UNIVERISITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE

CLUJ-NAPOCA DOCTORAL SCHOOL

FACULTY OF ANIMAL SCIENCES AND BIOTECHNOLOGY

Eng. TAOUTAOU Abdelmoumen  

DIFFERENTIAL GENE EXPRESSION WITH DIFFERENT TYPES OF RESISTANCE IN THE

PATHOSYSTEM P. INFESTANS-SOLANUM SPP.

Summary of Ph.D. Thesis  

                          

SUPERVISORS:

PROF. DR. SOCACIU CARMEN

PROF. DR.PAMFIL DORU

CLUJ-NAPOCA, OCT. 2010

Table of content

Introduction………………………………………………………………………………………… 2 Chapter 1: General Introduction………………………………………………………………... 3 1.2. Potato production, in the world, in Romania and in Algeria...………………………………... 3 1.3. Threat for potato production…………………………………………………………………... 3 Chapter 2: The Interaction Host-Pathogen……………………………………………………... 3 2.1. Introduction……………………………………………………………………………………. 3 2.2. Signal transduction and defense responses……………………………………………………. 4 2.3. Case study: P. infestans-Solanum spp. interaction……………………………………………. 4 2.3.1. Mechanisms of pathogenicity……………………………………………………………….. 4 2.3.2. Detection of P. infestans effectors…………………………………………………………... 5 2.3.3. Potato resistance genes……………………………………………………………………… 5 2.3.4. Resistance responses………………………………………………………………………. 5 Chapter 3: Differential Display………………………………………………………………….. 6 3.1 The original method……………………………………………………………………………. 6 Chapter 4: Transcriptomics……………………………………………………………………… 6 4.1.Introduction ……………………………………………………………………………………. 6 4.2. Material and Methods………………………………………………………………………… 7 4.3.Results and discussions……………………………………………………………………….. 7 4.3.1. Gene expression……………………………………………………………………………... 7 4.3.1.1. Compatible interactions………………………………………………………………….. 7 4.3.1.2. Incompatible interaction…………………………………………………………………... 8 Chapter 6: proteomics Resistance and pathogenesis-related proteins in the pathosystem p. infestans-solanum ssp…

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6.1. Introduction…………………………………………………………………………………… 9 6.2. Material and Methods………………………………………………………………………… 10 6.3. Results and Discussion……………………………………………………………………….. 10 6.3.1. Compatible interaction……………………………………………………………………… 10 6.3.2. Incompatible interaction…………………………………………………………………….. 11 Chapter 8: Metabolomics Fourier-Transform Infrared Spectroscopy for studying the pathosystem P. infestans-Solanum spp……………………………………………………………………………………….

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8.1. Introduction…………………………………………………………………………………… 14 8.2. Material and Methods………………………………………………………………………… 14 8.3. Results and discussions……………………………………………………………………….. 15 8.3.1. Effect of infection on the plant metabolism……………………………………………….. 15 8.3.2. Marker bands for late blight resistance and susceptibility…………………………………. 15

8.3.3. Discussions………………………………………………………………………………….. 17 Chapter 9

Preliminary results on diffential phenolic compounds synthesis in potato with different types of late blight resistance……………………………………………………………………..

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9.1. Introduction……………………………………………………………………………………. 22 9.2. Material and methods………………………………………………………………………… 22 9.3. Results and discussions………………………………………………………………………... 23 GENERAL CONCLUSIONS……………………………………………………………………. 26 Selective bibliography……………………………………………………………………………. 27

INTRODUCTION

Potato (Solanum tuberosum L. 1753) is a worldwide major culture (4th culture) with more than 300 million tonnes. However, this important agricultural production is threatened by the plant pathogen P. infestans (Mont.) de Bary. The last one has caused the famous Irish famine in the 1840’s.

The potato reaction to an invasion by a pathogen is determined by the type of resistance the plant has. The resistance response is genetically controlled. In the case of Phytophthora infestans two types of resistance are known; qualitative and quantitative. Both of them are highly controlled by the genetic expression of the gene (s) that the potato plant in question has.

Low progress in resistance breeding against P. infestans, which has been actively conducted over last 50 years in potato breeding, might be explained by limited knowledge on pathogen, host and their interaction (Zimnoch-Guzowska, 2008).

Gene pyramiding is proposed as o solution to late blight. Understanding the expression pattern in potato carrying several R genes facilitate the use of R gene pyramiding in late blight control.

In this thesis, we studied the resistance/susceptibility to late blight with different potato genotypes: free R gene potato (susceptible), represented by the cultivar Bintje; genotype with R2 (susceptible), as a the other case of susceptibility (other susceptible genotypes have been used also (R1, R3, and R5 as susceptible, in the part of metabolomics))

The objective of this thesis was estalished to answer to 2 questions

How potato plant reacts to P. infestans infection, in function of resistance type: monogenic, oligobenic, and quantitative?

What are the differences between S. tuberosum, S. demissum , and S. bulbucastanum reaction’s to P. infestans infection?

In this thesis we tryed to study the mechanism of resistance/susceptibility in three levels: genomic/transcriptomic, proteomic, and finaly metabolomic. For realising this scope, we used three complimentary methods: differential display, SDS-PAGE, and FTIR. We also used HPLC for identifing some phenolic compounds in case of resistant plants.

This work was realised in the depatment of Biotechnology in the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, for the part of Genomics-Transcriptomics, at the department of Molecular and Cellular Biology in the Univeristy of Medicine and Pharmacy of Cluj-Napoca, and in the department of Chemistry and Biochemistry at the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, for the part of Metabolomics.

 

 

 

 

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Chapter 1: General Introduction

1.1. The history of potato and its introduction in Romania and in Algeria 

The potato (Solanum tuberosum L.) is originated from the South America, in the Andes region. It was brought to Europe in the late 16th century.  The “cartof” was introduced to Transylvania from Germany in the 1700s. Potato arrived late in Africa, around the turn of the 20th century. After potato's introduction to Algeria, in the mid-1800s, potatoes were grown mainly for export to French markets. By national independence from France, in 1962, farmers were harvesting on average 250 000 tones/year, with about one third marked for export. 1.2. Potato production, in the world, in Romania and in Algeria Potato is the forth crop in the world after wheat, maize and rice. In Algeria, is the second after wheat. The same case is encountered in Romania. The consumption per capita per year is 53 kg and 96 kg in 2003 respectively (Faostat, 2008). Algeria is the 2nd potato producer in Africa after Egypt. Potato is grown over an area of 90 000 ha, and can be planted and harvested somewhere in Algeria in virtually any month of the year. The main fresh potato growing areas are along the Mediterranean coast, where a mild climate permits year-round production, also in internal regions, and the north of Sahara.

1.3. Threat for potato production Potato is a host for many plant pathogens, among 60 pathogens, ranged from phytoplasma,

viroids, viruses, bacteria, nematodes, fungi and oomycetes. The most important potato pathogens are: Phytophthora infestans, Streptomycesscabies, Ralstonia solanacearum, Alternaria solani, PVY, PVX, PLRV. Potato also is a host for many insects; the most important is the Colorado beetle (Leptinotarsa decemlineata) and other insects such as aphids, thrips, etc.

Phytophthora infestans is the most important and devastating disease on potato crop. It is also very destructive to tomatoes and some other members of the family Solanaceae (Agrios, 1997). In the years 1840, it caused the famous Irish famine. The direct monetary costs of control efforts and lost production are estimated at > $3 billion/year worldwide (CIP, 1996 in Fry, 2008). Today, although chemicals used against P. infestans provide some level of disease control, worldwide losses due to late blight and control measures, are estimated to exceed 5$ billions annually, P. infestans is thus regarded as a threat to global food security (Duncan, 1999).

Chapter 2

The Interaction Host-Pathogen 2.1. Introduction

Most plants are resistant to most plant pathogens. They have evolved sophisticated mechanisms to perceive pathogen attack and trigger an effective immune response (van der Hoorn & Kamoun, 2008). Passive protection against pathogens that are not specialized to attack a specific host is provided by waxy cuticular layer and performed by antimicrobial compounds (Dangl &Jones, 2001). Active resistance comprises a series of interconnected process which, following recognition, are induced in the host cell through its continuing irritation by structures or products of the parasite and which results in exclusion, inhibition or elimination of the potential pathogen (Heitefuss, 1982). Plant can defend themselves actively by neo-synthesizing of antimicrobial compounds, reinforcement of cell wall, formation of abscission layers, tyloses, and gum deposition, and finally by the hypersensitive response.

Early recognition of the pathogen by the plant is very important if the plant is to mobilize the available biochemical and structural defenses to protect itself from the pathogen. The plant apparently begins to receive signal molecules, which indicate the presence of a pathogen, as soon as the pathogen establishes physical contact with the plant (Agrios, 1997).

