humans lack this abilityand must obtain this vitamin from their diet

Today A gossypii is one of the main organisms used for biotech-nological riboflavin production Riboflavin production in Ashbya ishighly increased at the end of the growth phase during sporulationRecently Ashbya gossypii was isolated from mouth parts of heter-opteran insects feeding on milkweed and oleander which producetoxic alkaloids This led to the interesting hypothesis that ribofla-vin produced by A gossypii helps these insects to feed on theseplants (Dietrich et al 2013) Previously it was shown that ribofla-vin provides some UV-protection for the spores (Stahmann et al2001) On the molecular level recent studies found that Yap1 atranscription factor that regulates responses to oxidative stressalso provides a link to riboflavin production (Walther andWendland 2012) Engineering of strains to increase riboflavin pro-duction included eg increasing the pool of riboflavin precursorsGTP and glycine (Kato and Park 2012) The molecular details link-ing riboflavin production and sporulation are currently beinginvestigated

522 Hyphal growthBased on comparative biology studies between S cerevisiae and

Ashbya it was found that multiple Rho-protein modules areinvolved in processes of cell polarity establishment cell wall

integrity and maintenance of polarized hyphal growth (Wendlandand Philippsen 2001) Further studies characterized polarisomecomponents Spa2 and Bni1 and the Ras-like GTPase Bud1 and theirroles in maintaining hyphal cell polarity (Bauer et al 2004Schmitz et al 2006) Recently the transmembrane protein Axl2was found to be involved in polarity establishment polarity main-tenance and stress response (Anker and Gladfelter 2011) Hyphalmaturation in Ashbya results in increased growth speed and sup-presses lateral branching in favor of dichotomous branching atthe hyphal tips Tip splitting was shown to be dependent on theactivity of the p21-activated kinase Cla4 and its downstream effec-tor Pxl1 which may coordinate polarity factors and vesicle pro-cessing during tip splitting and fast hyphal elongation (Wendlandand Walther 2005) Comparative analyses between Eremotheciumspecies and S cerevisiae will elucidate gene expression differencestranscriptional circuitry the analysis of protein complexes andpolarized transport This will shed light on how Bauplan differencescan be generated from apparently the same set of genes

523 SeptationThe actin cytoskeleton plays a major role in polarized hyphal

growth Actin is found in three structures as linear actin filamentsemanating from the polarisome at hyphal tips as contractile actinrings at sites of septation and as actin patches at sites of endocyto-sis Generating linear actin filaments at sites of polarized growth isa function of the polarity establishment machinery and involvesCdc42 the formin Bni1 and the polarisome (Schmitz et al 2006Wendland and Walther 2005)

Localization of septal sites is directed via cortical cues so calledlsquolsquolandmarksrsquorsquo which usually are membrane-associated proteinsthat direct proteins or protein complexes to specific sites in thecell In A gossypii the yeast Bud3 landmark homolog was shownto be involved in septum formation Bud3 localizes to septal sitesand bud3 mutants are partially defective in actin ring formationdue to the mislocalization of Cyk1 (ScIqg1) which is essential foractin ring formation (Wendland and Walther 2005) Cyk2Hof1is also required for actin ring formation and its SH3-domain isrequired for actin ring integrity (Kaufmann and Philippsen2009) The coiled-coil domains of the Cdc11 and Cdc12 septinsare required for septin ring formation and phosphorylation of sep-tins plays an essential role for septin dynamics (Meseroll et al2013) The novel process of septin assembly termed lsquoannealingrsquowas found to depend on membranes which allows cells to use thisintrinsic property of septins at specific sites at the cell cortex(Bridges et al 2014)

524 Nuclear division and movementCellular compartments within Ashbya hyphae are generated via

septation These compartments are multi-nucleate and will betransformed into sporangia at a later developmental stage In con-trast to S cerevisiae nuclei in A gossypii divide asynchronously(Gladfelter et al 2006) The lengths of the nuclear division cyclesof individual nuclei can range from 45 min up to 200 min andasynchrony seems to be intrinsic since it returns rapidly after arti-ficial synchronization Recently specific localization of a G1 cyclintranscript by the Whi3 RNA-binding protein has been shown tocontribute to this asynchrony (Gladfelter et al 2006)

In S cerevisiae mitotic divisions require two regulatory net-works the Cdc fourteen early anaphase release (FEAR) and themitotic exit network (MEN) both of which drive exit from mitosisby activating the Cdc14 phosphatase While the A gossypii FEARnetwork seems to be conserved in regulating the MG1 transitionthe A gossypii MEN pathway was shown to have diverged signifi-cantly from its anticipated role in exit from mitosis and appearsto play a crucial role in septum formation (Finlayson et al 2011)


During hyphal growth nuclei need to migrate long distancestoward the growing tip using cytoplasmic microtubules In addi-tion to this long-range migration nuclei of A gossypii show alsoextensive oscillation rotation and occasional bypassing of oneanother Similar to S cerevisiae spindle pole bodies (SPBs) are theonly microtubule organizing centers in A gossypii The laminarSPB ultra structure is similar between S cerevisiae and A gossypiialthough there are differences at the cytoplasmic phase and addi-tional types of cMTs (long tangential cMTs) were identifiedAccordingly significant differences regarding the control of cMTdynamics and the role of MT-dependent motor proteins were iden-tified between S cerevisiae and A gossypii (Grava and Philippsen2010)

525 Genome evolutionComparative genomics are being employed to analyze evolu-

tionary trajectories in A gossypii and E cymbalariae Broad changescan be seen in terms of the size of clusters of synteny between bothspecies or to the yeast ancestor The E cymbalariae genome bearscloser resemblance to the yeast ancestor in terms of number ofchromosomes arrangement of mating-type loci conservation oftelomeric loci and GC content (Wendland and Walther 2011)Establishing other genome sequences within this genus will cer-tainly provide more detailed insight into Eremothecium genomeevolution This could lead for example to the compilation of thegenome of an Eremothecium ancestor at the base of the Eremothe-cium lineage that could be compared with other ancestral gen-omes eg that of the yeast ancestor just prior to the wholegenome duplication

53 Conclusions

Ashbya has a key role in the biotechnological production of ribo-flavin Molecular studies will further elucidate the co-regulation ofriboflavin production and sporulation Comparative biology with Agossypii and S cerevisiae will help to understand the regulatorymechanisms governing the Bauplan differences of yeast and hyphalcells This may also result in understanding the molecular mecha-nisms of lsquoyeast spot diseasersquo

6 Magnaporthe oryzae

The plant-pathogenic fungus Magnaporthe oryzae is a filamen-tous ascomycete that causes blast disease on rice (Oryza sativa)the principal disease of cultivated rice worldwide with yield lossesvarying between 10 and 30 (Talbot 2003) The pathogen affectsseveral other grasses such as wheat finger millet and barley(Talbot 2003) (Table 1) Magnaporthe oryzae BC Couch was firstisolated from rice (Oryza sativa) by F Cavara in 1892 as Pyriculariaoryzae (Couch and Kohn 2002 Rossman et al 1990) Rice blastdisease has however been known since 7000 BC when domestica-tion of O sativa started in the Yangtze Valley in China (Couch et al2005) (Supplementary Fig 1E) M oryzae was described as ricefever disease in China in 1637 and only later did it become morewidely known as the rice blast fungus (Ou 1985) The actual namelsquolsquoblastrsquorsquo refers to the fast expansion of the disease within the ricefields

The life cycle of M oryzae consists of a hemi-biotrophic infec-tion cycle and predominantly asexual mode of reproduction (Sup-plementary Fig 2E) The sexual stage exists but is rarely found innature in rice-infecting populations (Notteghem and Silueacute 1992Talbot et al 1993b) Analyses of M oryzae populations showedthat in most areas only one of the mating types MAT1-1 orMAT1-2 predominates or when both are present isolates lack fer-tility (Notteghem and Silueacute 1992) However it is possible to

initiate sexual reproduction by pairing fertile strains of oppositemating types (Notteghem and Silueacute 1992 Valent et al 1991) Thisleads to development of fruiting bodies (perithecia) which encloseasci filled with octads of ascospores (Valent et al 1991)

During its asexual cycle M oryzae produces three-cell conidiawhich are spread by dew drop splash (Talbot 2003) The infectioncycle starts when a conidium lands on a rice leaf (Hamer et al1988) A polarized germ tube emerges from the tip and elongatesbecoming flattened against the surface a phase known as hookingAt this stage physical cues such as hydrophobicity surface hard-ness and plant signals such as cutin monomers trigger the forma-tion of specialized infection structures called appressoria (Talbotet al 1993a) The appressorium is a single dome-shaped melanisedcell which is able to break the cuticle by translating high internalturgor into mechanical force (Dixon et al 1999) It contains a poreat its base from which a penetration peg emerges to break throughthe cuticle of the rice leaf initiating invasive growth Invasivehyphae are surrounded by the plant plasma membrane and ramifythrough plant tissue for 3ndash4 days until lesion formation occurs(Kankanala et al 2007) After 72 h almost 10 of biomass of a riceleaf can be M oryzae hyphae in heavy infections (Talbot et al1993b) After 4ndash5 days disease lesions are visible on the plant sur-face from which conidiophores develop to produce conidia thatspread to new plants (Talbot et al 1993a)

In the last 20 years M oryzae has emerged as a model organismin phytopathology especially as a system for the study of theplant-pathogen interaction The availability of methods forin vitro cultivation and stimulation of appressorium developmenttogether with the availability of the genome sequence and tools forclassical and molecular genetics makes this fungus an excellentexperimental model (Wilson and Talbot 2009)

61 Overview of the M oryzae genome

In 2002 the Fungal Genome Initiative (FGI) released a draft gen-ome sequence of M oryzae strain 70ndash15 The final genomesequence obtained by whole-genome shotgun sequencing waspublished in 2005 (Dean et al 2005) The total size of the M oryzaegenome is 417 Mb and is organized in 7 chromosomes (Table 2)The actual total number of genes is predicted to be between12827 and 16000 with a density of at least one gene every 4 KbRepetitive DNA is prevalent in the genome with 15 families oftransposable elements present There are 4734 M oryzae genes incommon with S cerevisiae

62 Assessing virulence of M oryzae

Magnaporthe oryzae has emerged as a major model organism forplant microbe interactions The fungus can be grown in vitro andinfection structures can be generated on artificial surfaces suchas glass or Teflon (Polytetrafluoroethylene) (Table 3) This charac-teristic and the availability of standard pathogenicity assays inboth rice and barley make M oryzae an excellent model to studyplant microbe interactions Among pathogenicity assays the mostwidely used method is spray-inoculation of 3ndash4 leaf staged riceseedlings Plants are stored for 1 day at high relative humidity(RH gt 95) and then transferred to growth chambers for 5ndash10 daysuntil symptoms are detectable (Fig 2H) (Valent et al 1991) Viru-lence can be quantified according to different criteria such as lesionnumber per leaf average lesion size and lesion sporulation rate(sporescm2) The rice leaf sheath assay readily allows observationof invasive hyphae inside cells (Kankanala et al 2007) (Fig 2I)Spray and drop-inoculation of barley are also used as pathogenicityassay (Fig 2J and K) Sterilized onion epidermis and detached riceleaves using a spot inoculation method can also be used to monitorpenetration and invasion (Fig 2K)



631 Mechanisms of fungal infectionThe appressorium is a specialized infection structure which

allows M oryzae to invade its host Generation of turgor insidethe appressorial dome results in production of a penetration pegThe penetration peg is formed following cytoskeletal repolariza-tion mediated by septin GTPases (Dagdas et al 2012) Septins forma toroidal ring structure at the base of the appressorial pore whichleads to the reorientation of the F-actin cytoskeleton and initiationof membrane curvature and peg development (Dagdas et al 2012)Regulation of cytoskeleton rearrangement is controlled throughthe regulated synthesis of reactive oxygen species by NADPH oxi-dases (Ryder et al 2013) Discovery of this toroidal structure atthe base of the appressorium has opened new research questionsregarding the remodeling of septins which lead to aggregation ofF-actin and how these processes are coordinated with turgor gen-eration The characterization of these intracellular mechanismswill be critical for understanding successful appressorium forma-tion and initial plant infection caused by M oryzae in the riceplants

632 Plant pathogen interactions in the rice blast fungus M oryzaeDuring plant tissue invasion M oryzae secretes a battery of

effector proteins to counteract plant defense responses and manip-ulate the host and facilitate fungal growth Effectors suppressPAMP (Pathogen-Associated Molecular Patterns) triggered immu-nity responses of the plant to allow a successful invasion and dis-ease M oryzae secretes both apoplastic and cytoplasmic effectorsduring infection Cytoplasmic effectors accumulate in thebiotrophic interfacial complex (BIC) A well-known example is Moryzae Avr-Pita effector that confers resistance to rice cultivarsexpressing the R gene Pita Avr-Pita is translocated and accumu-lated at the BIC however very little is known about its virulencefunction except that it appears to interact with its cognate resis-tance protein Pi-ta when present (Jia et al 2000) It has recentlybeen reported that cytoplasmic effectors may be secreted via anunconventional secretion system involving the exocyst complex(Giraldo et al 2013) Apoplastic effectors on the other hand aresecreted through the conventional tip secretion pathway (Giraldoet al 2013) Interesting ongoing studies aim to determine thetranslocation motif for effectors going through the BIC and whatdistinguishes these from effectors accumulating in the apoplast

633 SignalingSeveral signaling cascades are implicated in plant infection by

M oryzae including MAP kinase cAMP-signaling and the Ca2+cal-cineurin pathways All of these pathways mediate the intracellularresponse to environmental signals perceived from the plant andthe prevailing environmental conditions

M oryzae possesses three MAP kinase pathways including theMAPKs Osm1 Mps1 and Pmk1 (Dixon et al 1999 Xu andHamer 1996 Xu et al 1998) Null mutants of the Osm1 MAPkinase show defects in sporulation and increased sensitivity toosmotic stress but remain fully pathogenic indicating that appres-sorium turgor responses are unaffected by loss of hyperosmoticstress adaptation (Dixon et al 1999) The Mps1 MAP kinase isinvolved in cell wall integrity and M oryzae null mps1 mutantsare non-pathogenic because they are not able to form the penetra-tion peg responsible for cuticle penetration (Xu and Hamer 1996Xu et al 1998) Null mutants of Pmk1 MAP kinase are unable toproduce appressoria or invade the host (Xu and Hamer 1996)