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2.2. Signal transduction and defense responses

After pathogen detection, the signal of its presence must be transmitted to the defense genes. The defense reactions are induced by changes in membranes potential, ion fluxes, production of active oxygen species, lipid peroxidation and protein phosphorylation, which alter the cell physiology and generate many messengers involved in activation of local or of systemic defence (Dixon et al., 2002). More than one molecule is implicated in signal transmission: salicylic acid (Bennett & Wallsgrove, 1994), jasmonates (Bennett & Wallsgrove, 1994; Wasternack, 2007), ethylene (Nimchuck et al., 2003), nitric oxide (Neil et al., 2003), reactive oxygen species (Grant & Loake, 2000; Garcia-Olmedo, 2001), and calcium (Lecourieux et al., 2006).

2.3. Case study: P. infestans-Solanum spp. interaction

2.3.1. Mechanisms of pathogenicity Plant pathogens have developed sophisticated molecular strategies to evade and manipulate the

plant immune system (Qutob et al., 2006). P. infestans adopts a two-step infection style typical of hemibiotrophs: the first infection stage, in which the pathogen needs living host cells, is followed by extensive necrosis resulting in colonization and sporulation (Kamoun and Smart, 2005). For a successfully infection and colonization, a series of processes are needed. They include adhesion to plant surface, penetration and colonization (Huitema et al., 2004). It involves the secretion of proteins and other molecules (Huitema et al., 2004).

Some of these molecules participate to the pathogen attachment to plant surface, others to breaking physical obstacles to infection (cell wall and membranes), and several modify the plant physiology by suppressing plant defense (Kamoun and Smart, 2005). The suppression of host defenses can occur through the production of inhibitory proteins that target host enzyme (Kamoun, 2003). Effectors are molecules that manipulate host cell structure and function, thereby facilitating infection (virulence factors or toxin) and/or triggering defense responses (avirulence factors or elicitors) (Kamoun, 2006). A classification of P. infestans effectors is presented in table 1. It is based on the Kamoun’s (2006) oomycete effector classification.

Tab.1. P. infestans effectors and their function (Taoutaou et al., 2008).

Tab.1.

Efectori produse de P. infestans si rolul lor in patogeneitate (Taoutaou et al., 2008). Localisation Group Effector Function References Apoplastic Enzyme Inhibitors EPI1 and

EPI10 Inhibit and interact with P69B, protect proteins from degradation by P69B

Kamoun, 2006, and references in

EPIC1 and EPIC2

Target an apoplastic papain-like protease Kamoun 2006

Small Cysteine-rich protein

INF1, INF2A and INF2B

Condition avirulence in Nicotiana benthamiana Kamoun 2006 and references in

SCR74 and SCR91

unknown

Nep1-like (NLP) family

PiNPP1 Induce defense reponses in both susceptible and resistant plants

Kamoun, 2006

Cytoplasmic RxLR protein family AVR3a Induces HR in potato carrying R3a gene Cell death suppressor

Kamoun, 2007

CRN protein family CRN1, CRN2 and CRN8

Elicit cell death Kamoun 2007 and references in

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2.3.2. Detection of P. infestans effectors

Many of P. infestans effectors are described in literature. Some of these effectors are not detectable by the plant, and by consequences, the disease occurs. If the effector is detected, the plant activates its defense responses. In P. infestans until now, the effector characterized which induce defense responses is the Avr3a. The races which carry the gene Avr3a are avirulente on potato cultivars which carry R3a gene.

2.3.3. Potato resistance genes

Resistance to late blight is determined by dominant R genes and by quantitative resistance. The first type of resistance is race specific. The second is a nonspecific resistance controlled by unknown number of genes (Gebhardt &Valkonen, 2001). 20 R-genes conferring potato foliage resistance to late blight have been placed on a molecular map so far. 11 of these genes (R1, R2, R3a, R3b, R5-R11) from S. demissum, 5 genes (RB/Rpi-blb1, Rpi-blb2, Rpi-blb3, Rpi-abpt) from S. bulbocastanum, and one each from S. berthaultii (Rber/Rpi-ber), S. pinnatisectum (Rpi1), and S. mochiquense (Rpi-moc1) (Simko et al., 2007). R1 was cloned by Ballovora et al. (2002). It is situated in the chromosome 5. Huang et al. (2004) have found that in the R3 locus are 2 genes R3a and R3b. Both R3a and R3b are in the chromosome 11 and were cloned (Huang et al., 2004; Huang S. in Tae-Ho, 2005). R10 and R11 were found to be allelic versions of genes at the R3 locus on chromosome 11 (Bradshaw et al., 2006). Song et al. (2003) have cloned an R gene which was mapped by Naess et al. (2000) and was localized in the chromosome 8. The RB gene, cloned from the Mexican diploid potato species Solanum bulbocastanum, confers broad-spectrum resistance to potato late blight (Liu & Halterman, 2006; Staples, 2003). An RB-orthologous gene was cloned from Solanum verrucosum, and when introduced to S. tuberosum it confers resistance to P. infestans (Liu & Halterman, 2006). Recently, strains compatible with this gene were detected (Fry, 2008). Solis et al. (2007) have isolated a defensin gene, lm-def, isolated from the Andean crop ‘maca’ (Lepidium meyenii) with activity against the pathogen Phytophthora infestans.

The quantitative traits of potato late blight resistance are also known. Factors controlling quantitative resistance to P. infestans have been found on almost every potato chromosome (Gebhardt & Valkonen, 2001). Wang et al. (2005) has elucidated some mechanisms quantitative resistance and the genes involved in. a total of 348 P. infestans-responsive genes were identified. These functional genes are mostly related to metabolism, plant defense, signaling and transcription regulation, involving the whole process of plant defense response to pathogens. In addition, 114 novel genes with unknown functions were isolated (Wang et al., 2005). Abu-Nada et al. (2007) have found that these metabolites called as pathogenesis related (PR) metabolites play many roles: 1- homeostasis, 2- primary defense, 3- secondary defense and 4- collapse of primary and secondary defense responses. During the primary and secondary defence phases, dramatic changes in the amino acids, known precursors of several plant defence-related metabolites (Abu-Nada et al., 2007). The expression pattern differ in time, some genes are activated more rapidly than others (Tian et al., 2006)

2.3.4. Resistance responses

Plants respond to pathogen attack with a multicomponent defense response (Gobel et al., 2002).Elicitation results in the activation of a series of host defense responses, including cell wall reinforcement by deposition of callose and lignin, production of lytic enzymes such as chitinases and glucanases, biosynthesis of phytoalexins and of pathogenesis-related (PR) protein synthesis, and programmed cell death (Desender et al., 2007). Lipoxygenase (LOX) activity has been identified consistently during pathogen-induced defense responses. POTLX-3 (specific leaf LOX gene of potato) mRNA accumulation was induced in potato leaves infected with either virulent or avirulent strains of

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Phytophthora infestans (Kolomiets et al., 2000). Moreover the lipoxygenase, P. infestans elicits multiple defense responses, including an oxidative burst, accumulation of phenylpropanoid compounds, and sesquiterpenoid phytoalexins (lubimin and rishitin), which are accompanied by de novo synthesis of the enzymes that produce them (Nakane et al., 2003). The role of phenylpropanoid pathways in plant defense is discussed by Dixon et al. (2002).

Phenolic compounds with callose are the major components of the papilla, which is a wall apposition, the first wall against the invader (Schmelzer, 2002). The A crucial role in plant defending system is the reorganization of cytoplasm and cytoskeleton. Schelmelzer (2002) discusses the role and importance of the cytoskeleton in plant defence with exemples of the pathosystem P. infestans-S. tuberosum(fig.18). The last and the most effective mechanism of defence is the hypersensitive response (HR). The HR generally occurs as a rapid, localized necrosis, a form of programmed cell death (Kamoun et al., 1999). It prevents a further spread of the pathogen (Birch & Whisson, 2001).

Chapter 3

Differential Display

A fingerprinting technique to identify and compare mRNAs during different cell processes was first described by Liang and Pardee (1992) (Fig.1). The requirements of this protocol were; reproducibility, comparison of all mRNA species of interest, and the ability to isolate the corresponding cDNA (Sturtevant, 2000).

3.1 The original method

The procedure consists of 2 major steps:

Reverse transcription (RT) of RNAs isolated from different cell populations with a set of degenerate, anchored oligo(dT) primers to generate cDNA pools.

PCR amplification of random partial sequences from the cDNA pools with the original anchored dT primers and an upstream arbitrary primers. The annealing temperature of the PCR is low to maximize the number of amplified mRNA species. The α-35S-labeled radioisotopes are used for visualizing in sequencing gel. The same primer set had been used on multiple cell populations.

The primer sets are designed to amplify 50-100 mRNA (Sturtevant, 2000). The original primer set was formed by 12 oligo(dT): 11 are Ts to match with the 3’ polyadenylated end and 2 additional bases. For mammalian cells, it was calculated that 20 arbitrary 10-mers in conjunction with 12 anchored primers would statistically amplify all mRNA sequences (Sturtevant, 2000).

Once differentially displayed PCR products are identified, the cDNA species of interest are excised from the dried sequencing gel, extracted by boiling, and reamplified with the same original primer sets. These PCR products can then directly be subcloned, sequenced, and used as a probe in Northern analysis to confirm differential expression or to screen libraries.

Chapter 4

Transcriptomics

4.1.Introduction

Gene expression is the process by which the information stored in the genome is transformed to a phenotype. The genome, in the other hand, represents the whole genes in an organism. A gene is an ensemble of deoxyribonucleotides. In an organism, the spatial and temporal expression of different genes

6  

encoded by the genome is a highly regulated process that determines both the normal and pathological growth and developmental of an organism. Therefore, understanding the mechanisms of these normal and pathological processes requires identification, isolation, and characterization of differentially expressed genes (Sturtevant, 2000).

4.2. Material and Methods

For genomic DNA amplification by the RAPD technique, all potato plants have been used. The scope of genomic DNA amplification has been the selection of a set of primers which amplify potato DNA. In this study we used the cultivar Bintje as a control (susceptible, without any R gene). The susceptible plant carrying an R gene has been R2. As a resistant cultivated potato plant we took R4, 21. The last one is carrying three R genes: R2, R3, and R4. Two wild species of potato have been used also: S. demissum and S. bulbocastanum. The infection has been elaborated conform the CIP manual (1997) (see chapter: material and methods). All the plants have been grown in a green house. When used for experiment performing, they aged 5 weeks.

The infection process: a detached leaf assay was elaborated. Full developed leaves were recolted from healthy potato plants. The infection was made by 30 µl of P. Infestans suspension in the midrib of each leaf. As a support we used a water agar medium. The blank leaves were inoculated with sterile distilled water. The incubation was realized in a phytothron at 20°C, 16 h/8 h day/night and 70 % relative humidity. The t0 was put immediately at -80°C, t1 after 24 h, t2 after 48 h and t3 after 72 h, until used

Genomic DNA extraction was conforming to Lodhi et al (1994) improved by Pop et al (2004) (see material & methods). The mRNA isolation was carried with the extraction kit from Healthcare (see materials & methods). Reverse transcription was elaborated conform the kit ready to go from the same manufacturer. For PCR amplification, the same program used for genomic DNA was used. Sequencing: for cDNA sequencing, we call to Microsynth services.

4.3.Results and discussions

From 22 primers tested, 16 have amplified genomic potato DNA. 13 have been chosen for expression study: OPC-13, OPC-20, 70-03, 70-04, 70-08, Mic-07, Mic-13, Mic-14, 595, 270, 534, 563, 594.

4.3.1. Gene expression

For gene expression study, the method choosen has been the Differential Display. The mRNA was isolated and reverse transcripted. The obtained cDNA has been amplified with the different decamer primers above cited. The polymorfic bands have been excised from the gel and reamplified with same primer until obtention of a clean one band (the same), and then the band of interest is cloned, and sequenced. For sequencing we call for services of Microsynth (Swizerland).

4.3.1.1. Compatible interactions

In this case are included the cultivar Bintje and the potato plant carrying the resistance gene R2, noticed R2. Both plants have been susceptible when infested by the choosen P. infestans isolates.

Case of Bintje and R2

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In this case, many bands have been suppressed/induced. As it is demonstrated in the fig. 1. Infection with P. infestans has induced many bands which have not been detected in the control. However, many bands have been suppressed when infested with the mixture of isolates. In other cases, the induction/suppression is in function of time. Many bands have been induced/suppressed after some time from inoculation. This means that many corresponding to expressed or induced by the inoculation.

Fig. 1. Some cDNA profiles obtained by amplification with some decamer primers in the case of Bintje, 2: primer Mic-13, 3: primer 595, C: control, 0, 1, 2, and 3: 0 h, 24 h, 48 h, and 72 h post inoculation respectively. I: inoculated with the isolate A2.2, M: inoculated with the mixture of isolates.

 

Case 2: R2

In fig.2, we can see that also R2 has reacted with the same way to infestation as bintje

Fig. 2. Some cDNA profiles obtained by amplification with some decamer primers in the case of R2, 2: primer Mic-13, 3: primer 595, C: control, 0, 1, 2, and 3: 0 h, 24 h, 48 h, and 72 h post inoculation respectively. I: inoculated with the isolate A2.2, M: inoculated with the mixture of isolates.

The expression of many genes has been suppressed and for others induced. These bands have

been recuperated and cloned.

4.3.1.2. Incompatible interaction

Case R4:

8  

 

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Protein with 50-51 kDa molecular weight was identified as Beta-glucosidase 08 which belongs to the glycosyl hydrolase 17 family. It has a glycosidase and hydrolase activity. The protein with 27-28 Kd molecular weight was identified as Pathogenesis-related genes transcriptional activator PTI6 found in tomato (S. lycopersicum). It Acts as a transcriptional activator. Binds to the GCC-box pathogenesis-related promoter element. Activates the defense genes of plants. It is implicated in defence response, transcription and transcription regulation (http://www.uniprot.org/uniprot/O04682). It has been detected in only in the case of infested R2 potato and inoculated 21 which has been extremely resistant to infection by A2.2. With a moleuclar weight of 101 Kd, the protein detected in the case of R2A1.3 could be a

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R4:

R4 has been immune to A2.2. It did not show a HR. the figure 29 show the protein profile The table 23 is shown the molecular weight of different proteins detected. In this case, many pr

e implicated in the resistance of R4 to P. infestans: 112 kDa, 97, 65-67, 41-42, and 30 kDa.

Lipoxygenases (LOX; EC 1.13.11.12) are dioxygenases that catalyze the hydroperoxidation of polyunsaturated fatty acid or their esters that contains a cis, cis-1,4-pentadiene moiety (Kolomiets et al., 2000). Potato lipoxygenase (POTLX-3) is a polypeptide of 862 amino acids with a calculated molecular mass of 97.8 kD (Kolomiets et al., 2000). POTLX-3 transcripts accumulation was induced in leaves inoculated by both compatible and incompatible strains of P. infestans. They accumulated more rapidly, more consistently, and to greater levels during an incompatible interaction (Kolomiets et al

he case of R4P T0 and R4P T3, while in the case of bintje only in the case of BP T3.

The protein with molecular weight 37 kDa, was identified as Glucan endo-1,3-beta-glucosidase, basic isoform 1. Is thought to be an important plant defense-related product against fungal pathogens. It is implicated in Hydrolysis of (1->3)-beta-D-glucosidic linkages in (1->3)-beta-D-glucans (glucan endo-1,3-beta-D-glucosidase activity). In leaves, it is induced in response to infection, elicitor, ethylene, or wounding (http://www.uniprot.org/uniprot/P52400). In R4, it has been detected in all treatments, inoculated or not, with the exception of R4P T3. We think that it was induced after leaf dettachement, but it persisted in the case of inoculated leaves beacause of the late blight agent presence. The activity of β-1,3-glucanases and β-D-glucosidases increased in the leaves of potato plants infected by species of the root parasite genus Globodera. The reactions of cultivars to nematode infection differed, indicating that different pathotypes of the same species or different species of potato cyst nematode elicited the appeara

process. It has also a glycosidase and hydrolase activity (http://www.uniprot.org/uniprot/O04890

nce of different classes of β-1,3-glucanases (Rahimi et al., 1996).

The protein with 65-67 Kd, was identified as Endo-1,4-beta-glucanase (68.5 Kd). Is has been isolated from tomato (Solanum lycopersicon) with a cellulase activity. Also implicated in carbohydrate metabolic

). In our case it has been detected in the treatment R4P T0, R4P T1, R4P T3.

11  

Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid: One protein showed 1,3-β-glucanase activity, while the other one showed chitinase activity (Joosten & De Wit, 1989).

Case of 21

Potato plant 21 has been resistant to both isolates inoculated with, by the HR. in table 24 are shown different proteins detected. Also in this case, many proteins could be implicated: 243, 169, 156, 143, 98, 33-34, 24-25 kDa.

The protein detected in the case of 21A2.3 T3 is a CC-NBS-LRR protein (144,140 Kd). As all resistance protein it is a ATP-nucleotide binding protein, with a role in apoptosis (HR) and defence responses. However its expressed has been late, we think that it has an important role to play in resistance, because 21 has responsed to infection by a HR.