The cAMP signaling pathway is also involved in pathogenicityand required for formation of the appressoria (Adachi andHamer 1998) Cyclic AMP (cAMP) produced by the adenylatecyclase binds to the regulatory subunit of the the cAMP-dependent

protein kinase A (RPKA) releasing the catalytic subunit PKA (CPKA)Active CPKA activates by phosphorylation several target proteinsMutants of the adenylate cyclase gene MAC1 are not able to formappressoria a defect which can be restored by addition of exoge-nous cAMP (Adachi and Hamer 1998 Wilson and Talbot 2009)Constitutive activation of e CPKA by a mutation in RPKA1 leads torestoration of the appressorium development defect of Dmac1mutants (Choi and Dean 1997) Dcpka mutants are able to formappressoria but they are non-functional and fail to penetrate(Wilson and Talbot 2009) These results suggest that PKA medi-ated activation of the cAMP pathway is essential to form functionalappressoria

The Ca2+calcineurin pathway also plays an important role inthe growth and pathogenicity of M oryzae Environmental stressincreases intracellular Ca2+ level and leads to the binding of Ca2+

to calmodulin The Ca2+calmodulin complex activates calcineurina highly conserved protein phosphatase heterodimer which deph-oshorylates the transcription factor Crz1 to induce the expressionof stress responsive genes Deletion of the CRZ1 gene results inlower sporulation and highly reduced pathogenicity (Choi et al2009)

Studies have shown that there is an interaction between Pmk1MAPK pathway and cAMP signaling (Li et al 2012) and it is likelythat there is cross talk between Mps1 MAPK and Ca2+calcineurinpathways similar to that found in yeast (Li et al 2012) One cur-rent line of research is to establish the nature of these interactionsand to elucidate which processes they affect

64 Conclusions

Although the first biological studies on Magnaporthe oryzaewere performed more than 100 years ago it is only within the last20 years that it has attained model organism status With highlyefficient cells specialized in penetration (appressoria) and infection(biotrophic infectious hyphae) and the possibility to study thesestructures in vitro and in vivo M oryzae is a valuable model tostudy plant-pathogen interactions Several genetic tools includingtransformation by homologous recombination availability ofreporter genes for live cell imaging together with a fullysequenced genome and extensive expression data and mutant col-lections has led to new insight into the process of plant infection

Research on M oryzae has focused primarily on the molecularbiology of plant-pathogen interaction identification of virulencedeterminants by mutation fungal infection mechanisms and sig-naling pathways required for pathogenicity Applying this knowl-edge to disease control will be critical in future The currentspread of wheat blast disease in South America including regionsin Brazil Bolivia and Paraguay has opened up new research involv-ing comparative genomics and epidemiology of populationsaffecting rice wild grasses and wheat It will be critical to developnew and more durable control strategies for this devastatingdisease

7 Ustilago maydis

Corn smut disease caused by U maydis has interested biologistsand farmers alike for more than 250 years (Supplementary Fig 1F)But in spite of this the complete life cycle of U maydis remainedmysterious until the sexual stage was discovered in 1927(Stakman and Christensen 1927) Genetic studies in U maydiswere initiated with the isolation of biochemical mutants byPerkins (1949) With the help of mutants and diploid strains inwhich homologous recombination could be induced Hollidaywas able to deduce the formation of heteroduplex DNA duringcrossing over with a four-armed junction intermediate later


termed the Holliday junction yielding the first model for homolo-gous recombination (Holliday 1964)

With the advent of molecular genetics a wide range of toolswas developed allowing precise genome modifications (seeTable 3) Research in U maydis initially followed three main direc-tions (i) the characterization of genes involved in DNA recombina-tion (ii) the study of mating type loci and (iii) the so called Killerphenomenon related to the presence of virus-like particles Subse-quently researchers became interested in different aspects of thebiology ranging from gene regulation signaling and virulence tocell biology Community efforts led to a high quality genomesequence that now serves as a blueprint for comparative studies(Kamper et al 2006)

Nowadays U maydis is one of the best-characterized fungalplant pathogens although it does not cause an economicallyimportant disease Crop losses are usually below 2 and crop rota-tion is used as control strategy (Dean et al 2012) U maydis exhib-its a very narrow host range inducing disease only on corn and itsprogenitor teosinte (Zea mays ssp parviglumis) (Table 1) During itslife cycle U maydis undergoes a number of morphological transi-tions (Supplementary Fig 2F) In the field corn smut infectionsare spread by the air-borne diploid teliospores During their germi-nation meiosis occurs and haploid progeny is produced Haploidcells are yeast-like and divide by budding Transition to the patho-genic form the dikaryon requires fusion of two haploid cells ofopposite mating type (Feldbrugge et al 2004) Cells differing inthe a mating type locus recognize each other via lipopeptide pher-omones the cell cycle arrests in the G2 phase budding is stoppedand conjugation tubes are formed These structures develop at onecell tip and grow towards each other guided through the phero-mone gradient until they merge (Brefort et al 2009) The resultingdikaryon switches to polar growth if the two mating partners carrydifferent alleles of the b mating type locus The b locus codes for apair of homeodomain transcription factors that dimerize whenderived from different alleles and control subsequent sexual andpathogenic development In response to both chemical and physi-cal signals of the plant surface the dikaryotic filament formspoorly differentiated appressoria that penetrate the cuticle proba-bly via the action of lytic enzymes (Brefort et al 2009) After pen-etration the cell cycle is reactivated concomitantly with thedevelopment of clamp-like structures that allow the correct sortingof nuclei to maintain the dikaryotic status (see Brefort et al 2009)In this way the fungus proliferates forms a massive network ofhyphae and induces plant tumors Hyphal growth inside tumorsis followed by sporogenesis a poorly understood process thatincludes karyogamy hyphal fragmentation and differentiation intomelanized diploid teliospores Eventually the tumors dry up rup-ture and release the diploid spores which are dispersed by air Thiscloses the life cycle

71 Comparative genomics in U maydis

The genome size of U maydis is relatively small (205 Mb) incomparison to other plant pathogenic fungi (Table 2) An opensource manually curated MUMDB platform has been developedat Munich Information Center for Protein Sequences MIPS(httpmipshelmholtz-muenchendegenreprojustilago) Todate a total of 6788 protein-encoding genes are annotated Thesmall genome size is attributed to few introns (70 of the genescontain no introns) and the absence of large scale DNA duplica-tions Non-functional transposon-derived sequences constituteonly 11 of the genome assembly (Kamper et al 2006) RNA inter-ference (RNAi) components are absent and it has been hypothe-sized they may have been lost due to an incompatibility betweenRNAi and killer a viral double stranded RNA system encoding aprotein toxin providing a selective advantage (Drinnenberg et al

2011) Signs of gene inactivation by repeat induced point mutationare also absent (Laurie et al 2008)

The repertoire of genes coding for secreted hydrolytic enzymesis small and this has been correlated to the biotrophic lifestyle ofthe pathogen in which damage to the host that can trigger defenseresponses is minimized By contrast the genome contains a signif-icant expansion in novel genes encoding secreted proteins puta-tive effectors which are involved in the control and re-programming of the host during infection Comparative analysesbetween U maydis Sporisorium reilianum the causal agent of headsmut of maize (Schirawski et al 2010) and the barley coveredsmut Ustilago hordei (Laurie et al 2012) demonstrated that smutfungi display largely syntenic chromosomes although the overallsequence identity is only around 70 Sequence identity with therelated animal pathogens Malassezia globosa and M sympodialis(Xu et al 2007) and the biocontrol agent Pseudozyma flocculosa(Lefebvre et al 2013) is significantly lower The high level of syn-teny between U maydis and S reilianum allowed detection of 43lsquolsquodivergence clustersrsquorsquo in which the sequence conservation is lowerthan average and many of these clusters encode effectors Thisindicates that although both pathogens are able to infect the samehost and about 90 of the putative secreted effectors are sharedmany of these genes have undergone discrete specialization eventsduring evolution (Schirawski et al 2010) Moreover the clusters ofsecreted proteins typical of U maydis and S reilianum are absent inthe Malassezia and Pseudozyma genomes

72 Assessing virulence of U maydis

To assess virulence artificial syringe inoculations are carriedout on 7-day-old maize seedlings (Fig 2L) Scoring for symptomsis done usually 12 days post injection following a disease ratingdeveloped by Kamper et al (2006) The variety Early Golden Ban-tam is commonly used as host as symptoms are especially severeon sweet corn For infections of tassel or ears the dwarf varietyGaspe Flint has been adopted and other susceptible inbred maizelines have also been used (Skibbe et al 2010)

The availability of haploid strains causing disease without amating partner (termed solopathogenic) allows forward geneticapproaches to analyze virulence circumvents the need to intro-duce mutations into two compatible strains for virulence assaysand allows to assess gene contributions to virulence independentlyof their involvement in mating (see Brefort et al 2009)


731 Host perception and plant responseSurface recognition and penetration are among the most critical

points during plant infection In U maydis as well as in other fun-gal plant pathogens surface perception involves the signalingmucin Msb2 and the tetraspanin protein Sho1 that regulate theactivity of a downstream MAP kinase module via an as yetunknown mechanism (Lanver et al 2010) For activity Msb2 needsto be O-mannosylated by the mannosyltransferase Pmt4(Fernandez-Alvarez et al 2012) Already on the leaf surface Umaydis is recognized by the plant via a non-specific PAMP-trig-gered reaction However with the establishment of biotrophyplant defenses are suppressed This includes various cell death-related processes such as ROS production and salicylic acid signal-ing (Djamei et al 2011 Hemetsberger et al 2012 Rabe et al2013) At later stages of the infection photosynthesis and C4

metabolism are significantly down-regulated (Brefort et al2009) illustrating metabolic reprogramming of the host


732 Effector functionThe U maydis genome codes for more than 300 putatively

secreted effector proteins mostly lacking any predicted enzymaticfunction or domain Many effectors are encoded by gene clusterswhich are transcriptionally induced during plant colonization(Kamper et al 2006) While some effectors show redundancy infunction (Schirawski et al 2010) others such as pep1 and pit2are crucial virulence determinants Pep1 acts as inhibitor of apo-plastic plant peroxidases involved in H2O2 production(Hemetsberger et al 2012) while Pit2 functions as an inhibitorof a set of apoplastic maize cysteine proteases whose activity isdirectly linked with the salicylate pathway (Muumlller et al 2013)The chorismate mutase Cmu1 was shown to enter plant cells afterbeing secreted and re-routes the chorismate flow in maize cellstowards phenylpropanoid compounds instead of salicylate(Djamei et al 2011) How Cmu1 and probably additional effectorsenter plant cells is currently unknown A future challenge will be toelucidate the function of the U maydis effector battery which islikely to consist not only of conventionally secreted proteins butalso of proteins secreted by unconventional routes (Reissmann Sand Kahmann R unpublished) and to elucidate their mode ofuptake by host cells

733 Cell cycle dimorphism and pathogenic developmentA critical step in the life cycle of U maydis is the morphological

transition from the non-pathogenic haploid state to the dikaryoticinfectious hypha Many aspects of this transition remain poorlyunderstood Since pathogenic development is intimately coupledto cell cycle several proteins controlling the cell cycle have beencharacterized Among these are elements of the DNA damageresponse such as Chk1 and Atr1 (de Sena-Tomas et al 2011Mielnichuk et al 2009) and the protein Clp1 necessary for the cor-rect nuclear distribution in the dikaryotic filament (Heimel et al2010) Recently the cell wall in budding cells and filamentoushyphae has been analyzed Proteins involved in b-16-glucan syn-thesis N-glycosylation and glycosyl transfer seem to affect notonly the morphological switch but also virulence (Robledo-Briones and Ruiz-Herrera 2013) Finally the production of anti-sense transcripts has been shown to have a regulatory role duringpathogenic development (Donaldson and Saville 2013) Thedetailed understanding of the function of proteins and RNAs cont-roling cell cycle dimorphism and virulence of U maydis will beinstrumental for the full understanding of the morpho-physiologi-cal transitions intimately coupled to discrete steps throughout thelife cycle (Supplementary Fig 1F)

734 Signaling and MAP kinase targets involved in virulenceControl of biotrophic development and virulence in U maydis

involves a complex cross-talk of regulatory and signaling mainlybelonging to both the cAMP pathway and the pheromone MAPKcascade The cAMP pathway is required for many aspects of the lifecycle including nutritional sensing morphogenesis and mating(Egan et al 2009) The protein kinase A (PKA) activates among oth-ers the central regulator Prf1 which is responsible for expression ofa large set of genes including the a and b mating type genesRecently two phosphodiesterases UmPde1 and UmPde2 also act-ing on the c-AMP-dependent PKA pathway have been implicatedin pathogenic development as well as dimorphic growth control(Agarwal et al 2013) cAMP signaling is connected with the pher-omone responsive MAPK cascade the best characterized signalingpathway in U maydis Mutants in components of this cascade areseverely affected in their ability to infect corn plants The coreplayers in the MAPK pheromone pathway are the three hierarchi-cal kinases Kpp4 Fuz7 and Kpp2 Two alternative MAPKs Crk1and Kpp6 necessary for full virulence are also present (seeBrefort et al 2009)

As in other plant pathogenic fungi two additional MAPK cas-cades exist in U maydis the cell wall integrity (CWI) pathway andthe osmostress responsive MAPK cascade The main componentsof these cascades although not all characterized are similar to thosedescribed in yeast as well as the plethora of stimuli to which theyrespond In spite of the central role played by the CWI pathway inseveral pathogenic fungi in U maydis strains lacking elements ofthe CWI cascade no apparent defects in the ability to infect maizeplants have been detected (Carbo and Perez-Martin 2010)

735 Understanding the molecular basis of fundamental eukaryoticprocesses

Due to its unexpected high level of genetic similarity sharedwith metazoans U maydis has recently been proposed as a modelfor the study of essential cellular processes of higher eukaryoteswhich are not present in S cerevisiae or Schizosaccharomyces pombe(Steinberg and Perez-Martin 2008) For instance several homo-logues of human proteins involved in DNA repair homologousrecombination and genomic stability such as the Breast CancerType 2 susceptibility protein (BRCA2) or in long-distance transportsuch as kinesis-3 have been found in U maydis (Kojic et al 2002Vollmeister et al 2012) Surprisingly U maydis hyphae have sim-ilarities to mammalian neurons with respect to the elongation andorientation of microtubules and the transport of mRNA to thegrowth region (see Steinberg and Perez-Martin 2008Vollmeister et al 2012) Moreover the disassembly-assemblymachinery of the nuclear pores during open mitosis appears con-served between U maydis and animal cells (see Steinberg andPerez-Martin 2008) Uniparental mitochondrial inheritance a pro-cess dominating in sexual species including mammals has alsobeen described in U maydis (Fedler et al 2009) Furthermore ithas recently been demonstrated in U maydis that several coreenzymes for glycolysis reside in both the cytoplasm and in peroxi-somes and that the peroxisomal targeting is achieved throughpost-transcriptional regulation The presence of peroxisomal tar-geting sequences in glycolytic enzymes of mammals suggests thatdual localization may be a general feature (Freitag et al 2012)

74 Conclusions

In the years ahead we expect U maydis to continue to serve as anattractive model for all aspects of microbial development whereshape and morphological transitions are involved We also expectthis organism to continue to play a pioneering role for the study ofcomplex biological processes involving genes for which no orthologsexist in yeast And last but not least we expect the U maydis systemto reveal many more of the intricacies that have endowed this fun-gus with the ability to colonize a plant host and establish a biotroph-ic relationship On the basis of comparative approaches we alsoexpect to learn much more on the evolution of virulence traits andhow they evolved to generate host specificity

8 Zymoseptoria tritici

Zymoseptoria tritici (Desm) is responsible for Septoria tritici leafblotch (STB) on bread and durum wheat The asexual form of thefungus was first described in 1842 by Desmazieres while its sexualstage was only identified in 1972 (Quaedvlieg et al 2011) (Supple-mentary Fig 1G) Both asexual and sexual stages are currentlyreported in all wheat growing countries STB has a severe economicimpact in wheat-producing areas leading up to 30ndash50 of annualyield losses (Suffert et al 2011) The disease is controlled mainlyby fungicide treatments however Z tritici populations resistantto fungicides have appeared worldwide and only a few resistantwheat cultivars are currently used to control this disease (Orton


et al 2011) This might result from the fact that all (Stb 1-16) butone (Stb16) of the major resistance genes are overcome by virulentisolates (Ghaffary et al 2012 Orton et al 2011)

Z tritici is a haploid ascomycete from the Dothideomycetes(order Capnodiales family Zymoseptoriaceae) (Table 1) This hetero-thallic bipolar fungus has two alleles (MAT1-1 MAT1-2) at the mat-ing type locus equally distributed in populations (Zhan et al2002) Z tritici causes polycyclic epidemics on wheat leaves Inoc-ula are asexual spores (pycnidiospores) released from pycnidiafound on leaves and sexual spores (ascospores) released from ascicontained in fruiting bodies (pseudothecia) found on infectedleaves and plant debris (Supplementary Fig 2G) Wind-dispersedascospores are the main source of primary inoculum and the peakof their release coincides with the growing season of wheatSplash-dispersed pycnidiospores are the main source of secondaryinoculum and spread of the disease from lower to upper leaves(Suffert et al 2011) As a consequence of the high frequency ofsexual reproduction of Z tritici in wheat fields its populationsare characterized by a high level of genetic diversity at field regio-nal and continental scales (Orton et al 2011)

Zymoseptoria tritici is a hemi-biotrophic pathogen with a bio-trophic-like symptomless phase of about 10-days duration fol-lowed by a necrotrophic phase (11ndash21 days) The funguspenetrates wheat leaves through stomata about 3 days after sporegermination (Orton et al 2011) Appressorium-like structures dis-pensable for penetration are formed at the point of stomatal entryOnce in the sub-stomatal cavities the fungus develops through theintercellular space in close contact with mesophyll cells withoutforming specialized feeding structures During the asymptomaticphase the fungal biomass is low and increases exponentiallyduring the early stages of the necrotrophic phase During thenecrotrophic phase the fungus differentiates black pycnidia insub-stomatal cavities which produce pycnodiospores that arereleased by the extrusion of mucilage (cirrus) through the stomata

In the last decades the development of genomics in this speciesand relatives has contributed to establishment of Z tritici as a modelorganism for the study of effectors and their role in fungal infectionand of evolutionary trends associated with speciation and host plantadaptation (Marshall et al 2011 Stukenbrock 2013)

81 Overview of the Z tritici genome

Z tritici IPO-323 has a genome size of approximately 40 Mb and21 chromosomes 13 of which are core chromosomes (CCs 1ndash13)while the other 8 are dispensable (DCs 14ndash21 referred to as lsquolsquodis-pensosomersquorsquo) (Table 2) This dispensosome displays a high varia-tion among isolates and a low density of genes among whichonly a few have paralogs on CCs (Croll et al 2013 Goodwinet al 2011) In particular DCs carry only a few genes encodingsecreted proteins (Morais do Amaral et al 2012) The origin andevolutionary benefit of the DCs is still unclear although they haveevolved differently from CCs (Goodwin et al 2011) Howeverthere are no natural isolates lacking all DCs suggesting that theyhave an important role in nature Z tritici IPO-323 genome contains10933 genes and a significant level of repeated sequences (21)Around 60 of the genes have a functional annotation of which45 encode proteins likely secreted (Morais do Amaral et al2012) Other particular aspects of Z tritici genome are the absenceof methylation (Goodwin et al 2011) and the reduced number ofgenes encoding plant cell wall degrading enzymes (Ohm et al2012)

82 Assessing virulence of Z tritici

Several pathogenicity assays have been established for Z tri-tici Field experiments conducted to evaluate yield losses caused

by Z tritici are traditionally carried out under natural infectionconditions which owing to the high diversity of Z tritici popula-tions hide the effect of single fungal isolates (Fig 2M) Morerecently the development of protocols for field experimentsusing artificial inoculations with a single isolate have allowed amore precise evaluation of wheat resistance (Ghaffary et al2011) (Fig 2N and O) Several assays have also been developedunder controlled conditions either in greenhouses or in growthchambers Such assays are necessary to standardize environmen-tal parameters such as temperature relative humidity and lightwhich strongly influence the outcome of wheat-Z tritici interac-tions (Suffert et al 2013) (Fig 2P) Pathogenicity assays on youngplants in controlled conditions are also used to rapidly character-ize the virulence spectrum of Z tritici isolates and identify candi-date genes associated with pathogenicity (Ghaffary et al 2011) Aprotocol to perform disease assays on detached leaves from adultplants is also available for cultivar resistance assessment (Ortonet al 2011) (Fig 2Q1ndash2)


831 Signaling pathways and protein secretionIn recent years genomics of the Z tritici-wheat pathosystem has

allowed a better understanding of its infectious process Severalstudies have identified intracellular signaling pathways that arecrucial for pathogenicity Some components of the fungal mito-gen-activated protein kinase (MAPK) signaling cascades have beenfunctionally characterized in Z tritici They play a role either in thepenetration of wheat leaves (MgFus3 MgHog1) or in leaf coloniza-tion (MgSlt2) (Cousin et al 2006 Mehrabi et al 2006a 2006b)These signaling pathways likely activate transcription factors reg-ulating the expression of downstream genes essential for infectionFor instance the transcription factor Ste12 downstream of MgFus3MAPK is involved in fungal colonization of wheat leaves (Krameret al 2009) Other regulatory networks involved in pathogenicityhave been identified in Z tritici MgWOR1 encodes a transcriptionfactor involved in morphological switches (yeastndashmycelium) andpathogenicity that is orthologous to WOR1 from Candida albicansand SGE1 from Fusarium oxysporum (Mirzadi Gohari et al 2014)WOR1 null mutants display some of the phenotypes of MgGPB1 nullmutants (Mehrabi and Kema 2006) MgGPB1 belongs to the G-pro-teins complex which acts upstream the cAMP-pathway Compo-nents of the cAMP pathway downstream of the adenylatecyclase such as the protein kinase A catalytic subunit encodinggene MgTPK2 are also important for yeastndashmycelium transitionand infection Inactivation of the cyclin MgMCC1 orthologous toFusarium verticillioides FCC1 is associated with a reduced radialgrowth a delayed filamentous growth unusual hyphal swellingsand increased melanin biosynthesis and stress tolerance as wellas reduced pathogenicity (Choi and Goodwin 2011a) Deletion ofMgMVE1 encoding one essential component of the regulatory com-plex Velvet leads to pleiotropic phenotypes including defects inyeastndashmycelial transition and the effect of light on this processHowever it has no role in pathogenicity (Choi and Goodwin2011b) Several other genes important for infection have beenfunctionally characterized Deletion of MgALG2 involved in glyco-sylation of secreted proteins led to a mutant unable to infectwheat This mutant is also impaired in switching from yeast to fil-amentous growth and in protein secretion (Motteram et al 2011)Among several ABC transporters studied only MgATR4 had a quan-titative effect on pathogenicity (Stergiopoulos et al 2003)

832 Plant-pathogen interaction effectorsCurrently comparative genomic and proteomic studies are

applied to identify secreted proteins (SPs) Within the putativesecretome of Z tritici about 500 small SPs (lt30 kD) have been


identified (Morais do Amaral et al 2012) Two of the three Z triticiLysM effector encoding genes (Mg3LYSM and Mg1LYSM) whichbind to chitin are strongly up-regulated during the symptomlessphase of infection One of them Mg3LysM is required for wheatleaf colonization and sporulation (Lee et al 2014 Marshall et al2011) The secreted MgNEP1 gene encoding a protein related tonecrosis and ethylene-inducing peptide is up-regulated duringthe symptomless phase of disease However it is not essential forinfection (Motteram et al 2009) Tandem Repeat secreted Proteins(MgTRPs) have been also characterized and shown to be expressedspecifically during the symptomless phase of infection (Rudd et al2010)

833 Disease controlMolecular strategies have been used also to elucidate the mech-

anisms of resistance of the pathogen to most frequently appliedfungicides In the last years European Z tritici populations havebecome resistant to azoles by a combination of single point muta-tions in the CYP51 gene and increased active efflux through ABCtransporters (Cools and Fraaije 2013) To circumvent these prob-lems a new-generation of carboxamide fungicides have beenintroduced for the control of STB (Fraaije et al 2012) Major effortsare focused on identifying genetic determinants of resistance to Ztritici in wheat Quantitative trait loci (QTLs) implicated in partialresistance and susceptibility to STB have been identified in addi-tion to new Stb major resistance genes (Ghaffary et al 2012Ghaffary et al 2011) Some Stb genes have been introduced incommercial bread wheat cultivars (Stb1 Stb5 Stb6 and Stb9) butall these resistance genes have been defeated by virulent isolatesAlternative strategies based on the pyramiding of Stb genes andor resistance QTLs are being tested for their efficiency against Z tri-tici (Orton et al 2011)

834 Evolutionary genomicsIn recent years studies conducted on different populations of Z

tritici and related species has brought major inputs on the evolu-tionary trends of fungal pathogens (Stukenbrock 2013) It has beenshown for example that adaptation of Zymoseptoria species to dif-ferent host plants is associated with positive selection signatures ina few genes some of which encode candidate effectors(Stukenbrock et al 2011 2012) as well as plant cell wall degrad-ing enzymes (Brunner et al 2013) Due to its efficient sexualreproduction Z tritici is a good model for fungal genetics and ithas been successfully used to study the behavior of accessory chro-mosomes during meiosis (Croll et al 2013 Wittenberg et al2009) Rapid identification of genes involved in adaptation to hostplants will be aided by association genetics

84 Conclusions

At present Z tritici is the main fungal disease of wheat How-ever the molecular mechanisms involved in its pathogenicity onwheat are still poorly understood Deciphering these mechanismsat different stages of infection will be of general interest for plantpathology and fungal development To date Z tritici overcomesmost control methods even in a single growing season For thisreason studying its biology population dynamics and geneticstructure is important to propose successful control methods

9 General conclusions

The establishment of fungal model systems in the last 30 yearshas enabled researchers to begin to address fundamental questionsof fungal pathogenesis In this review we have critically evaluatedrecent progress in seven fungal species which are well-studied

models for elucidation of pathogenicity determinants of animaland plant hosts We have presented an overview of the historybiology and current state of research for each model species inorder to stimulate further interest and study of these pathosys-tems In this context Supplementary Fig 1 provides a detailedaccount of the main steps relevant for establishment of each organ-ism as a model system We have also provided up-to-date repre-sentations of the life cycles of each of the species inSupplementary Fig 2 Additionally we have presented a compara-tive analysis of general biological features (Table 1) informationgained from genome projects (Table 2) and available moleculartools (Table 3) for each of the seven fungal species When consid-ered together this will provide a means of rapidly comparing someof the the most studied fungal pathogens

Overall our comparative analysis has highlighted a number ofshared characteristics but also key differences between each path-ogen investigated First of all five of the seven species possess sex-ual (or parasexual) cycles whereas no sexual cycle has yet beenidentified in F oxysporum and mating is not required for sporula-tion in A gossyppi However in spite of the ability to undertakesexual reproduction the frequency with which this occurs also var-ies greatly between species from being an absolute pre-requisitefor plant infection as in Ustilago maydis to being almost absentfrom clonally propagating asexual populations of M oryzae Theabsence of sexually recombining populations clearly has conse-quences for the manner in which genetic variability is generatedas seen most obviously in F oxysporum with its evidence of dis-pensable chromosomes and horizontal gene transfer Comparativegenome analysis now possible on a very large-scale in populationsof pathogens therefore promises new insight into the generation ofvariation in fungal populations and the degree to which selectivepressures imposed since the onset of organized agriculture andmonoculture has influenced plant pathogen populations in com-parison to the distinct pressures imposed on human pathogenpopulations

Secondly it is noteworthy that conserved signaling pathwaysfor instance those composed of cAMP-dependent protein kinaseA and MAP kinases are essential in a very wide range of pathogensincluding Magnaporthe oryzae Ustilago maydis and Fusarium oxy-sporum (Di Pietro et al 2001 Hamer and Talbot 1998 Kahmannet al 1999) and for regulating morphological changes observedin human pathogens such as Candida albicans (Sanchez-Martinezand Perez-Martin 2001 Whiteway 2000) Thus although inputsignals receptors and indeed downstream target genes differ verysignificantly among the pathogens we compared there appears tobe evidence of ancient conserved signaling modules at the core ofthese pathways that underpin fungal pathogenesis and in particu-lar the infection-associated morphogenetic transitions exhibitedby the pathogens represented here Other conserved traits includethe importance of secondary metabolic pathways in pathogenicfungi often in linked clusters in Aspergillus (Bergmann et al2007 Perrin et al 2007) and M oryzae (Dean et al 2005) forexample and the considerable secreted proteomes associated withpathogens (Ma et al 2010 Wilson and Talbot 2009)