The protein (34-36 kd) identified as Endochitinase. Implicated in Defense against chitin containing fungal pathogens, by Random hydrolysis of N-acetyl-beta-D-glucosaminide (1->4)-beta-linkages in chitin and chitodextrins. It is implicated in different biological process: carbohydrate metabolism, chitin degradation, plant defence, and polysaccharide degradation. It is induced by ethylene (http://www.uniprot.org/uniprot/P05315). Ethylene is a phytohormone which play also a role in signaling, and also in HR. And this is the reason which this protein is present only in this case, where the resistance as a HR. Endochitinase is implicated in plant defense especially against chitin containing organism like P. Infestans, by chitin degradation. (http://www.uniprot.org/uniprot/P52403). Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3-beta-glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens (Beerhues L, Kombrink E, 1994).

The serine protease inhibitor with 24.009 Kd, has been found in the case of infested 21 by the both isolates A2.2 and A2.3 until the second day inclusive (24-25 Kd). It has been induced by the pathogen P. infestans. Protects the plant by inhibiting proteases of invading organisms, decreasing both hyphal growth and zoospores germination of P. Infestans (http://www.uniprot.org/uniprot/P58514). Serine protease inhibitor 1 (it has 2 chains, chain A and chain B), it protects the plant by inhibiting proteases of invading organisms, decreasing both hyphal growth and zoospores germination of P. infestans. Induced by P. Infestans infection. It was isolated from potato tubers infected by P. infestans (Valueva et al., 1998). P. infestans has two specific effectors EPI1 and EPI10, with the role to inhibit in the degradation of proteins by the tomato protease P69B (Kamoun, 2006).

Three proteins could be interesting, with the molecular weight with 243 Kd and 169 Kd in the case of 21A2.2 T1. And the third with 156 Kd detected in the case 21A2.2 T2.

The hypersensitive reaction to a pathogen is one of the most efficient defense mechanisms in nature and leads to the induction of numerous plant genes encoding defense proteins. These proteins include:

ϒ Structural proteins that are incorporated into the extracellular matrix and participate in the confinement of the pathogen;

ϒ Enzymes of secondary metabolism, for instance those of the biosynthesis of plant antibiotics; ϒ Pathogenesis-related (PR) proteins which represent major quantitative changes in soluble protein

during the defense response.

12  

Case of S. demissum

S. demissum also has been resistant to infection by P. infestans, it did not show any symptoms,neither the HR. in this case, 3 proteins could be implicated: 40-43, 24-25, and 20-21 kDa. In table 25 are shown proteins detected in this case.

The protein with the molecular weight 149 kDa was identified as CC-NBS-LRR protein (150.789 Kd). It is an ATP-binding, nucleotide binding, and protein binding protein (http://www.uniprot.org/uniprot/A6YP83). It is implicated in apoptosis and plant defence. It has been detected in the case of DMSP T0. Or it could be Disease resistance protein R3a-like protein. Both of these proteins have the same function and role in plant defence. We think it has been induced by P. Infestans. So the pathogen has been early detected. And the resistance reaction has started early, which resulted in a non-succesful infection by the pathogen. The protein which has a molecular weight of 17 kDa, was identified as Late blight resistance protein (putative). It has been deteted only in the case of DMSP T3. The protein identified in the case of DMSP T1 (45 kDa) was identified as Putative late blight resistance protein (identical) (45.335 kDa). It is implicated in apoptosis (HR), with the function of ATP binding (http://www.uniprot.org/uniprot/Q6L3N0). The protein (33-34 kDa) which has been detected in all inoculated treatments and only in DMS T0 and DMS T1 (control). The inoculation by P. Infestans has induced a persistance of this protein in inoculated leaves. It was identified as Putative Pto-like serine/threonine kinase. It belongs to the protein kinase superfamily. It is a kinase, serine/threonine-protein kinase, and a transferase. It is implicated in protein amino acid phosphorilation, and also an ATP binding protein (http://www.uniprot.org/uniprot/Q947Q3). The protein with a molecular weight of 61-63 Kd detected in the case of DMSP T1 si DMSP T2 was identified as Late blight resistance protein (putative) (60,405 Kd) (http://www.uniprot.org/uniprot/Q6L405).

Protein detected in the case of DMSP T1 was identified as Putative disease resistance protein CC-NBS-LRR. It is implicated in plant defence and apoptosis (HR), with an ATP-protein binding function (http://www.uniprot.org/uniprot/B3VI88).

Case of S. bulbocastanum

The protein with the molecular weight 146 Kd corresponds to the Late blight resistance protein Rpi-blb2. It is implicated in apoptosis (HR) and in defence responses. It is like all potato late blight resistance gene a NBS-LRR (van der Vossen et al., 2005; http://www.uniprot.org/uniprot/Q38QB6). It has been detedted only in the third day after inoculation. The protein with the molecular weight 38 kd, and which has been suppressed in all inoculated S. bulbocastanum plants corresponds to a putative uncharacterized protein (http://www.uniprot.org/uniprot/Q0ILF9).

The 24-25 Kd protein has been detected in the non-inoculate S. bulbocastanum and the inoculated one at T0. It has been suppresseb by P. Infestans. We identified it as 30S ribosomal protein S3, chloroplastic. It is part of the 30S ribosomal subunit in the chloroplast. It is implicated in translation, rRNA binding and it has a structural role in ribosome constitution (http://www.uniprot.org/uniprot/Q2MIE9).

13  

CHAPTER 8: METABOLOMICS

FOURIER-TRANSFORM INFRARED SPECTROSCOPY FOR STUDYING THE PATHOSYSTEM P. infestans-Solanum spp.

8.1. Introduction

Spectroscopy can be defined as the study of the interaction of an electromagnetic wave with matter (Dufour, 2009). Infrared spectroscopy is one of the most important and widely used analytical techniques available to scientists working in a whole range of fields (Stuart, 2004).

8.2. Material and Methods

Potato plant: we used three types of plant materials: the first was plant material R gene free, represented by the cultivars Bintje characterized by its susceptibility to late blight agent P. infestans and the cultivar Desiree known as a medium resistant to late blight. The second type is represented by the potato plant with resistance genes. Two subgroups have been formed, the first with a single R gene, and the second with multiple R genes (tab. 1). The third group was represented by other potato species: S. demissum and S. bulbocastanum. All plant material has been grown in green house under controlled conditions.

Tab. 1. Potato plants used in FTIR studies

Tab. 1. Materialul biologic utilizat în FTIR.

Free R genes plants Plante fără gene R

With R genes plants Plante cu gena R

Other species Alte specii

Bintje R1 S. demissum Desiree R2 S. bulbocastanum

R3 R4 R5 21 (R2R3R4)

Pathogen: 5 preparations of P. Infestans have been used for potato infection. A mixture of 6 races (see material & methods) and 4 preparations, each one containing a single race. The preparation of infection solution was as indicated in the CIP manual (1997) (see material & methods).

Infection: the CIP instructions for detached leaves infection have been followed for infection procedure (see material & methods). Two treatments have been designed: with the same pathogen isolate (genotype) and different host genotypes, and the second has been the same host genotype and different pathogen races (genotypes). In the case of cultivars bintje and desiree, two treatments have designed, inoculation by the isolate A2.2 and by the mixture of the six isolates.

Spectroscopy procedure: The leaves have been ground into a fine powder in a mortar and pestle under liquid nitrogen, and then transferred to an eppendorf 2 ml tube. 1 ml of 70 % methanol was added, and mixed by sonification for 15 minutes, then centrifuged. 100 µl of supernatant was used for analysis. Infrared profile was realized using the Shimatzu Prestige 2, Apodization: Happ-Genzel spectrophotometer, the profile was recorded in the wavelength range of 4000-500 cm-1.

14  

8.3. Results and discussions

All the plant material used in this test was susceptible to P. infestans. However the degree of susceptibility/resistance differs in function of the host and pathogen genotype.

8.3.1. Effect of infection on the plant metabolism

The total number of peaks has not been affected by the infection in the case of R2 (Fig.6.). In the case of R1, the infection has decreased the number of peaks significantly from 43 in the case of non infested potato leaves to 12when it has been infested by the race A1.1, and to 21, 19, and 15 peaks when infested by A1.3, A2.2, and A2.3 respectively. The inverse has been encountered in the case of R3; the infection increased the number of absorbance bands, from 19 in the case of non-infested to 34, 29, 33, and 31 if infested by A1.1, A1.3, A2.2 and A2.3 respectively. The case of R5 is somehow special; the infection by A2.3 has not affected significantly the number of peaks, but using the races A1.1, A1.3, and A2.2 as pathogen has increased the number of peaks.

Fig. 6. Effect of P. infestans infection on the potato metabolism, as a criteria the total number of peaks has been taken. R1, R2, R3, R5 are potato plant carrying the same R resistance gene to P. infestnas. H: control, sound potato leaves inoculate with sterile distilled water. A1.1, A1.3, A2.2, and A2.3 are P. infestans isolates used for this test.