There are however also considerable differences among thepathogens explored in this review It is striking for instance howthe plant pathogens examined ndash which are highly diverse phyloge-netically ndash all contain large repertoires of secreted effector-encod-ing genes These effectors play diverse roles in the suppression andevasion of plant immune responses LysM effectors can for exam-ple suppress PAMP-triggered immunity by competitively inhibit-ing pattern recognition receptors such as those that perceivechitin monomer released from the walls of invading fungal patho-gens such as M oryzae (Mentlak et al 2012) and M graminicola(Lee et al 2014) or modulate plant defense-associated metabolicpathways such as phenylpropanoid metabolism (Djamei et al


2011) Other effectors are delivered directly into host plant cellswhere they appear to bind components of plant immune responsepathways thereby suppressing the plantrsquos defenses (Giraldo et al2013) although the precise mechanisms are not yet clear in mostfungal pathogens The presence of large batteries of secreted effec-tor proteins encoded by gene specifically expressed during hostinvasion is however in marked contrast to the human pathogensstudied here Although evasion of host recognition may be a fea-ture of some human pathogenic fungi and pattern recognitionreceptors of the innate immunity response of animals can perceivefungal PAMPs such as chitin there is much less evidence of activeeffector-mediated suppression of immunity as exhibited by humanpathogenic bacteria or plant pathogenic fungi This may be a con-sequence of most human pathogens being opportunists that canonly cause systemic infections in immuno-compromised hosts asis the case for A fumigates C albicans and the emerging pathogenF oxysporum It will therefore be interesting to investigate humanpathogenic fungi of immune-competent hosts (such as Histoplasmacapsulatum for example) to determine if there are significant num-bers of fungal effectors in such species

An overriding consideration in compiling this review is theurgent need to identify novel targets for the development of anti-fungal agents that are broad-spectrum in terms of the diseasesthey control but fungi-specific This is a considerable challengebut comparative genome analysis has already revealed fungal-spe-cific genes and signaling pathways that are important in pathogen-esis in diverse species while targeting further intervention intofungal cell wall biogenesis also offers considerable promise Inhuman pathogens there is a requirement for new antifungal drugsfor treatment of fungal infections in both animal and humanpatients showing less toxicity and better efficacy than currentdrugs By contrast in fungal plant pathogens especially cereal croppathogens a deeper understanding of the host-pathogen relation-ship is going to be required in order to identify novel drug targetswhich do not adversely affect plant health and can lead to moreefficient crop protection strategies This is coupled with a require-ment for any new fungicide to be rapidly degraded in the environ-ment such that residues do not accumulate in the environmentwhile being systemic and potent within plant tissue ndash a challeng-ing combination The powerful genetic systems developed for thefungal pathogens described in this review and elsewhere (seePotts et al 2013) will be invaluable in discovering the next gener-ation of chemical compounds for combating fungal diseases

Acknowledgments

The authors would like to thank Ashley Bird from the MedicalIllustration (MedIT) unit at the University of Aberdeen for herassistance with digitization and edition of the life cycle figuresThis review was carried out by members of the EU-Initial TrainingNetwork Ariadne (PITN-GA-2009-237936) which provided finan-cial support for CB SD MEG EG EM PVM MM VNMFAN TO MOR KS and LW

Appendix A Supplementary material

Supplementary data associated with this article can be found inthe online version at httpdxdoiorg101016jfgb201406011

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Aspergillus fumigatus a successful pathogen Genes and molecules involved ininvasive aspergillosis Rev Iberoam Micol 27 155ndash182

Adachi K Hamer JE 1998 Divergent cAMP signaling pathways regulate growthand pathogenesis in the rice blast fungus Magnaporthe grisea Plant Cell 101361ndash1374

Agarwal C Aulakh KB Edelen K Cooper M Wallen RM Adams S Schultz DJPerlin MH 2013 Ustilago maydis phosphodiesterases play a role in thedimorphic switch and in pathogenicity Microbiology 159 857ndash868

Agrios GN 2005 Plant Pathology Academic Press Inc New York USAAimanianda V Bayry J Bozza S Kniemeyer O Perruccio K Elluru SR Clavaud

C Paris S Brakhage AA Kaveri SV Romani L Latge JP 2009 Surfacehydrophobin prevents immune recognition of airborne fungal spores Nature460 1117ndash1121

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Anker JF Gladfelter AS 2011 Axl2 integrates polarity establishmentmaintenance and environmental stress response in the filamentous fungusAshbya gossypii Eukaryot Cell 10 1679ndash1693

Arie T Kaneko I Yoshida T Noguchi M Nomura Y Yamaguchi I 2000 Mating-type genes from asexual phytopathogenic ascomycetes Fusarium oxysporum andAlternaria alternata Mol Plant Microbe Interact 13 1330ndash1339

Balloy V Chignard M 2009 The innate immune response to Aspergillus fumigatusMicrobes Infect 11 919ndash927

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Bauer Y Knechtle P Wendland J Helfer H Philippsen P 2004 A Ras-likeGTPase is involved in hyphal growth guidance in the filamentous fungus Ashbyagossypii Mol Biol Cell 15 4622ndash4632

Behnsen J Hartmann A Schmaler J Gehrke A Brakhage AA Zipfel PF 2008The opportunistic human pathogenic fungus Aspergillus fumigatus evades thehost complement system Infect Immun 76 820ndash827

Behnsen J Lessing F Schindler S Wartenberg D Jacobsen ID Thoen M ZipfelPF Brakhage AA 2010 Secreted Aspergillus fumigatus protease Alp1 degradeshuman complement proteins C3 C4 and C5 Infect Immun 78 3585ndash3594

Bergmann S Schumann J Scherlach K Lange C Brakhage AA Hertweck C2007 Genomics-driven discovery of PKS-NRPS hybrid metabolites fromAspergillus nidulans Nat Chem Biol 3 213ndash217

Brakhage AA 2005 Systemic fungal infections caused by Aspergillus speciesepidemiology infection process and virulence determinants Curr Drug Targets6 875ndash886

Brakhage AA 2013 Regulation of fungal secondary metabolism Nat RevMicrobiol 11 21ndash32

Brakhage AA Bruns S Thywissen A Zipfel PF Behnsen J 2010 Interaction ofphagocytes with filamentous fungi Curr Opin Microbiol 13 409ndash415

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Brand A MacCallum DM Brown AJ Gow NA Odds FC 2004 Ectopicexpression of URA3 can influence the virulence phenotypes and proteome ofCandida albicans but can be overcome by targeted reintegration of URA3 at theRPS10 locus Eukaryot Cell 3 900ndash909

Brasier CM 2008 The biosecurity threat to the UK and global environment frominternational trade in plants Plant Pathol 57 792ndash808

Braun BR van Het Hoog M drsquoEnfert C Martchenko M Dungan J Kuo A InglisDO Uhl MA Hogues H Berriman M et al 2005 A human-curatedannotation of the Candida albicans genome PLoS Genet 1 (1) 36ndash57

Brefort T Doehlemann G Mendoza-Mendoza A Reissmann S Djamei AKahmann R 2009 Ustilago maydis as a Pathogen Annu Rev Phytopathol 47423ndash445

Bridges AA Zhang H Mehta SB Occhipinti P Tani T Gladfelter AS 2014Septin assemblies form by diffusion-driven annealing on membranes ProcNatl Acad Sci USA 111 2146ndash2151

Brown GD Denning DW Gow NA Levitz SM Netea MG White TC 2012Hidden killers human fungal infections Sci Transl Med 4 165rv13

Brunner PC Torriani SF Croll D Stukenbrock EH McDonald BA 2013Coevolution and life cycle specialization of plant cell wall degrading enzymes ina hemibiotrophic pathogen Mol Biol Evol 30 1337ndash1347

Bruns S Kniemeyer O Hasenberg M Aimanianda V Nietzsche S Thywissen AJeron A Latge JP Brakhage AA Gunzer M 2010 Production of extracellulartraps against Aspergillus fumigatus in vitro and in infected lung tissue isdependent on invading neutrophils and influenced by hydrophobin RodA PLoSPathog 6 e1000873

Butler G Rasmussen MD Lin MF Santos MA Sakthikumar S Munro CARheinbay E Grabherr M Forche A Reedy JL Agrafioti I Arnaud MBBates S Brown AJ Brunke S Costanzo MC Fitzpatrick DA de Groot PWHarris D Hoyer LL Hube B Klis FM Kodira C Lennard N Logue MEMartin R Neiman AM Nikolaou E Quail MA Quinn J Santos MCSchmitzberger FF Sherlock G Shah P Silverstein KA Skrzypek MS SollD Staggs R Stansfield I Stumpf MP Sudbery PE Srikantha T Zeng QBerman J Berriman M Heitman J Gow NA Lorenz MC Birren BW KellisM Cuomo CA 2009 Evolution of pathogenicity and sexual reproduction ineight Candida genomes Nature 459 657ndash662

Calderone RA Clancy CJ 2012 In Candida and Candidiasis ASM Press USACalo S Billmyre RB Heitman J 2013 Generators of phenotypic diversity in the

evolution of pathogenic microorganisms PLoS Pathog 9 e1003181


Caracuel Z Casanova C Roncero MI Di Pietro A Ramos J 2003 PHresponse transcription factor PacC controls salt stress tolerance and expressionof the P-Type Na+ -ATPase Ena1 in Fusarium oxysporum Eukaryot Cell 2 1246ndash1252

Carbo N Perez-Martin J 2010 Activation of the cell wall integrity pathwaypromotes escape from G2 in the fungus Ustilago maydis PLoS Genet 6e1001009

Clemons KV Stevens DA 2005 The contribution of animal models ofaspergillosis to understanding pathogenesis therapy and virulence MedMycol 43 (Suppl 1) pp S101-10

Cools HJ Fraaije BA 2013 Update on mechanisms of azole resistance inMycosphaerella graminicola and implications for future control Pest Manag Sci69 150ndash155

Couch BC Fudal I Lebrun MH Tharreau D Valent B van Kim P NotteghemJL Kohn LM 2005 Origins of host-specific populations of the blast pathogenMagnaporthe oryzae in crop domestication with subsequent expansion ofpandemic clones on rice and weeds of rice Genetics 170 613ndash630

Couch BC Kohn LM 2002 A multilocus gene genealogy concordant with hostpreference indicates segregation of a new species Magnaporthe oryzae from Mgrisea Mycologia 94 683ndash693

Cousin A Mehrabi R Guilleroux M Dufresne M Van der Lee T Waalwijk CLangin T Kema GHJ 2006 The MAP kinase-encoding gene MgFus3 of thenon-appressorium phytopathogen Mycosphaerella graminicola is required forpenetration and in vitro pycnidia formation Mol Plant Pathol 7 269ndash278

Cramer Jr RA Perfect BZ Pinchai N Park S Perlin DS Asfaw YG Heitman JPerfect JR Steinbach WJ 2008 Calcineurin target CrzA regulates conidialgermination hyphal growth and pathogenesis of Aspergillus fumigatusEukaryot Cell 7 1085ndash1097

Croll D McDonald BA 2012 The accessory genome as a cradle for adaptiveevolution in pathogens PLoS Pathog 8 e1002608

Croll D Zala M McDonald BA 2013 Breakage-fusion-bridge cycles and largeinsertions contribute to the rapid evolution of accessory chromosomes in afungal pathogen PLoS Genet 9 e1003567

Chevenet F Brun C Banuls AL Jacq B Christen R 2006 TreeDyn towardsdynamic graphics and annotations for analyses of trees BMC Bioinformatics 7439

Choi J Kim Y Kim S Park J Lee YH 2009 MoCRZ1 a gene encoding acalcineurin-responsive transcription factor regulates fungal growth andpathogenicity of Magnaporthe oryzae Fungal Genet Biol 46 243ndash254

Choi W Dean RA 1997 The adenylate cyclase gene MAC1 of Magnaporthe griseacontrols appressorium formation and other aspects of growth anddevelopment Plant Cell 9 1973ndash1983

Choi YE Goodwin SB 2011a Gene encoding a c-type cyclin in Mycosphaerellagraminicola is involved in aerial mycelium formation filamentous growthhyphal swelling melanin biosynthesis stress response and pathogenicity MolPlant Microbe Interact 24 469ndash477

Choi YE Goodwin SB 2011b MVE1 encoding the velvet gene product homologin Mycosphaerella graminicola is associated with aerial mycelium formationmelanin biosynthesis hyphal swelling and light signaling Appl EnvironMicrobiol 77 942ndash953

da Silva Ferreira ME Heinekamp T Hartl A Brakhage AA Semighini CPHarris SD Savoldi M de Gouvea PF de Souza Goldman MH GoldmanGH 2007 Functional characterization of the Aspergillus fumigatus calcineurinFungal Genet Biol 44 219ndash230

Dagdas YF Yoshino K Dagdas G Ryder LS Bielska E Steinberg G Talbot NJ2012 Septin-mediated plant cell invasion by the rice blast fungus Magnaportheoryzae Science 336 1590ndash1595

de Sena-Tomas C Fernandez-Alvarez A Holloman WK Perez-Martin J 2011The DNA damage response signaling cascade regulates proliferation of thephytopathogenic fungus Ustilago maydis in planta Plant Cell 23 1654ndash1665

Dean R Van Kan JAL Pretorius ZA Hammond-Kosack KE Di Pietro A SpanuPD Rudd JJ Dickman M Kahmann R Ellis J Foster GD 2012 The Top 10fungal pathogens in molecular plant pathology Mol Plant Pathol 13 414ndash430

Dean RA Talbot NJ Ebbole DJ Farman ML Mitchell TK Orbach MJ ThonM Kulkarni R Xu JR Pan H Read ND Lee YH Carbone I Brown D OhYY Donofrio N Jeong JS Soanes DM Djonovic S Kolomiets E RehmeyerC Li W Harding M Kim S Lebrun MH Bohnert H Coughlan S Butler JCalvo S Ma LJ Nicol R Purcell S Nusbaum C Galagan JE Birren BW2005 The genome sequence of the rice blast fungus Magnaporthe grisea Nature434 980ndash986

Denning DW Pleuvry A Cole DC 2013 Global burden of allergicbronchopulmonary aspergillosis with asthma and its complication chronicpulmonary aspergillosis in adults Med Mycol 51 361ndash370

Di Pietro A Garcia-MacEira FI Meglecz E Roncero MI 2001 A MAP kinase ofthe vascular wilt fungus Fusarium oxysporum is essential for root penetrationand pathogenesis Mol Microbiol 39 1140ndash1152

Di Pietro A Roncero MI 1998 Cloning expression and role in pathogenicityof pg1 encoding the major extracellular endopolygalacturonase of thevascular wilt pathogen Fusarium oxysporum Mol Plant Microbe Interact 1191ndash98

Dichtl K Helmschrott C Dirr F Wagener J 2012 Deciphering cell wall integritysignalling in Aspergillus fumigatus identification and functional characterizationof cell wall stress sensors and relevant Rho GTPases Mol Microbiol 83 506ndash519

Dietrich FS Voegeli S Brachat S Lerch A Gates K Steiner S Mohr CPohlmann R Luedi P Choi S Wing RA Flavier A Gaffney TD Philippsen

P 2004 The Ashbya gossypii genome as a tool for mapping the ancientSaccharomyces cerevisiae genome Science 304 304ndash307

Dietrich FS Voegeli S Kuo S Philippsen P 2013 Genomes of Ashbya fungiisolated from insects reveal four mating-type loci numerous translocationslack of transposons and distinct gene duplications G3

Dixon KP Xu JR Smirnoff N Talbot NJ 1999 Independent signaling pathwaysregulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea Plant Cell 11 2045ndash2058

Djamei A Schipper K Rabe F Ghosh A Vincon V Kahnt J Osorio S Tohge TFernie AR Feussner I Feussner K Meinicke P Stierhof Y-D Schwarz HMacek B Mann M Kahmann R 2011 Metabolic priming by a secreted fungaleffector Nature 478 395ndash398

Donaldson ME Saville BJ 2013 Ustilago maydis natural antisense transcriptexpression alters mRNA stability and pathogenesis Mol Microbiol 89 29ndash51

Drinnenberg IA Fink GR Bartel DP 2011 Compatibility with killer explains therise of RNAi-deficient fungi Science 333 pp 1592ndash1592

Egan JD Garcia-Pedrajas MD Andrews DL Gold SE 2009 Calcineurin is anantagonist to PKA protein phosphorylation required for postmatingfilamentation and virulence while PP2A is required for viability in Ustilagomaydis Mol Plant Microbe Interact 22 1293ndash1301

Fedler M Luh K-S Stelter K Nieto-Jacobo F Basse CW 2009 The a2 mating-type locus genes lga2 and rga2 direct uniparental mitochondrial DNA (mtDNA)inheritance and constrain mtDNA recombination during sexual development ofUstilago maydis Genetics 181 847ndash860

Fedorova ND Khaldi N Joardar VS Maiti R Amedeo P Anderson MJCrabtree J Silva JC Badger JH Albarraq A Angiuoli S Bussey H BowyerP Cotty PJ Dyer PS Egan A Galens K Fraser-Liggett CM Haas BJInman JM Kent R Lemieux S Malavazi I Orvis J Roemer T Ronning CMSundaram JP Sutton G Turner G Venter JC White OR Whitty BRYoungman P Wolfe KH Goldman GH Wortman JR Jiang B DenningDW Nierman WC 2008 Genomic islands in the pathogenic filamentousfungus Aspergillus fumigatus PLoS Genet 4 e1000046

Feldbrugge M Kamper J Steinberg G Kahmann R 2004 Regulation of matingand pathogenic development in Ustilago maydis Curr Opin Microbiol 7 666ndash672

Fernandez-Alvarez A Marin-Menguiano M Lanver D Jimenez-Martin A Elias-Villalobos A Perez-Pulido AJ Kahmann R Ibeas JI 2012 Identification ofO-mannosylated virulence factors in Ustilago maydis PLoS Pathog 8 e1002563

Finlayson MR Helfer-Hungerbuhler AK Philippsen P 2011 Regulation of exitfrom mitosis in multinucleate Ashbya gossypii cells relies on a minimal networkof genes Mol Biol Cell 22 3081ndash3093

Fischer R Zekert N Takeshita N 2008 Polarized growth in fungindashinterplaybetween the cytoskeleton positional markers and membrane domains MolMicrobiol 68 813ndash826

Fisher MC Henk DA Briggs CJ Brownstein JS Madoff LC McCraw SL GurrSJ 2012 Emerging fungal threats to animal plant and ecosystem healthNature 484 186ndash194

Fonzi WA Irwin MY 1993 Isogenic strain construction and gene mapping inCandida albicans Genetics 134 717ndash728

Fraaije BA Bayon C Atkins S Cools HJ Lucas JA Fraaije MW 2012 Riskassessment studies on succinate dehydrogenase inhibitors the new weapons inthe battle to control Septoria leaf blotch in wheat Mol Plant Pathol 13 263ndash275

Freitag J Ast J Bolker M 2012 Cryptic peroxisomal targeting via alternativesplicing and stop codon read-through in fungi Nature 485 522ndash525

Gange AC Gange EG Sparks TH Boddy L 2007 Rapid and recent changes infungal fruiting patterns Science 316 71

Garcia-Vidal C Upton A Kirby KA Marr KA 2008 Epidemiology of invasivemold infections in allogeneic stem cell transplant recipients biological riskfactors for infection according to time after transplantation Clin Infect Dis 471041ndash1050

Gehrke A Heinekamp T Jacobsen ID Brakhage AA 2010 Heptahelicalreceptors GprC and GprD of Aspergillus fumigatus Are essential regulators ofcolony growth hyphal morphogenesis and virulence Appl Environ Microbiol76 3989ndash3998

Gerami-Nejad M Dulmage K Berman J 2009 Additional cassettes for epitopeand fluorescent fusion proteins in Candida albicans Yeast 26 399ndash406

Ghaffary SM Faris JD Friesen TL Visser RG van der Lee TA Robert O KemaGH 2012 New broad-spectrum resistance to Septoria tritici blotch derivedfrom synthetic hexaploid wheat Theor Appl Genet 124 125ndash142

Ghaffary SM Robert O Laurent V Lonnet P Margale E van der Lee TA VisserRG Kema GH 2011 Genetic analysis of resistance to Septoria tritici blotch inthe French winter wheat cultivars Balance and Apache Theor Appl Genet 123741ndash754

Giraldo MC Dagdas YF Gupta YK Mentlak TA Yi M Martinez-Rocha ALSaitoh H Terauchi R Talbot NJ Valent B 2013 Two distinct secretionsystems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzaeNat Commun 4 1996

Gladfelter AS Hungerbuehler AK Philippsen P 2006 Asynchronous nucleardivision cycles in multinucleated cells J Cell Biol 172 347ndash362

Goecks J Nekrutenko A Taylor J 2010 Galaxy a comprehensive approach forsupporting accessible reproducible and transparent computational research inthe life sciences Genome Biol 11 R86

Goodwin SB MrsquoBarek SB Dhillon B Wittenberg AH Crane CF Hane JKFoster AJ Van der Lee TA Grimwood J Aerts A Antoniw J Bailey ABluhm B Bowler J Bristow J van der Burgt A Canto-Canche B Churchill


AC Conde-Ferraez L Cools HJ Coutinho PM Csukai M Dehal P De WitP Donzelli B van de Geest HC van Ham RC Hammond-Kosack KEHenrissat B Kilian A Kobayashi AK Koopmann E Kourmpetis Y KuzniarA Lindquist E Lombard V Maliepaard C Martins N Mehrabi R Nap JPPonomarenko A Rudd JJ Salamov A Schmutz J Schouten HJ Shapiro HStergiopoulos I Torriani SF Tu H de Vries RP Waalwijk C Ware SBWiebenga A Zwiers LH Oliver RP Grigoriev IV Kema GH 2011 Finishedgenome of the fungal wheat pathogen Mycosphaerella graminicola revealsdispensome structure chromosome plasticity and stealth pathogenesis PLoSGenet 7 e10020

Gordon JL Byrne KP Wolfe KH 2011 Mechanisms of chromosome numberevolution in yeast PLoS Genet 7 e1002190

Gow NA Hube B 2012 Importance of the Candida albicans cell wall duringcommensalism and infection Curr Opin Microbiol

Gow NA Knox Y Munro CA Thompson WD 2003 Infection of chickchorioallantoic membrane (CAM) as a model for invasive hyphal growth andpathogenesis of Candida albicans Med Mycol 41 (4) 331ndash338

Gow NA van de Veerdonk FL Brown AJ Netea MG 2011 Candida albicansmorphogenesis and host defence discriminating invasion from colonizationNat Rev Microbiol 10 112ndash122

Grava S Philippsen P 2010 Dynamics of multiple nuclei in Ashbya gossypiihyphae depend on the control of cytoplasmic microtubules length by Bik1Kip2 Kip3 and not on a captureshrinkage mechanism Mol Biol Cell 21 3680ndash3692

Grice CM Bertuzzi M Bignell EM 2013 Receptor-mediated signaling inAspergillus fumigatus Front Microbiol 4 26

Grosse C Heinekamp T Kniemeyer O Gehrke A Brakhage AA 2008 Proteinkinase A regulates growth sporulation and pigment formation in Aspergillusfumigatus Appl Environ Microbiol 74 4923ndash4933

Guindon S Dufayard JF Lefort V Anisimova M Hordijk W Gascuel O 2010New algorithms and methods to estimate maximum-likelihood phylogeniesassessing the performance of PhyML 30 Syst Biol 59 307ndash321

Hamer JE Howard RJ Chumley FG Valent B 1988 A mechanism for surfaceattachment in spores of a plant pathogenic fungus Science 239 288ndash290

Hamer JE Talbot NJ 1998 Infection-related development in the rice blast fungusMagnaporthe grisea Curr Opin Microbiol 1 693ndash697

Harvell CD Kim K Burkholder JM Colwell RR Epstein PR Grimes DJHofmann EE Lipp EK Osterhaus AD Overstreet RM Porter JW SmithGW Vasta GR 1999 Emerging marine diseases-climate links andanthropogenic factors Science 285 1505ndash1510

Heimel K Scherer M Schuler D Kamper J 2010 The Ustilago maydis Clp1protein orchestrates pheromone and b-dependent signaling pathways tocoordinate the cell cycle and pathogenic development Plant Cell 22 2908ndash2922

Heinekamp T Thywissen A Macheleidt J Keller S Valiante V Brakhage AA2012 Aspergillus fumigatus melanins interference with the host endocytosispathway and impact on virulence Front Microbiol 3 440

Hemetsberger C Herrberger C Zechmann B Hillmer M Doehlemann G 2012The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition ofhost peroxidase activity PLoS Pathog 8 e1002684

Herbrecht R Denning DW Patterson TF Bennett JE Greene RE OestmannJW Kern WV Marr KA Ribaud P Lortholary O Sylvester R Rubin RHWingard JR Stark P Durand C Caillot D Thiel E Chandrasekar PHHodges MR Schlamm HT Troke PF de Pauw B 2002 Voriconazole versusamphotericin B for primary therapy of invasive aspergillosis N Engl J Med347 408ndash415

Hickman MA Zeng G Forche A Hirakawa MP Abbey D Harrison BD WangYM Su CH Bennett RJ Wang Y Berman J 2013 The lsquoobligate diploidrsquoCandida albicans forms mating-competent haploids Nature 494 55ndash59

Holliday R 1964 A mechanism for gene conversion in fungi Genetics Res 5 282ndash304

Horn F Heinekamp T Kniemeyer O Pollmacher J Valiante V Brakhage AA2012 Systems biology of fungal infection Front Microbiol 3 108

Houterman PM Cornelissen BJ Rep M 2008 Suppression of plant resistancegene-based immunity by a fungal effector PLoS Pathog 4 e1000061

Hull CM Johnson AD 1999 Identification of a mating type-like locus in theasexual pathogenic yeast Candida albicans Science 285 1271ndash1275

Hull CM Raisner RM Johnson AD 2000 Evidence for mating of the lsquolsquoasexualrsquorsquoyeast Candida albicans in a mammalian host Science 289 307ndash310

Inglis DO Binkley J Skrzypek MS Arnaud MB Cerqueira GC Shah PWymore F Wortman JR Sherlock G 2013 Comprehensive annotation ofsecondary metabolite biosynthetic genes and gene clusters of Aspergillusnidulans A fumigatus A niger and A oryzae BMC Microbiol 13 91

Jackson AP Gamble JA Yeomans T Moran GP Saunders D Harris D AslettM Barrell JF Butler G Citiulo F Coleman DC de Groot PW Goodwin TJQuail MA McQuillan J Munro CA Pain A Poulter RT Rajandream MARenauld H Spiering MJ Tivey A Gow NA Barrell B Sullivan DJBerriman M 2009 Comparative genomics of the fungal pathogens Candidadubliniensis and Candida albicans Genome Res 19 2231ndash2244

Jacobsen ID Grosse K Slesiona S Hube B Berndt A Brock M 2010Embryonated eggs as an alternative infection model to investigate Aspergillusfumigatus virulence Infect Immun 78 2995ndash3006

Jain R Valiante V Remme N Docimo T Heinekamp T Hertweck CGershenzon J Haas H Brakhage AA 2011 The MAP kinase MpkA controlscell wall integrity oxidative stress response gliotoxin production and ironadaptation in Aspergillus fumigatus Mol Microbiol 82 39ndash53

Jia Y McAdams SA Bryan GT Hershey HP Valent B 2000 Direct interactionof resistance gene and avirulence gene products confers rice blast resistanceEMBO J 19 4004ndash4014

Juvvadi PR Gehrke C Fortwendel JR Lamoth F Soderblom EJ Cook EC HastMA Asfaw YG Moseley MA Creamer TP Steinbach WJ 2013Phosphorylation of Calcineurin at a novel serine-proline rich regionorchestrates hyphal growth and virulence in Aspergillus fumigatus PLoSPathog 9 e1003564

Kahmann R Basse C Feldbrugge M 1999 Fungal-plant signalling in the Ustilagomaydis-maize pathosystem Curr Opin Microbiol 2 647ndash650