Beside the total number of peaks, which can give as a high number of information about the reaction of potato to late blight agent infection, it is not sufficient. The response to infection is clearly more evident if we take in consideration the number of peaks induced/suppressed by the infection, or the number of peaks in common between the sound control and the infested. The infection by the pathogen P. infestans perturbs totally the metabolism; the peaks in common in the same plant differing only by the infection, is minimum: in the case of R1 only 7 peaks have been found in both infected and healthy, 11 in the case of R2, and 9 for R5. The rest of peaks has been suppressed (case of the control) or induced (case of infested leaves).

8.3.2. Marker bands for late blight resistance and susceptibility

In this part, we try to find some peaks which are in common between the resistant plants, and we did not find them in the susceptible ones. The behavior of all resistant plant is aproximatively the same, no matter of the species. Also the susceptible plant taken in this case, have had the same behavior of absorption in the IR interval. However, in comparison with the susceptible potato plants especially, the cultivar desiree and R2, the last have a delay in their absorption bands. In this scope, we looked after absorbance bands that are specific to resistant potato. Many bands have been identified. The band 631-637 cm-1, this band has been detected in the infested R4 and S. demissum, all the 21 treatments, and the control and infested S. bulbocastanum. Also the band 683-770 cm-1, with the exception of the peak at 691 cm-1, detected in the case of healthy bintje. The band 949-1016 cm-1, was also a band specific to the resistant potato. Near to the last band, the susceptible plants absorb at 1072-1076cm-1, while the resistant ones had absorbed at 1078-1109 cm-1. The band 1196-1200 has been in common for the aproximatively all the plant indifferent of their status in matter of resistance or susceptibility. An interesting band which has been specific to S. tuberosum, is 1396-1400 cm-1. Another resistant plant specific band is 1238-1265 cm-1. While the resistant S. tuberosum has absorbed at 1506 cm-1, and the also the infested R1, the susceptible plants has absorbed in the intervals 1524-1553 cm-1 and 1558-1572 cm-1. More other resistance band have been found: 1576-1589 cm-1, 2291-2313 cm-1, 2590-2932 cm-1 (for the last interval,

15  

some exceptions have been found with the control R1 at 2590 cm-1, the infested R3 at 2617 cm-1 and at 2855 cm-1), 3015-3262 cm-1, 3271-3275 cm-1. Other susceptibility bands have been detected at 1603-1630 cm-1, 1969-2041 cm-1, 2062-2152 cm-1, 2270-2284 cm-1 (Fig.7).

According to Sakhamuri et al., (2004), the peaks identified in all spectra can be grouped in 5 regions (I-V) and represent absorptions of IR energy by different groups of molecules. Carbohydrate specific region is located between 1000-1200 cm-1, while proteins and amino acids absorb in regions 3 and 4. Region 5 corresponds to O-H containing products (e.g. water) or to cholesterol and carotenoids (double bonds) (Tab. 2).

     

4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

1 . 2

1 . 4

1 . 6

1 . 8

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Inte

nsity

A b s o r b a n c e B a n d 1 / c m

2 1 A 2 . 2 2 1 A 2 . 3 D e s i r e e A 2 . 2 R 2 A 2 . 2 R 4 A 2 . 2

941

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1336

1483

1703

1056

1180

1294

1442

1585

1832

Tab. 2.

Assignement of IR wavenumbers identified in the 5 regions of the fTIR spectra.

Fig.7. FTIR spectra with characteristic bands of resistant and susceptible potato plant to late blight agent. All the spectra show the same behavior, however, for susceptible plant there is a delay for these bands. Resistant: 941-1180 cm-1, susceptible: 1056-1294 cm-1; resistant: 1336-1483 cm-1, susceptible: 1442-1585 cm-1; resistant: 1483-1703 cm-1, susceptible: 1585-1832 cm-1.

Tab. 2. Atribuiera lunigimi de unda identificate in 5 regiuni ale spectrului FTIR Frequency regions cm-1 Characteristic

frequencys cm-1 Assignment ( according to Sakhamuri et al., 2004)

Region 1 <1000 cm-1 976 964 961

Phosphates (P=O) Compoud with double conjugated bonds: polyinsaturated fatty acids and carotenoids

Region 2 : 1000-1200 cm-1 1035 1065 1079 1103

Glucose Fructose Galactose,Oligo-and Starch

Region 3: 1200-1500 cm-1 1120 1240, 1341 1400

Phosphorilated glycosides Secondary amines Carboxyl groups, specific cu organic acids and amino acids

Region 4: 1500-1700 cm-1 1566, 1574 1595 1650, 1662 1655

Proteins, peptides, Phenolics, chlorophylls Amides I, II

Region 5: 2800-3000 cm-1 2854 2920 2934

Fatty acids Cholesterol Carotenoid pigments

16  

8.3.3. Discussions The mid-infrared spectrum (4000–400 cm−1) can be approximately divided into four regions and

the nature of a group frequency may generally be determined by the region in which it is located. The regions are generalized as follows: the X–H stretching region (4000–2500 cm−1), the triple-bond region (2500–2000 cm−1), the double-bond region (2000–1500 cm−1) and the fingerprint region (1500–600 cm−1) (Stuart, 2004).

The bending frequency of C–H bond is close to 1340 cm-1, whereas its stretching frequency is observed at about 3000 cm-1. The intensity of the bands is related to the nature and polarity of the bond. Indeed, the C-O bond, formed by different atoms and highly polarized, strongly absorbs in the MIR region, while C-C bond absorbance in the MIR region is much weaker (Dufour, 2009). C–H bonds, which are found in large quantities in organic molecules, show stretching vibrations between 2750 and 3320 cm-1 in the MIR region (Dufour, 2009). The presence of methyl or methylene function can be assessed by the observation between 1465 and 1370 cm-1 of the bending vibrations of C–H bonds. For alcenes, the deformation outside of the plan of the C–H bond is characterized by a relatively intense absorption band between 650 and 1000 cm-1. The stretching vibration of the double bond C-C (non-conjugated) is observed between 1640 and 1666 cm-1 (Dufour, 2009). The carbonyl group, found in aldehydes, ketones, acids, esters, and amides, strongly absorbs in the MIR between 1650 and 1850 cm-1. The precise location of the stretching vibration ν C-O depends on resonance effects and hydrogen bonding. Infection of R2

The absorbance peak at 662 cm-1, has been found only in the case of healthy and A2.2 treatment, but suppressed in the case of A1.1 and A1.3. However, the peak intensity has been bigger in the case of healthy leaves (0.2561 for non-infested and 0.1812 for infested R2). This peak was assigned by Wolpert & Helling (2006) to amidazole group, which can be incorporated into many important biological molecules, such as amino acid histidine. The peak at 775 cm-1, which has been induced by A2.2, has been assigned to the amino acid histidine (Wolpert & Helling, 2006). Many other peaks have been attributed to amino acids, e.g. 1319 cm-1 to leucine, found only after infection by A1.3 and A2.2. The same amino acid has been found in the healthy treatment but at 1341 cm-1, the same peak has been observed also after infection by A2.2. Leucine with valine and isoleucine are important amino acids for the synthesis of plant defence secondary metabolites such as cyanigenetic glycosides, and glucosinolates (Coruzzi & Last, 2000).

The peak at 1072 cm-1, which has been detected in the infested R2 by A1.1 and A2.2, was assigned to methionine (Wolpert & Helling, 2006). The intensity of absorbance is has been doubled, passing from 0.1611 to 0.3354. The same amino acid has been found in the case of healthy R2, but at 1281 cm-1 with the lowest intensity (0.1398), indicating the degradation of cell membranes and/or defense enzymes used by potato for defense. The amount of released methionine is proportional to the evolution of symptoms. The band at 1341 cm-1 was attributed to serine (Pawlukojc et al., 2001), and the one at 1339 cm-1 they has assigned to threonine. While serine has been detected in the case of healthy R2, threonine was identified at infested R2 by A2.2. The same authors have attributed the peaks at 1387 and 1319 cm-1 to threonine observed after infection by A1.1 and A1.3 respectively. So, the amino acid threonine has been detected in all the infested potato R2 and not in the case of the healthy one. The peak at 1363 cm-1, was attributed to glutamine (Ramirez et al., 1998), which has been induced by the A1.1. Recently, Abu-Nada et al. (2007) have found that nine amino acids (L-aspartic, L-threonine, L-alanine, L-proline, L-valine, L-isoleucine, L-tyrosine, glutamine and L-phenylalanine) are up-regulated when potato was infested by P. infestans, in early stage of infection (after two days) and after four days, the induction became not significant compared to the control. These data indicates a significant role of amino acids in primary defence response against P. infestans (Abu-Nada et al., 2007).