Kamper J Kahmann R Bolker M Ma LJ Brefort T Saville BJ Banuett FKronstad JW Gold SE Muller O Perlin MH Wosten HA de Vries RRuiz-Herrera J Reynaga-Pena CG Snetselaar K McCann M Perez-MartinJ Feldbrugge M Basse CW Steinberg G Ibeas JI Holloman W GuzmanP Farman M Stajich JE Sentandreu R Gonzalez-Prieto JM Kennell JCMolina L Schirawski J Mendoza-Mendoza A Greilinger D Munch KRossel N Scherer M Vranes M Ladendorf O Vincon V Fuchs USandrock B Meng S Ho EC Cahill MJ Boyce KJ Klose J KlostermanSJ Deelstra HJ Ortiz-Castellanos L Li W Sanchez-Alonso P Schreier PHHauser-Hahn I Vaupel M Koopmann E Friedrich G Voss H Schluter TMargolis J Platt D Swimmer C Gnirke A Chen F Vysotskaia VMannhaupt G Guldener U Munsterkotter M Haase D Oesterheld MMewes HW Mauceli EW DeCaprio D Wade CM Butler J Young SJaffe DB Calvo S Nusbaum C Galagan J Birren BW 2006 Insights fromthe genome of the biotrophic fungal plant pathogen Ustilago maydis Nature444 97ndash101

Kankanala P Czymmek K Valent B 2007 Roles for rice membrane dynamics andplasmodesmata during biotrophic invasion by the blast fungus Plant Cell 19706ndash724

Kato T Park EY 2012 Riboflavin production by Ashbya gossypii Biotechnol Lett34 611ndash618

Kaufmann A Philippsen P 2009 Of bars and rings Hof1-dependent cytokinesis inmultiseptated hyphae of Ashbya gossypii Mol Cell Biol 29 771ndash783

Kaur R Ma B Cormack BP 2007 A family of glycosylphosphatidylinositol-linkedaspartyl proteases is required for virulence of Candida glabrata Proc Natl AcadSci USA 104 7628ndash7633

Keane TM Creevey CJ Pentony MM Naughton TJ McLnerney JO 2006Assessment of methods for amino acid matrix selection and their use onempirical data shows that ad hoc assumptions for choice of matrix are notjustified BMC Evol Biol 6 29

Kohli M Galati V Boudier K Roberson RW Philippsen P 2008 Growth-speed-correlated localization of exocyst and polarisome components in growth zonesof Ashbya gossypii hyphal tips J Cell Sci 121 3878ndash3889

Kojic M Kostrub CF Buchman AR Holloman WK 2002 BRCA2 homologrequired for proficiency in DNA repair recombination and genome stability inUstilago maydis Mol Cell 10 683ndash691

Kramer B Thines E Foster AJ 2009 MAP kinase signalling pathway componentsand targets conserved between the distantly related plant pathogenic fungiMycosphaerella graminicola and Magnaporthe grisea Fungal Genet Biol 46 667ndash681

Kroll K Pahtz V Kniemeyer O 2014 Elucidating the fungal stress response byproteomics J Proteomics 97 151ndash163

Langfelder K Jahn B Gehringer H Schmidt A Wanner G Brakhage AA 1998Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatusinvolved in conidial pigment biosynthesis and virulence Med MicrobiolImmunol 187 79ndash89

Lanver D Mendoza-Mendoza A Brachmann A Kahmann R 2010 Sho1 andMsb2-related proteins regulate appressorium development in the smut fungusUstilago maydis Plant Cell 22 2085ndash2101

Larkin MA Blackshields G Brown NP Chenna R McGettigan PA McWilliamH Valentin F Wallace IM Wilm A Lopez R Thompson JD Gibson TJHiggins DG 2007 Clustal W and Clustal X version 20 Bioinformatics 232947ndash2948

Latge JP 2001 The pathobiology of Aspergillus fumigatus Trends Microbiol 9 382ndash389

Laurie JD Ali S Linning R Mannhaupt G Wong P Guldener U MunsterkotterM Moore R Kahmann R Bakkeren G Schirawski J 2012 Genomecomparison of barley and maize smut fungi reveals targeted loss of RNAsilencing components and species-specific presence of transposable elementsPlant Cell 24 1733ndash1745

Laurie JD Linning R Bakkeren G 2008 Hallmarks of RNA silencing are found inthe smut fungus Ustilago hordei but not in its close relative Ustilago maydis CurrGenet 53 49ndash58

Lee WS Rudd JJ Hammond-Kosack KE Kanyuka K 2014 Mycosphaerellagraminicola LysM effector-mediated stealth pathogenesis subverts recognitionthrough both CERK1 and CEBiP homologues in wheat Mol Plant MicrobeInteract 27 236ndash243

Lefebvre F Joly DL Labbe C Teichmann B Linning R Belzile F Bakkeren GBelanger RR 2013 The transition from a phytopathogenic smut ancestor to ananamorphic biocontrol agent deciphered by comparative whole-genomeanalysis Plant Cell 25 1946ndash1959

Lehrnbecher T Kalkum M Champer J Tramsen L Schmidt S Klingebiel T2013 Immunotherapy in invasive fungal infection-focus on invasiveaspergillosis Curr Pharm Des 19 3689ndash3712

Li G Zhou X Xu JR 2012 Genetic control of infection-related development inMagnaporthe oryzae Curr Opin Microbiol 15 678ndash684


Liebmann B Muhleisen TW Muller M Hecht M Weidner G Braun A BrockM Brakhage AA 2004a Deletion of the Aspergillus fumigatus lysinebiosynthesis gene lysF encoding homoaconitase leads to attenuated virulencein a low-dose mouse infection model of invasive aspergillosis Arch Microbiol181 378ndash383

Liebmann B Muller M Braun A Brakhage AA 2004b The cyclic AMP-dependent protein kinase a network regulates development and virulence inAspergillus fumigatus Infect Immun 72 5193ndash5203

Lionakis MS Netea MG 2013 Candida and host determinants of susceptibility toinvasive candidiasis PLoS Pathog 9 e1003079

Lockhart SR Daniels KJ Zhao R Wessels D Soll DR 2003 Cell biology ofmating in Candida albicans Eukaryot Cell 2 49ndash61

Lopez-Berges MS Capilla J Turra D Schafferer L Matthijs S Jochl C CornelisP Guarro J Haas H Di Pietro A 2012 HapX-mediated iron homeostasis isessential for rhizosphere competence and virulence of the soilborne pathogenFusarium oxysporum Plant Cell 24 3805ndash3822

Lopez-Berges MS Hera C Sulyok M Schafer K Capilla J Guarro J Di Pietro A2013 The velvet complex governs mycotoxin production and virulence ofFusarium oxysporum on plant and mammalian hosts Mol Microbiol 87 49ndash65

Lopez-Berges MS Rispail N Prados-Rosales RC Di Pietro A 2010 A nitrogenresponse pathway regulates virulence functions in Fusarium oxysporum via theprotein kinase TOR and the bZIP protein MeaB Plant Cell 22 2459ndash2475

Ma LJ van der Does HC Borkovich KA Coleman JJ Daboussi MJ Di Pietro ADufresne M Freitag M Grabherr M Henrissat B Houterman PM Kang SShim WB Woloshuk C Xie X Xu JR Antoniw J Baker SE Bluhm BHBreakspear A Brown DW Butchko RA Chapman S Coulson R CoutinhoPM Danchin EG Diener A Gale LR Gardiner DM Goff S Hammond-Kosack KE Hilburn K Hua-Van A Jonkers W Kazan K Kodira CDKoehrsen M Kumar L Lee YH Li L Manners JM Miranda-Saavedra DMukherjee M Park G Park J Park SY Proctor RH Regev A Ruiz-RoldanMC Sain D Sakthikumar S Sykes S Schwartz DC Turgeon BG WapinskiI Yoder O Young S Zeng Q Zhou S Galagan J Cuomo CA Kistler HCRep M 2010 Comparative genomics reveals mobile pathogenicitychromosomes in Fusarium Nature 464 367ndash373

MacCallum DM 2012 Hosting infection experimental models to assay Candidavirulence Int J Microbiol 2012 363764

Magee BB Magee PT 2000 Induction of mating in Candida albicans byconstruction of MTLa and MTLalpha strains Science 289 310ndash313

Marshall R Kombrink A Motteram J Loza-Reyes E Lucas J Hammond-KosackKE Thomma BP Rudd JJ 2011 Analysis of two in planta expressed LysMeffector homologs from the fungus Mycosphaerella graminicola reveals novelfunctional properties and varying contributions to virulence on wheat PlantPhysiol 156 756ndash769

Martinez-Rocha AL Roncero MI Lopez-Ramirez A Marine M Guarro JMartinez-Cadena G Di Pietro A 2008 Rho1 has distinct functions inmorphogenesis cell wall biosynthesis and virulence of Fusarium oxysporumCell Microbiol 10 1339ndash1351

May GS Xue T Kontoyiannis DP Gustin MC 2005 Mitogen activated proteinkinases of Aspergillus fumigatus Med Mycol 43 (Suppl 1) pp S83-6

McCormick A Jacobsen ID Broniszewska M Beck J Heesemann J Ebel F2012 The two-component sensor kinase TcsC and its role in stress resistance ofthe human-pathogenic mold Aspergillus fumigatus PLoS ONE 7 e38262

Mehrabi R Kema GHJ 2006 Protein kinase A subunits of the ascomycetepathogen Mycosphaerella graminicola regulate asexual fructificationfilamentation melanization and osmosensing Mol Plant Pathol 7 565ndash577

Mehrabi R Van der Lee T Waalwijk C Gert HJ 2006a MgSlt2 a cellularintegrity MAP kinase gene of the fungal wheat pathogen Mycosphaerellagraminicola is dispensable for penetration but essential for invasive growthMol Plant Microbe Interact 19 389ndash398

Mehrabi R Zwiers LH de Waard MA Kema GHJ 2006b MgHog1 regulatesdimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerellagraminicola Mol Plant Microbe Interact 19 1262ndash1269

Mentlak TA Kombrink A Shinya T Ryder LS Otomo I Saitoh H Terauchi RNishizawa Y Shibuya N Thomma BP Talbot NJ 2012 Effector-mediatedsuppression of chitin-triggered immunity by Magnaporthe oryzae is necessaryfor rice blast disease Plant Cell 24 322ndash335

Meseroll RA Occhipinti P Gladfelter AS 2013 Septin phosphorylation andcoiled-coil domains function in cell and septin ring morphology in thefilamentous fungus Ashbya gossypii Eukaryot Cell 12 182ndash193

Michielse CB Rep M 2009 Pathogen profile update Fusarium oxysporum MolPlant Pathol 10 311ndash324

Michielse CB van Wijk R Reijnen L Cornelissen BJ Rep M 2009Insight into the molecular requirements for pathogenicity of Fusariumoxysporum f sp lycopersici through large-scale insertional mutagenesisGenome Biol 10 R4

Mielnichuk N Sgarlata C Perez-Martin J 2009 A role for the DNA-damagecheckpoint kinase Chk1 in the virulence program of the fungus Ustilago maydisJ Cell Sci 122 4130ndash4140

Miller MG Johnson AD 2002 White-opaque switching in Candida albicans iscontrolled by mating-type locus homeodomain proteins and allows efficientmating Cell 110 293ndash302

Mirzadi Gohari A Mehrabi R Robert O Ince IA Boeren S Schuster MSteinberg G de Wit PJ Kema GH 2014 Molecular characterization andfunctional analyses of ZtWor1 a transcriptional regulator of the fungal wheatpathogen Zymoseptoria tritici Mol Plant Pathol 15 394ndash405

Mitrovich QM Tuch BB Guthrie C Johnson AD 2007 Computational andexperimental approaches double the number of known introns in thepathogenic yeast Candida albicans Genome Res 17 (4) 492ndash502

Morais do Amaral A Antoniw J Rudd JJ Hammond-Kosack KE 2012 Definingthe predicted protein secretome of the fungal wheat leaf pathogenMycosphaerella graminicola PLoS ONE 7 e49904

Motteram J Kufner I Deller S Brunner F Hammond-Kosack KE NurnbergerT Rudd JJ 2009 Molecular characterization and functional analysis of MgNLPthe sole NPP1 domain-containing protein from the fungal wheat leaf pathogenMycosphaerella graminicola Mol Plant Microbe Interact 22 790ndash799

Motteram J Lovegrove A Pirie E Marsh J Devonshire J van de Meene AHammond-Kosack K Rudd JJ 2011 Aberrant protein N-glycosylation impactsupon infection-related growth transitions of the haploid plant-pathogenicfungus Mycosphaerella graminicola Mol Microbiol 81 415ndash433

Muumlller AN Ziemann S Treitschke S Assmann D Doehlemann G 2013Compatibility in the Ustilago maydis-maize interaction requires inhibition ofhost cysteine proteases by the fungal effector Pit2 PLoS Pathog 9 e1003177

Mylonakis E 2008 Galleria mellonella and the study of fungal pathogenesismaking the case for another genetically tractable model host Mycopathologia165 1ndash3

Navarro-Velasco GY Prados-Rosales RC Ortiz-Urquiza A Quesada-Moraga EDi Pietro A 2011 Galleria mellonella as model host for the trans-kingdompathogen Fusarium oxysporum Fungal Genet Biol 48 1124ndash1129

Nierman WC Pain A Anderson MJ Wortman JR Kim HS Arroyo J BerrimanM Abe K Archer DB Bermejo C Bennett J Bowyer P Chen D Collins MCoulsen R Davies R Dyer PS Farman M Fedorova N Fedorova NFeldblyum TV Fischer R Fosker N Fraser A Garcia JL Garcia MJ GobleA Goldman GH Gomi K Griffith-Jones S Gwilliam R Haas B Haas HHarris D Horiuchi H Huang J Humphray S Jimenez J Keller N Khouri HKitamoto K Kobayashi T Konzack S Kulkarni R Kumagai T Lafon ALatge JP Li W Lord A Lu C Majoros WH May GS Miller BLMohamoud Y Molina M Monod M Mouyna I Mulligan S Murphy LOrsquoNeil S Paulsen I Penalva MA Pertea M Price C Pritchard BL QuailMA Rabbinowitsch E Rawlins N Rajandream MA Reichard U Renauld HRobson GD Rodriguez de Cordoba S Rodriguez-Pena JM Ronning CMRutter S Salzberg SL Sanchez M Sanchez-Ferrero JC Saunders D SeegerK Squares R Squares S Takeuchi M Tekaia F Turner G Vazquez deAldana CR Weidman J White O Woodward J Yu JH Fraser C GalaganJE Asai K Machida M Hall N Barrell B Denning DW 2005 Genomicsequence of the pathogenic and allergenic filamentous fungus Aspergillusfumigatus Nature 438 1151ndash1156

Notteghem JL Silueacute D 1992 Distribution of the Mating Type Alleles inMagnaporthe grisea Population Pathogenic on Rice Phytopathology 82 421ndash424

OrsquoDonnell K Kistler HC Cigelnik E Ploetz RC 1998 Multiple evolutionaryorigins of the fungus causing Panama disease of banana concordant evidencefrom nuclear and mitochondrial gene genealogies Proc Natl Acad Sci USA95 2044ndash2049

Ohm RA Feau N Henrissat B Schoch CL Horwitz BA Barry KW CondonBJ Copeland AC Dhillon B Glaser F Hesse CN Kosti I LaButti KLindquist EA Lucas S Salamov AA Bradshaw RE Ciuffetti L HamelinRC Kema GH Lawrence C Scott JA Spatafora JW Turgeon BG de WitPJ Zhong S Goodwin SB Grigoriev IV 2012 Diverse lifestyles andstrategies of plant pathogenesis encoded in the genomes of eighteenDothideomycetes fungi PLoS Pathog 8 e1003037

Orton ES Deller S Brown JK 2011 Mycosphaerella graminicola from genomicsto disease control Mol Plant Pathol 12 413ndash424

Ortoneda M Guarro J Madrid MP Caracuel Z Roncero MI Mayayo E DiPietro A 2004 Fusarium oxysporum as a multihost model for the geneticdissection of fungal virulence in plants and mammals Infect Immun 72 1760ndash1766

Ou SH 1985 Rice Diseases Commonwealth Mycological Institute UKPareja-Jaime Y Martin-Urdiroz M Roncero MI Gonzalez-Reyes JA Roldan

Mdel C 2010 Chitin synthase-deficient mutant of Fusarium oxysporum elicitstomato plant defence response and protects against wild-type infection MolPlant Pathol 11 479ndash493

Perez-Nadales E Di Pietro A 2011 The membrane mucin Msb2 regulates invasivegrowth and plant infection in Fusarium oxysporum Plant Cell 23 1171ndash1185

Perkins DD 1949 Biochemical Mutants in the Smut Fungus Ustilago maydisGenetics 34 607ndash626

Perrin RM Fedorova ND Bok JW Cramer RA Wortman JR Kim HSNierman WC Keller NP 2007 Transcriptional regulation of chemicaldiversity in Aspergillus fumigatus by LaeA PLoS Pathog 3 e50

Pfaller MA Diekema DJ Gibbs DL Newell VA Bijie H Dzierzanowska DKlimko NN Letscher-Bru V Lisalova M Muehlethaler K Rennison C ZaidiM Group GAS 2009 Results from the ARTEMIS DISK Global AntifungalSurveillance Study 1997 to 2007 105-year analysis of susceptibilities ofnoncandidal yeast species to fluconazole and voriconazole determined by CLSIstandardized disk diffusion testing J Clin Microbiol 47 117ndash123

Plantinga TS Johnson MD Scott WK Joosten LA van der Meer JW PerfectJR Kullberg BJ Netea MG 2012 Human genetic susceptibility to Candidainfections Medical Mycology Official Publication of the International Societyfor Human and Animal Mycology 50 785ndash794

Pontecorvo G 1956 The parasexual cycle in fungi Annu Rev Microbiol 10 393ndash400


Potts MB Kim HS Fisher KW Hu Y Carrasco YP Bulut GB Ou YHHerrera-Herrera ML Cubillos F Mendiratta S Xiao G Hofree M Ideker TXie Y Huang LJ Lewis RE Macmillan JB White MA 2013 Usingfunctional signature ontology (FUSION) to identify mechanisms of action fornatural products Sci Signal 6 ra90

Prados-Rosales RC Roldan-Rodriguez R Serena C Lopez-Berges MS Guarro JMartinez-del-Pozo A Di Pietro A 2012 A PR-1-like protein of Fusariumoxysporum functions in virulence on mammalian hosts J Biol Chem 28721970ndash21979

Prados Rosales RC Di Pietro A 2008 Vegetative hyphal fusion is not essential forplant infection by Fusarium oxysporum Eukaryot Cell 7 162ndash171

Quaedvlieg W Kema GH Groenewald JZ Verkley GJ Seifbarghi S Razavi MMirzadi Gohari A Mehrabi R Crous PW 2011 Zymoseptoria gen nov anew genus to accommodate Septoria-like species occurring on graminicoloushosts Persoonia 26 57ndash69

Rabe F Ajami-Rashidi Z Doehlemann G Kahmann R Djamei A 2013Degradation of the plant defence hormone salicylic acid by the biotrophicfungus Ustilago maydis Mol Microbiol 89 179ndash188

Ratnieks FL Carreck NL 2010 Ecology Clarity on honey bee collapse Science327 152ndash153

Rep M Kistler HC 2010 The genomic organization of plant pathogenicity inFusarium species Curr Opin Plant Biol 13 (4) 420ndash426

Rep M van der Does HC Meijer M van Wijk R Houterman PM Dekker HLde Koster CG Cornelissen BJ 2004 A small cysteine-rich protein secreted byFusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato Mol Microbiol 53 1373ndash1383

Richards TA Soanes DM Jones MD Vasieva O Leonard G Paszkiewicz KFoster PG Hall N Talbot NJ 2011 Horizontal gene transfer facilitated theevolution of plant parasitic mechanisms in the oomycetes Proc Natl Acad SciUSA 108 15258ndash15263

Riscili BP Wood KL 2009 Noninvasive pulmonary Aspergillus infections ClinChest Med 30 pp 315-35 vii

Rispail N Di Pietro A 2009 Fusarium oxysporum Ste12 controls invasive growthand virulence downstream of the Fmk1 MAPK cascade Mol Plant MicrobeInteract 22 830ndash839

Rispail N Soanes DM Ant C Czajkowski R Grunler A Huguet R Perez-Nadales E Poli A Sartorel E Valiante V Yang M Beffa R Brakhage AAGow NA Kahmann R Lebrun MH Lenasi H Perez-Martin J Talbot NJWendland J Di Pietro A 2009 Comparative genomics of MAP kinase andcalcium-calcineurin signalling components in plant and human pathogenicfungi Fungal Genet Biol 46 287ndash298

Robledo-Briones M Ruiz-Herrera J 2013 Regulation of genes involved in cell wallsynthesis and structure during Ustilago maydis dimorphism FEMS Yeast Res 1374ndash84

Rossman AY Howard RJ Valent B 1990 Pyricularia grisea the correct name forthe rice blast disease fungus Mycologia 82 509ndash512

Rudd JJ Antoniw J Marshall R Motteram J Fraaije B Hammond-Kosack K2010 Identification and characterisation of Mycosphaerella graminicola secretedor surface-associated proteins with variable intragenic coding repeats FungalGenet Biol 47 19ndash32

Ryder LS Dagdas YF Mentlak TA Kershaw MJ Thornton CR Schuster MChen J Wang Z Talbot NJ 2013 NADPH oxidases regulate septin-mediatedcytoskeletal remodeling during plant infection by the rice blast fungus ProcNatl Acad Sci USA 110 3179ndash3184

Samaranayake DP Hanes SD 2011 Milestones in Candida albicans genemanipulation Fungal Gen Biol FG amp B 48 858ndash865

Sanchez-Martinez C Perez-Martin J 2001 Dimorphism in fungal pathogensCandida albicans and Ustilago maydis ndash similar inputs different outputs CurrOpin Microbiol 4 214ndash221

Santos MA Tuite MF 1995 The CUG codon is decoded in vivo as serine and notleucine in Candida albicans Nucleic Acids Res 23 1481ndash1486

Schaumlfer K Di Pietro A Gow NA MacCallum D 2014 Murine model for Fusariumoxysporum invasive fusariosis reveals organ-specific structures fordissemination and long-term persistence PLoS One 9 (2) e89920

Scharf DH Heinekamp T Brakhage AA 2014 Human and plant fungalpathogens the role of secondary metabolites PLoS Pathog 10 e1003859

Schirawski J Mannhaupt G Munch K Brefort T Schipper K Doehlemann G DiStasio M Rossel N Mendoza-Mendoza A Pester D Muller O WinterbergB Meyer E Ghareeb H Wollenberg T Munsterkotter M Wong P WalterM Stukenbrock E Guldener U Kahmann R 2010 Pathogenicitydeterminants in smut fungi revealed by genome comparison Science 3301546ndash1548

Schmitz HP Kaufmann A Kohli M Laissue PP Philippsen P 2006 Fromfunction to shape a novel role of a formin in morphogenesis of the fungusAshbya gossypii Mol Biol Cell 17 130ndash145

Schrettl M Bignell E Kragl C Joechl C Rogers T Arst Jr HN Haynes K HaasH 2004 Siderophore biosynthesis but not reductive iron assimilation isessential for Aspergillus fumigatus virulence J Exp Med 200 1213ndash1219

Selmecki A Forche A Berman J 2006 Aneuploidy and isochromosome formationin drug-resistant Candida albicans Science 313 367ndash370

Seok J Warren HS Cuenca AG Mindrinos MN Baker HV Xu W RichardsDR McDonald-Smith GP Gao H Hennessy L Finnerty CC Lopez CMHonari S Moore EE Minei JP Cuschieri J Bankey PE Johnson JL SperryJ Nathens AB Billiar TR West MA Jeschke MG Klein MB Gamelli RLGibran NS Brownstein BH Miller-Graziano C Calvano SE Mason PHCobb JP Rahme LG Lowry SF Maier RV Moldawer LL Herndon DN

Davis Xiao W Tompkins RG 2013 Genomic responses in mouse modelspoorly mimic human inflammatory diseases Proc Natl Acad Sci USA 1103507ndash3512

Skibbe DS Doehlemann G Fernandes J Walbot V 2010 Maize tumors causedby Ustilago maydis require organ-specific genes in host and pathogen Science328 89ndash92

Slutsky B Buffo J Soll DR 1985 High-frequency switching of colonymorphology in Candida albicans Science 230 666ndash669

Slutsky B Staebell M Anderson J Risen L Pfaller M Soll DR 1987 lsquolsquoWhite-opaque transitionrsquorsquo a second high-frequency switching system in Candidaalbicans J Bacteriol 169 189ndash197

Snyder WC Hansen HN 1940 The species concept in Fusarium Am J Bot 2764ndash67

Stahmann KP Arst Jr HN Althofer H Revuelta JL Monschau N Schlupen CGatgens C Wiesenburg A Schlosser T 2001 Riboflavin overproduced duringsporulation of Ashbya gossypii protects its hyaline spores against ultravioletlight Environ Microbiol 3 545ndash550

Stahmann KP Revuelta JL Seulberger H 2000 Three biotechnical processesusing Ashbya gossypii Candida famata or Bacillus subtilis compete with chemicalriboflavin production Appl Microbiol Biotechnol 53 509ndash516

Stakman EC Christensen JJ 1927 Heterothallism in Ustilago zeaePhytopathology 17 827ndash834

Starnes JH Thornbury DW Novikova OS Rehmeyer CJ Farman ML 2012Telomere-targeted retrotransposons in the rice blast fungus Magnaportheoryzae agents of telomere instability Genetics 191 389ndash406

Steele C Rapaka RR Metz A Pop SM Williams DL Gordon S Kolls JKBrown GD 2005 The beta-glucan receptor dectin-1 recognizes specificmorphologies of Aspergillus fumigatus PLoS Pathog 1 e42

Steinbach WJ 2013 Are we there yet Recent progress in the molecular diagnosisand novel antifungal targeting of Aspergillus fumigatus and invasiveaspergillosis PLoS Pathog 9 e1003642

Steinberg G Perez-Martin J 2008 Ustilago maydis a new fungal model system forcell biology Trends Cell Biol 18 61ndash67

Steiner S Wendland J Wright MC Philippsen P 1995 Homologousrecombination as the main mechanism for DNA integration and cause ofrearrangements in the filamentous ascomycete Ashbya gossypii Genetics 140973ndash987

Stergiopoulos I Zwiers LH De Waard MA 2003 The ABC transporter MgAtr4 isa virulence factor of Mycosphaerella graminicola that affects colonization ofsubstomatal cavities in wheat leaves Mol Plant Microbe Interact 16 689ndash698

Stukenbrock Bataillon Dutheil Hansen Li Zala McDonald Wang Schierup 2011The making of a new pathogen insights from comparative population genomicsof the domesticated wheat pathogen Mycosphaerella graminicola and its wildsister species Genome Res 21 2157ndash2166

Stukenbrock EH 2013 Evolution selection and isolation a genomic view ofspeciation in fungal plant pathogens New Phytol 199 895ndash907

Stukenbrock EH Christiansen FB Hansen TT Dutheil JY Schierup MH 2012Fusion of two divergent fungal individuals led to the recent emergence of aunique widespread pathogen species Proc Natl Acad Sci USA 109 10954ndash10959

Sudbery P Gow N Berman J 2004 The distinct morphogenic states of Candidaalbicans Trends Microbiol 12 317ndash324

Sudbery PE 2011 Growth of Candida albicans hyphae Nat Rev Microbiol 9 737ndash748

Suffert F Sache I Lannou C 2013 Assessment of quantitative traits ofaggressiveness in Mycosphaerella graminicola on adult wheat plants PlantPathol

Suffert F Sache I Lannou C 2011 Early stages of septoria tritici blotch epidemicsof winter wheat build-up overseasoning and release of primary inoculumPlant Pathol 60 166ndash177

Talavera G Castresana J 2007 Improvement of phylogenies after removingdivergent and ambiguously aligned blocks from protein sequence alignmentsSyst Biol 56 564ndash577

Talbot NJ 2003 On the trail of a cereal killer Exploring the biology ofMagnaporthe grisea Annu Rev Microbiol 57 177ndash202

Talbot NJ Ebbole DJ Hamer JE 1993a Identification and characterization ofMPG1 a gene involved in pathogenicity from the rice blast fungus Magnaporthegrisea Plant Cell 5 1575ndash1590

Talbot NJ Salch YP Ma M Hamer JE 1993b Karyotypic variation within clonallineages of the rice blast fungus Magnaporthe grisea Appl Environ Microbiol59 585ndash593