17  

The absorption bands between 1200-950 cm-1 have been assigned to the deformation modes of C-O and O-H groups of secondary alcohols, and can be regarded as a spectroscopic marker of the cellulosic skeleton of the leaves (cellulosic domain); therefore, this region can be indicative of the presence of polysaccharides and of their structural modification (Bertoluzza et al., 1999). The peak at 1062 cm-1 was attributed to the P-O-C terminal phosphate by Cagnasso et al. (2010) found in R2 infested by A2.2 as the band at 1063 cm-1. Also, Bertoluzza el al. (1999), considered the band at 1300 cm-1 as a marker for healthy horse chestnut, absent in diseased or abiotic stressed leaves. In our studies, the peak at 1302 cm-1

was identified in the case of healthy R2 and absent in all infested R2. However we are not sure that this band could be considered as a marker for healthy plant specially potato, because we did not find this band in the case of the other healthy treatment (Bintje, Desiree, R1, and R5).

The peak at 1400 cm-1 was attributed to COO-, found in amino acids and fatty acids chains (Quilles et al., 2010). This peak (1396 cm-1) has been induced by the infection of potato by P. infestans. Abu-Nada et al., (2007) identified many fatty acids which were up regulated when potato was infected by P. infestans: palmitic acid, linoleic acid, and oleic acid, one day after infection. In the second day after infection, three down regulated and three up-regulated fatty acids: 9-octadecanoic acid, hexadecanoic acid, eicosanoic acid, 9-hexadecenoic acid, 7, 10, 13-hexadecatrienoic acid, 7,10-hexadecadienoic acid respectively. In the fifth day post infection, only eicosanoic acid has been up-regulated, when 9-hexadecenoic acid, 9, 12, 15-octadecatrienoic acid, 7, 10, 13-hexadecatrienoic acid, and 7, 10-hexadecadienoic acid have been down-regulated. Fatty acids are major components of triglycerides, cutin, suberin, and waxes, plasma, plastid and mitochondrial membranes (Somervilles et al., 2000). About 70 % of the membrane lipids in chloroplasts are represented by unsaturated fatty acids (Yaeno et al., 2004). Abu-Nada et al. (2007) noticed that following pathogen invasion, over time, their total abundance was reduced. In our case, for R1, the infection by A2.2 suppressed the peak. In the same way, in the infested R3, the peak did not appear. In case of Desiree and Bintje, the infection has induced a diminution of peak intensity by 30 %. In these cases, either the production of fatty acids was reduced, as a result of the chloroplasts dysfunction, due to pathogen infection (Soulie et al., 1989) or due to the use of unsaturated fatty acids was increased (Abu-Nada et al., 2007). However, infection of R2 has induced the peak. In the case of R5, the absorbance has doubled after infection (from 0.2324 to 0.4218). Wolpert & Helling (2006) assigned this peak to valine.

The peak at 1547 cm-1 which has been observed in the non-infested R2, this peak was identified to belong to the amid II band (Quilles et al., 2010). The increased hydroxyproline content of cucumber cell walls were associated with resistant, but not susceptible, responses of ten cucumber cultivars to Cladosporium cucumerinum (Hammerschmidt et al., 1984). The cell wall hydroxyproline increased beginning within 12–18 h after inoculation of a resistant cultivar but not until after 48 h in a susceptible cultivar. This initial increase in hydroxyproline content of the resistant cultivar corresponded with the time of initial penetration of the pathogen into all cultivars (Hammerschmidt et al., 1984). The set of bands between 1650 and 1500 cm-1 is due to vibration modes of aromatic rings probably in aromatic derivatives, unsaturated conjugated compounds, phenols, chlorophyll, etc (Ramirez et al., 1992). Bertoluzza et al. (1999) have noticed a gradual weakening of the intensity of the aromatic domain band (1500-1650 cm-1) passing from healthy to pathological and to physiologically stressed leaves. The R2 has not respected the model of Bertoluzza et al (1999), as we can see in the table 1 and figure 2 and 3. The absorbance peak has been the most intense in the case of R2 infested by A2.2, which has been the most virulent (Figure 1). The weakest intensity has been occurred in the case of R2 infested by A1.1, even the healthy R2 leaves taken as control. If we take the number of peaks as a criteria, the healthy R2 has had 5, 3 for infested by A1.1, 2 for infested by A1.3 and 5 for infested by A2.2. In the case of A1.1, all peaks are common with R2. In the case of A1.3 no one and only the peak at 1572 cm-1 has been common between R2 and infested R2 by A2.2. In function of infection progression, P. infestans has suppressed

18  

two peaks (case of A1.1), and induced two others (case of A1.3), in the case of A2.2, four peaks have been suppressed and other four have been induced. The same reaction was seen in the interval 600-950 cm-1. The peak at 1508 cm-1, was attributed to lignin (Stuart, 2004). This has been induced after infection by A1.3 and A2.2, the amount has been bigger in the case of A2.2. The accumulation of lignin after infection is a known phenomenon in plant reaction after infection (Agrios, 1997; Schmelzer, 2002). The lignification of the cell wall is a particularly important response by plants to pathogens (Stewart et al., 1994).The peak at 1601 cm-1 was assigned by Stuart (2004) to pectin; however this peak was detected only in the case of A1.3. In the case of A2.2 we have found it at 1597 cm-1. In the study of wheat resistance to Puccinia graminis f.sp. trici, Moerschbacher et al. (1988) have demonstrated that  the resistant and the susceptible iso-line both exhibit a first maximum in the coordinately regulated enzyme activities of phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) at a time when the fungus is still growing on the surface of the leaves. This maximum is followed by a decrease to the levels of controls. In the resistant isoline, a second increase is observed at the time of the hypersensitive resistant reaction. In contrast, enzyme activities in susceptible plants continue to decline, even falling below control levels until the onset of sporulation when a second, late increase can be detected.

In tomato, under salinity stress, the region 2100-2300 cm-1 was attributed to saturated and unsaturated nitrile compounds (Schulz & Baranska, 2009). The increased occurrence of these substances is attributed to the detoxification of hydrogen cyanide, a by-product produced during the biosynthesis of ethylene, which is enhanced in response to stress conditions, such as salinity (Mizrahi, 1982). In infested R2 by A2.2, two peaks have been suppressed. However, the infection by A1.1 has induced three peaks and no one has been in common with the infested R2 by A2.2. The infection by A1.3 has induced two new peaks. A peak has been identified as being in common with the healthy infested R2 by A1.1 and A1.3.

The double peaks at 2925 and 2850 cm-1 are attributable mainly to the asymmetric and symmetric stretching modes of the CH2 methylene group, the most abundant structural unit in plant products (Bertoluzza et al., 1999). However, Quilles et al. (2010) have assigned these bands to fatty acids in the membranes. In the case of the infestation by A1.1 one band has been induced at 2940 cm-1 while peaks at 2859 and 2938 cm-1 have been suppressed. When infested by A1.3, the peak at 2859 cm-1 has been suppressed. The infection by A2.2 suppressed all the peaks cited above, and induced a new peak at 2934 cm-1. If we link the apparition and suppression of bands with the symptoms evolution, this phenomenon could be explained by the destruction of membranes, especially in the case of A2.2. This is in concordance with the apparition of the peak at 1396 cm-1 in all the infested R2, which was assigned to amino acids and fatty acids chains, resulting from membrane degradation.

Resistant or susceptible, where is the difference?

As it has been expected, the cultivars Bintje and Desiree, which are both a free R genes have been infested by the isolate tested with and the mixture. The plants R1, R2, R3, and R5 have been susceptible to the infection by P. infestans (isolates tested). On the other hand, R4, 21, dms, and blb have been resistant. However, the resistance types have been different. P. infestans isolate used for this test has not been able to infest R4. We think that R4 has been immune to this isolate. The plant 21 has been resistant to the two late blight agent isolates tested. It has developed a HR. the wild species dms, has not developed any symptoms after infection. The species blb has developed another type of reaction; the zone of infection has been destroyed, but the healthy zone has still green.

Proteins and Amico-acids

19  

The peak at ~ 660 cm-1 was assigned by Dufour (2009) to the amide V. This peak has been detected in the case of R2, R4, and 21A2.2 at 662 cm-1. 21A2.2 absorbance at this peak has been at least three times more than in the other treatment. At the 664 cm-1 has absorbed R2A2.2, with the lowest amount. For the amid A, the characteristic peak is situated at ~ 3300 cm-1, all the susceptible potatoes have absorbed in this zone. The exception has been made by sound bintje and R1A2.2. The amount of absorbance has been aproximatively the same between the infested and control treatments. Wolpert and Helling (2006) assigned this peak to amidazole (see case of R2). Two other peaks have been assigned to imidazole by Stuart (2004) at 1610 cm-1 and at 1578 cm-1. The peak at 1722 cm-1 found only in the case of infested wild species S. bulbocastanum, is assigned to aspartic acid (Stuart, 1997). The peak at 1560 cm-1, detected in the case of sound R2 and desiree, has been assigned to glutamic acid. At 1558 cm-1, many other treatments have absorbed sound bintje, R5 A2.2, D A2.2, and both sound and infested S. bulbocastanum. The peak at 1586 cm-1 has been attributed to arginine (Stuart, 1997), found only in the case of 21 A2.2, at 1585cm-1, and at 1587 cm-1 in the case of inoculated S. demissum. Another peak was attributed to arginine by the same author at 1608 cm-1. In this case, only the susceptible treatment have absorbed; both sound and infested bintje, R2, R3 A2.2, and infested desiree. But the amount is bigger in the case of and S. demissum 21 A2.2 (two to four times more, respectively). For tyrosine, the peak is indicated at 1600 cm-1 by Stuart (1997), in our case, we found that 21 A2.3 and sound S. demissum has absorbed at 1599 cm-1, at 1603 cm-1, R1A2.2 and R5 have absorbed. The peak at 1304 cm-1 has been observed in the case of R4 A2.2, for R2, it has been situated at 1302 cm-1, another peak at 1306 cm-1 for R5 A2.2, it has been attributed to alanine (Pawlukojc et al. 2003). The peak at 1287 cm-1 assigned to isoleucine (Herlinger and Long, 1974). The same amino-acid has been detected in the case of 21 and R3 A2.2, for the peak at 1310 cm-1 (Herlinger and Long, 1974). In the case of R1 and R4, they have had peaks at 1308 cm-1. 21 has had the peak at 1312 cm-1. The same authors assigned the peak at 1339 cm-1 to alanine. This has been found in the case of R2A2.2, sound bintje, and R4 A2.2. Wolpert and Hellwing (2006) assigned the peak at 1340 cm-1 to leucine. The peak at 1375 cm-1, associated to leucine by Stuart (1997), has been found only in the case of infested desiree. The peak at 1396 cm-1 was assigned to valine (Stepanian et al., 1999). It has been identified in R1, R2 A2.2, R5, R5 A2.2, desiree. The peak at 1395 cm-1, in case of bintje, infested desiree, R4, R4 A2.2, 21 A2.3, 21 A2.2. An important note is that the amount in the case of infested 21 has been the highest (0.4536 for 21 A2.3 and 0.8863 for 21 A2.2). Leucine, valine, and isoleucine play an important role in the production of plant defence secondary metabolites such as cyanigenic glycosides, and glucosinolates (Corruzii & Last, 2000). This is illustrated by the amount of absorbance in the case of treatment which they expressed a high level resistance, by the mean of HR.

Many susceptible treatments have had the peak at 1341 cm-1, R1, R2, R3 A2.2, R5 A2.2, sound desiree, and R4. This peak has been assigned to serine (Pawlukojc et al. 2001). Pawlukojc et al. (2003) identified alanine in the peak at 1391 cm-1. In our case, this peak has been found in both sound and infested R2. But after infection the amount has increased (from 0.2421 to 0.3057). The amino acid proline has been found in the case of 21A2.2 at 1238 cm-1. The last peak was assigned to proline by (Wolpert & Helling, 2006).

The amino acid methionine has been detected in the case of R2 A2.2, B A2.2, and blb A2.2 by the peak at 1072 cm-1 (Cao & Fischer, 2002). At 1281 cm-1 in the case of 21. The peak at 993 cm-1, represents cysteine (Li et al., 1992). It has been detected in the case of infested blb only. Also the peak at 1066 cm-1 represents cysteine (Li et al., 1992), but has been detected in the case of R1at 1067 cm-1, and both sound and inoculated R4, at 1065 cm-1. The peak 1304 cm-1, which represents cysteine (Li et al., 1992) has been found only in the case of inoculated R4. The peak at 1400 cm-1 was attributed to COO-, found in amino acids and fatty acids chains (Quilles et al., 2010). It has been identified in the case of

20  

infested bintje. Very close to this peak, we identified another at 1398 cm-1 in the case of R4. Taoutaou et al., (submitted) have considered also the peak at 1396 cm-1 as a peak for COO- found in the amino acids and fatty acids chains. Abu-Nada et al., (2007), Taoutaou et al., (submitted) have discussed the role of amino acids and in the resistance to late blight (see the case of R2). As we can see, these peaks have not been identified in the case of resistant treatments, only in the case of sound R4.

Nucleic acids

The peak at 1015 cm-1 is assigned to DNA/RNA ribose (Stuart, 1997). At this peak, sound S. bulbocastanum has absorbed. At 1016 cm-1, the peak has been detected in the case of R4A2.2. The inoculation has induced a high level of nucleic acid expression in the case of R4, but a suppression of the normal nucleic activity in the case of potato wild relative S. bulbocastanum (blb). Another peak assigned to ribose (Benedetti et al., 1997), has been detected in the snound blb, at 1049 cm-1. Another peak for the same molecule is situated at 1060 cm-1 (Benedetti et al., 1997). In our case we faound two peaks near to the indicated by the author, at 1059 cm-1, peaks have been found in the case of sound desiree, 21, and inoculated blb. At 1063 cm-1, in the case of 21 A2.2, and R2 A2.2. what we did notice, is the important amount of absorbance in the case of both sound and inoculated 21 (0.8552, 0.8271 respectively), in comparison with the others.

Glucides and Polysaccharides

The peak at 993 cm-1 was assigned to trehalose by Yamanaka et al. (2007), this peak has been detected only in the case of infested S. bulbocastanum. Fructose has been detected in the case of R4 both infested and healthy, and in aproximatively the same amount. The peak which has been assigned to fructose by Yamanaka et al. (2007) is at 1065 cm-1. Peaks at 1063 cm-1 and 1067 cm-1 have been detected in R2 A2.2, 21 A2.2, and R1 respectively. The most important amount of absorbance has been seen in the case of 21A2.2. The peak at 1070 cm-1, has been assigned to mannose. And it has been detected in R1 A2.2, bintje, 21, and S. bulbocastanum. Galactose, at 1076 cm-1, has been detected only in the case of infested R5, desiree, and R4. The peak at 1103 cm-1, assigned to starch by Dufour (2009). This peak has been found only in the case of R4 A2.2. At 1105 cm-1, the peak is detected in the case of blb, assigned by Dufour (2009) to glucose. Plotnikovo & Ausubel (2007) mentioned that powdery mildews, rusts and viral infections result in general increased levels of apoplastic monosaccharides due to the activity of cell invertases, which are enzymes that catalyze the cleavage of sucrose to glucose and fructose. In our cases, we could not find a correlation between resistance/susceptibility and monosaccharides.

The peak at 1260 cm-1 has been observed in the case of R4, 21 A2.2, S. demissum (dms), dms A2.2, and blb A2.2. For dms and blb A2.2, the peak has been found at 1063 cm-1. Mascarenhas et al., (2000) attributed to pectin this peak. The role of pectin in disease resistance has been well discussed by Schmelzer (2002) and Agrios (1997). The inability of P. infestans to colonize potato leaves is explained by the rapidity of reaction to the invasion. Generally the accumulation of pectin has been induced by the infection, or it amount has increased after infection. The case of R4 made an exception because the peak has been suppressed. It could be explained by the fact that R4 has been immune to A2.2. Its resistance was not a HR, as it was in the case of 21. Being a hemibiotrophic, P. infestans has the same behavior as necrotrphic plant pathogens in the second stage of infection. Pectins in the plant wall appear to be an early target of fungal digestion, pectolytic enzymes degrade pectins of middle lamellae, primary and secondary walls and create acces for other enzymes like cellulases, proteases, and phospholipases (Plotnikovo & Ausubel, 2007).

21  

Phenolic compounds

The peak at 1595 cm-1 has been detected in the case of control R1, R4, and desiree and infested desiree. The absorbance amount has been the most important in the case of healthy R4 (0.3225, 0.4713, 0.3584, and 0.531 for healthy R1 and desiree, infested desiree, and healthy R4, respectively). This peak is associated with aromatic ring (Stewart et al., 1994). In terms of plant cell wall this is normally associated with lignin (Stewart and Morrison, 1992). However, Stewart et al. (1994) associate it with chlorogenic acid. In the susceptible cases, this peak has not been detected, but when detected it has been suppressed or its amount decreased. In the case of resistant plant R4, another peak at 1593 cm-1, has been detect and the amount has still high (0.4507) which is aproximatively the same with the residual amount in the case of desiree. Also, a residual amount has been detected in the case of sound S. bulbocastanum. Another peak has been associated with lignin, is the peak at 1240 cm-1 (Stewart et al., 1994). This peak, in our case, has only been detected in 21 A2.2, also with a high amount (0.4901). When it is detected, in the case of resistant plant, the amount is high than in the case of susceptible plant, no matter if it is induced or residual. In the case of desiree, the residual amount has been high, but after infection, it has decrease a lot. It could be explained by the degradation of cell wall. This is in concordance with the symptoms.