Thywissen A Heinekamp T Dahse HM Schmaler-Ripcke J Nietzsche S ZipfelPF Brakhage AA 2011 Conidial dihydroxynaphthalene melanin of thehuman pathogenic fungus Aspergillus fumigatus interferes with the hostendocytosis pathway Front Microbiol 2 96

Valent B Farrall L Chumley FG 1991 Magnaporthe grisea genes forpathogenicity and virulence identified through a series of backcrossesGenetics 127 87ndash101

Verweij PE Snelders E Kema GH Mellado E Melchers WJ 2009 Azoleresistance in Aspergillus fumigatus a side-effect of environmental fungicide useLancet Infect Dis 9 789ndash795

Volling K Thywissen A Brakhage AA Saluz HP 2011 Phagocytosis ofmelanized Aspergillus conidia by macrophages exerts cytoprotective effectsby sustained PI3KAkt signalling Cell Microbiol 13 1130ndash1148

Vollmeister E Schipper K Feldbrugge M 2012 Microtubule-dependent mRNAtransport in the model microorganism Ustilago maydis RNA Biol 9 261ndash268


Walther A Wendland J 2012 Yap1-dependent oxidative stress response providesa link to riboflavin production in Ashbya gossypii Fungal Genet Biol 49 697ndash707

Wendland J Dunkler A Walther A 2011 Characterization of alpha-factorpheromone and pheromone receptor genes of Ashbya gossypii FEMS Yeast Res11 418ndash429

Wendland J Philippsen P 2001 Cell polarity and hyphal morphogenesis arecontrolled by multiple rho-protein modules in the filamentous ascomyceteAshbya gossypii Genetics 157 601ndash610

Wendland J Walther A 2005 Ashbya gossypii a model for fungal developmentalbiology Nat Rev Microbiol 3 421ndash429

Wendland J Walther A 2011 Genome evolution in the eremothecium clade ofthe Saccharomyces complex revealed by comparative genomics G3 539ndash548

Whiteway M 2000 Transcriptional control of cell type and morphogenesis inCandida albicans Curr Opin Microbiol 3 582ndash588

Wilson RA Talbot NJ 2009 Under pressure investigating the biology of plantinfection by Magnaporthe oryzae Nat Rev Microbiol 7 185ndash195

Wittenberg AH van der Lee TA Ben Mrsquobarek S Ware SB Goodwin SB KilianA Visser RG Kema GH Schouten HJ 2009 Meiosis drives extraordinarygenome plasticity in the haploid fungal plant pathogen Mycosphaerellagraminicola PLoS ONE 4 e5863

Xu J Saunders CW Hu P Grant RA Boekhout T Kuramae EE Kronstad JWDeangelis YM Reeder NL Johnstone KR Leland M Fieno AM Begley

WM Sun Y Lacey MP Chaudhary T Keough T Chu L Sears R Yuan BDawson Jr TL 2007 Dandruff-associated Malassezia genomes revealconvergent and divergent virulence traits shared with plant and humanfungal pathogens Proc Natl Acad Sci USA 104 18730ndash18735

Xu JR Hamer JE 1996 MAP kinase and cAMP signaling regulate infectionstructure formation and pathogenic growth in the rice blast fungusMagnaporthe grisea Genes Dev 10 2696ndash2706

Xu JR Staiger CJ Hamer JE 1998 Inactivation of the mitogen-activated proteinkinase Mps1 from the rice blast fungus prevents penetration of host cells butallows activation of plant defense responses Proc Natl Acad Sci USA 9512713ndash12718

Zhan J Kema GH Waalwijk C McDonald BA 2002 Distribution of mating typealleles in the wheat pathogen Mycosphaerella graminicola over spatial scalesfrom lesions to continents Fungal Genet Biol 36 128ndash136

Zhao W Panepinto JC Fortwendel JR Fox L Oliver BG Askew DS RhodesJC 2006 Deletion of the regulatory subunit of protein kinase A in Aspergillusfumigatus alters morphology sensitivity to oxidative damage and virulenceInfect Immun 74 4865ndash4874

Zordan RE Galgoczy DJ Johnson AD 2006 Epigenetic properties of white-opaque switching in Candida albicans are based on a self-sustainingtranscriptional feedback loop Proc Natl Acad Sci USA 103 12807ndash12812


1 Introduction





231 Virulence determinants

232 Signaling involved in virulence

233 Host perception and response

234 Development of anti-infective strategies

235 Contribution of fungal secondary metabolism to virulence

24 Conclusions

3 Candida species




331 Genome functional analysis

332 Altered host responses

333 Yeast-to-hypha transition

334 Importance of the cell wall in immune recognition

34 Conclusions





431 Role of MAPK cascades in virulence

432 Lineage specific (LS) chromosomes

433 Secreted effectors and gene-for-gene system

434 F oxysporum as a model for fungal trans-kingdom pathogenicity

44 Conclusions

5 Ashbya gossypii



521 Riboflavin production

522 Hyphal growth

523 Septation

524 Nuclear division and movement

525 Genome evolution

53 Conclusions





631 Mechanisms of fungal infection

632 Plant pathogen interactions in the rice blast fungus M oryzae

633 Signaling

64 Conclusions

7 Ustilago maydis




731 Host perception and plant response

732 Effector function

733 Cell cycle dimorphism and pathogenic development

734 Signaling and MAP kinase targets involved in virulence

735 Understanding the molecular basis of fundamental eukaryotic processes

74 Conclusions





831 Signaling pathways and protein secretion

832 Plant-pathogen interaction effectors

833 Disease control

834 Evolutionary genomics

84 Conclusions


Acknowledgments


References


24 Conclusions 49

3 Candida species 50

31 Comparative genomics in Candida species 5032 The toolbox for C albicans 5033 Current research interests 52

331 Genome functional analysis 52332 Altered host responses 52333 Yeast-to-hypha transition 52334 Importance of the cell wall in immune recognition 52

34 Conclusions 52

4 Fusarium oxysporum 52

41 Overview of the F oxysporum genome 5342 Assessing virulence of F oxysporum 5343 Current research interests 53

431 Role of MAPK cascades in virulence 53432 Lineage specific (LS) chromosomes 54433 Secreted effectors and gene-for-gene system 54434 F oxysporum as a model for fungal trans-kingdom pathogenicity 54

44 Conclusions 54

5 Ashbya gossypii 54

51 Comparative genomics in Eremothecium 5552 Current research interests 55

521 Riboflavin production 55522 Hyphal growth 55523 Septation 55524 Nuclear division and movement 55525 Genome evolution 56

53 Conclusions 56

6 Magnaporthe oryzae 56

61 Overview of the M oryzae genome 5662 Assessing virulence of M oryzae 5663 Current research interests 57

631 Mechanisms of fungal infection 57632 Plant pathogen interactions in the rice blast fungus M oryzae 57633 Signaling 57

64 Conclusions 57

7 Ustilago maydis 57

71 Comparative genomics in U maydis 5872 Assessing virulence of U maydis 5873 Current research interests 58

731 Host perception and plant response 58732 Effector function 58733 Cell cycle dimorphism and pathogenic development 59734 Signaling and MAP kinase targets involved in virulence 59735 Understanding the molecular basis of fundamental eukaryotic processes 59

74 Conclusions 59

8 Zymoseptoria tritici 59

81 Overview of the Z tritici genome 6082 Assessing virulence of Z tritici 6083 Current research interests 60

831 Signaling pathways and protein secretion 60832 Plant-pathogen interaction effectors 60833 Disease control 61834 Evolutionary genomics 61

84 Conclusions 61

9 General conclusions 61

Acknowledgments 62Appendix A Supplementary material 62

References 62

1 Introduction

A small fraction of the estimated 5 million fungal species areresponsible for devastating diseases affecting agriculture andhuman health Emerging infectious diseases (EIDs) caused by fungiare increasingly recognized as major threats to food security andanimal health (Brown et al 2012 Fischer et al 2008) Dispersaland emergence of fungal diseases are furthermore promoted by

human activity primarily through global trade which lacks suffi-cient biosecurity measures and may be exacerbated by the impactof climate change (Brasier 2008 Fisher et al 2012 Gange et al2007 Harvell et al 1999 Ratnieks and Carreck 2010 Verweijet al 2009) Fungal pathogens are characterized by a remarkablegenetic flexibility that facilitates rapid evolution and adaptationto the host or environment (Calo et al 2013) These features whencombined with Darwinian selection support the emergence of new


































not identified






bipolar MATaa
















EPerez-Nadales

etalFungal

Genetics

andBiology

70(2014)

42ndash67

45















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alFungalG

eneticsand

Biology70

(2014)42ndash

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24 Conclusions





3 Candida species






































34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References


































not identified






bipolar MATaa
















EPerez-Nadales

etalFungal

Genetics

andBiology

70(2014)

42ndash67

45















no -








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adaleset

alFungalG

eneticsand

Biology70

(2014)42ndash

67
























Episomalelements


































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Pathogenicitymodels






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adaleset

alFungalG

eneticsand

Biology70

(2014)42ndash

67

Stra

ins

CEA

10C

EA17

aku

BSC

5314

NG

Y15

2C

AI4

R

M10

00W

P17

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SN

95S

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2

Gu

y11

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15P

H14

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4287

(FG

SC99

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24 Conclusions





3 Candida species






































34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References















no -








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Biology70

(2014)42ndash

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24 Conclusions





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34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References
























Episomalelements


































Does not apply


Pathogenicitymodels






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eneticsand

Biology70

(2014)42ndash

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ins

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cope

rsic

iw

ild

type

4287

(FG

SC99

35)

IPO

323

FB1

(a1b

1)F

B2

(a2b

2)

FB6a

(a2b

1)F

B6b

(a1b

2)

SG20

0(a

1mfa

2bE

1bW

2)

FBD

11(a

1a2

b1b2

)A

B31

(a2

P crg

1b

E1P c

rg1b

W2)

AB

33(a

2P n

ar1b

E1P n

ar1b

W2)

ATC

C10

895

CB

S102

347

and

deri

vati

ves

Bio

info

rmat

ic

gen

ome

data

base

s

Asp

ergi

llu

sG

enom

eD

atab

ase

Can

dida

Gen

ome

Dat

abas

eM

agn

apor

the

com

para

tive

Dat

abas

eFu

sari

um

Com

para

tive

Dat

abas

eM

ycos

phae

rella

gram

inic

ola

(an

amor

phS

epto

ria

trit

ici)

Ust

ilag

om

aydi

sD

atab

ase

Ash

bya

Gen

ome

Dat

abas

e

htt

p

ww

wa

spgd

org

h

ttp

w

ww

ca

ndi

dage

nom

eor

gh

ttp

w

ww

br

oadi

nst

itu

teo

rg

ann

otat

ion

gen

ome

mag

nap

orth

e_co

mpa

rati

ve

Mu

ltiH

ome

htm

l

htt

p

ww

w

broa

din

stit

ute

org

an

not

atio

ng

enom

efu

sari

um

_gro

up

Mu

ltiH

ome

htm

l

htt

p

gen

ome

jgid

oeg

ov

Myc

gr3

Myc

gr3

hom

eh

tml

htt

p

mip

sh

elm

hol

tz-

mu

ench

end

ege

nre

pro

ju

stil

ago

htt

p

agd

vita

l-it

ch

in

dex

htm

l

htt

p

ww

wb

road

inst

itu

teo

rg

ann

otat

ion

gen

ome

ust

ilag

o_m

aydi

s2

Hom

eh

tml








24 Conclusions





3 Candida species






































34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References

Stra

ins

CEA

10C

EA17

aku

BSC

5314

NG

Y15

2C

AI4

R

M10

00W

P17

SN87

SN

95S

N15

2

Gu

y11

P1-2

70-

15P

H14

TH

3FR

13B

R88

Fox

yspo

rum

fsp

ly

cope

rsic

iw

ild

type

4287

(FG

SC99

35)

IPO

323

FB1

(a1b

1)F

B2

(a2b

2)

FB6a

(a2b

1)F

B6b

(a1b

2)

SG20

0(a

1mfa

2bE

1bW

2)

FBD

11(a

1a2

b1b2

)A

B31

(a2

P crg

1b

E1P c

rg1b

W2)

AB

33(a

2P n

ar1b

E1P n

ar1b

W2)

ATC

C10

895

CB

S102

347

and

deri

vati

ves

Bio

info

rmat

ic

gen

ome

data

base

s

Asp

ergi

llu

sG

enom

eD

atab

ase

Can

dida

Gen

ome

Dat

abas

eM

agn

apor

the

com

para

tive

Dat

abas

eFu

sari

um

Com

para

tive

Dat

abas

eM

ycos

phae

rella

gram

inic

ola

(an

amor

phS

epto

ria

trit

ici)

Ust

ilag

om

aydi

sD

atab

ase

Ash

bya

Gen

ome

Dat

abas

e

htt

p

ww

wa

spgd

org

h

ttp

w

ww

ca

ndi

dage

nom

eor

gh

ttp

w

ww

br

oadi

nst

itu

teo

rg

ann

otat

ion

gen

ome

mag

nap

orth

e_co

mpa

rati

ve

Mu

ltiH

ome

htm

l

htt

p

ww

w

broa

din

stit

ute

org

an

not

atio

ng

enom

efu

sari

um

_gro

up

Mu

ltiH

ome

htm

l

htt

p

gen

ome

jgid

oeg

ov

Myc

gr3

Myc

gr3

hom

eh

tml

htt

p

mip

sh

elm

hol

tz-

mu

ench

end

ege

nre

pro

ju

stil

ago

htt

p

agd

vita

l-it

ch

in

dex

htm

l

htt

p

ww

wb

road

inst

itu

teo

rg

ann

otat

ion

gen

ome

ust

ilag

o_m

aydi

s2

Hom

eh

tml








24 Conclusions





3 Candida species






































34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References




3 Candida species






































34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References


























34 Conclusions


























44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References






















44 Conclusions



5 Ashbya gossypii



























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References
























53 Conclusions


























64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References















64 Conclusions



7 Ustilago maydis



























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References


























74 Conclusions

























84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References






















84 Conclusions











Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References




Acknowledgments




References






























































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References
































































































































































































































1 Introduction










24 Conclusions

3 Candida species








34 Conclusions









44 Conclusions

5 Ashbya gossypii




522 Hyphal growth

523 Septation



53 Conclusions







633 Signaling

64 Conclusions

7 Ustilago maydis









74 Conclusions







833 Disease control


84 Conclusions


Acknowledgments


References

fungal genetics and biology - core · review fungal model systems and the elucidation of...

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