The difference spectrum susceptible potato plant leaves subtracted from resistant potato plant after infection show positive bands in the interval 1650-1500 cm-1. This indicates a higher relative concentraction of phenolics or proteins expressed in the case of resistant potatoes. This is related to the fact that increased absorbances at 1650 cm-1 (protein) occurs, indicating higher degradation during infection (Steward et al., 1994). See the next chapter for more details on phenols and potato late blight resistance.

In both compatible and incompatible interactions similar plant responses are observed but their timing is different (Tonon et al., 1998). In compatible interactions, P. infestans is using many specific metabolic ways to secrete proteins and other defense molecules (Huitema et al., 2004). Some of these molecules participate to the pathogen attachment to plant surface, others as obstacles to break physically the infection (cell wall and membranes), or modifying the physiological paths by suppressing plant defense (Kamoun & Smart, 2005). The suppression of host defenses can occur through the production on inhibitory proteins that target host enzymes (Kamoun, 2003). These molecules are known as effectors. They are molecules that manipulate host cell structure and function, thereby facilitating infection (virulence factors or toxin) and/or triggering defence responses (avirulence factors or elicitors) (Kamoun, 2006).

Chapter 9

Preliminary results on diffential phenolic compounds synthesis in potato with different types of late

blight resistance

9.1. Introduction

Interaction of the secondary messengers with the genome of an infected cell results in a change of the genome activity: expression of some genes will be suppressed, and of the others, will be strongly activated. This leads to accumulation of new products (stress metabolites) in the cell, and many of them are toxic for the parasites (Dzhavakhiya et al., 2007).

9.2. Material and methods

Plant: in this test we used three potato genotypes: the cultivar bintje as a R0 cultivar, and two potato genotypes each with a different gene, R2 and R5 respectively. All these plants are susceptible to late

22  

blight. As resistant S. tuberosum plants, two plants have been tested, the first carrying the R4 resistance gene and the second a plant with 3 R genes (21). Two wild species have been tested also, S. demissum and S. bulbucastanum.

Pathogen: four late blight agent isolates have been used; A1.1, A1.3, A2.2, and A2.3. For inoculums preparation, see materials and methods.

Total phenol extraction:

The leaves have been ground into a fine powder in a mortar and pestle under liquid nitrogen, and then transferred to an eppendorf 2 ml tube. 1 ml of 70 % methanol was added, and mixed by sonification for 15 minutes, then centrifuged. 50 µl of supernatant was used for total polyphenol determination. For a microplate with 24 wells were used following quantities of reagents: 50 µl sample; 150 µl Folin Ciocalteu Reagent; 450 µl Na2CO3 (7.5%), 2350 µl bidistilled water. For data reading, we used the multidetection spectrophotometer BIOTEK Synergy HT

Standard curve was done using different concentrations of galic acid (mg/ml). Absorption at 765 nm was measured. Total phenol contents were expressed in gallic acid equivalents (mg galic acid/g DW).

HPLC method

For HPLC analysis only resistant plant have been chosen, in the scope to see where the difference between the resistance forms R4 is, 21 and the wild species S. demissum. The isolate A2.2 has been chosen for infection. The infection procedure has been the same used before in this thesis.

Preparation of HPLC samples: 0.2-0.5 g infested and healthy leaves have been used for extraction, with 1 ml methanol 70 %. The extract was filtered and 20µl were injected in HPLC system Agilent 1200 with UV-Vis detector.

HPLC protocol of phenolic compounds separation: 5µm Supelcosil LC18 column (250x4,6 mm) using a gradient A (methanol: acetic acid: water 10:2:88) and B (methanol: acetic acid: water 90:3:7) for 55 min, registered at λ =280 nm, at 25°C.

The statistical analysis was performed with the software Statgraphics.

9.3. Results and discussions

The content of total phenol of different susceptible potato is aproximatively the same (214, 225, 239 mg of polyphenol/100 g plant bintje, R2, and R5 respectively) (Fig.8). However, the reaction of potato to infection differs. The infection of cultivar bintje has induced an increase of total phenol content, immediately after infection. However, this amount has decreased in the 5th day post inoculation, even less than the residual amount before infection. In the case of R2, the infection has induced a decrease of polyphenol total content. After 5 days, the polyphenol content has increased until 212 mg/100 g when the R2 has been inoculated by the isolate A1.1. When infested by A2.2, the content has not changed. In the case of R5, immediate after inoculation, there was a decrease of the total content of polyphenol from 239 mg/100 g to 165 mg/g, after 5 days, the total content did not change. When infected by the isolate A2.3, the content of polyphenol has increased but it did overpass the control.

214

332

180

0

100

200

300

400

Bintje B+P.t0 B+P.t5

Fig. 8. total polyphenol content of the susceptible potato cultivar bintje.

23  

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24

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4

two molecules of farnesyl pyrophosphate condense under the effect of the enzyme squalene synthetase, with the production of C30 squalene, from which phytosterols are produced, while in the resistant varieties under the effect of another enzyme-cyclase-the farnesyl pyrophosphate molecules close up in two rings and produce bicyclic sesquiterpenes, which are very toxic phytoalexins. Thus, instead of providing the required nutrient, the plant palms off a toxin on the parasite (Dyakov & Dzhavakhiya, 2007).

The role played by phenols in plant resistance to pathogens, herbivores, and in allelopathy has been well discussed in many reviews (Field et al. (2006), Dixon et al. (2002), Bennett & Wallsgrove (1994)). Many studies outline this important role. Dzhavakhiya et al. (2007) found that Transgenic tobacco plants with a lower level of phenol compounds showed a much higher growth rate of Phytophthora parasitica compared with the control. The application of exogenous phenol increase resistance of Ulmus minor to Dutch elm disease caused by Ophiostoma novo-ulmi through formation of suberin-like compounds on xylem tissues (Martin et al., 2008). Martin et al. (2010) have found that the soil application of phenol increases the resistance of elms to Dutch elm disease, but the mechanism is still unknown. In the case of the pathosystem Capsicum annum-Fusarium pallidoroseum, Generally total phenols ortho-dihydroxy phenols and the enzyme activity were invariably high in resistant parents and hybrids irrespective of growth stages, while, in case of susceptible parents the phenols content and enzyme activities were comparatively less. There existed a positive correlation between the host resistance and the amount of phenols and increased enzyme activities while it was almost the opposite in susceptible lines (Jabeen et al., 2009).

However the ability to detoxify these compounds is a good weapon for a successful colonization. The pathogen of dry rot of potato Gibberella pulcaris (ana. Fusarium sambucinum) is able to decompose the main potato phytoalexins rishitin and lubimin to less toxic metabolites. The strain ability for phytoalexin metabolization correlates with their pathogenicity (Dyakov & Ozeretskovskaya, 2007).

The production the high level of phenolic compounds however is not always associated with resistance to the pathogen attack. Pannecoucque & Höfte (2009) demonstrated the pronounced deposition of phenolic compounds and callose against weak and non-aggressive AGs which resulted in a delay or complete block of the host colonization in the interaction between cauliflower and Rhizoctonia anastomosis. This is clearly seen in the case of infection of R2 by A1.1, the colonization is only delayed.

In our opinion it is not the amount of the total pylephenol which affects resistance/susceptibility, but the composition. In our case, the both resistant potato S. tuberosum plants synthesized huge amount of chlorogenic acid. Even in the wild species S. demissum, inoculation has induced the synthesis of chlorogenic acid. Choi et al., 2004 found that the infection by phytoplasma in Catharanthus roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Leiss et al., (2009) showed that thrips-resistant and -susceptible chrysanthemums can be discriminated on basis of their metabolomic profiles. Thrips-resistant chrysanthemums contained higher amounts of the phenylpropanoids chlorogenic acid and feruloyl quinic acid. Both phenylpropanoids are known for their inhibitory effect on herbivores as well as pathogens. In our case, ferulic acid has been detected only in the case of S. demissum. After inoculation it has been induced in high amounts. Sarma & Singh (2003) studied the reaction of Cicer arietinum to the infection by Sclerotium rolfsii. They examined the status of phenolic compounds. Three major peaks that appeared consistently were identified as gallic, vanillic and ferulic acids. Gallic acid concentrations were increased in the leaves and stems of infected plants compared to healthy ones. Vanillic acid detected in stems and leaves of healthy seedlings was not detected in infected seedlings. There was a significant increase of ferulic acid in those stem portions located above the infected collar region compared to minimal amounts in the roots of healthy seedlings. In vitro studies of ferulic acid showed significant antifungal activity against S. rolfsii. Complete inhibition of mycelial growth was observed with 1000 µg of ferulic acid/ml. Lower concentrations (250, 500 and 750 µg/ml) were also inhibitory and colony growth was compact in comparison with the fluffy growth of normal mycelium. Higher amounts of phenolics were found in the

25  

stems and leaves of S. rolfsii-infected seedlings in comparison to the healthy ones. A role for ferulic acid in preventing infections by S. rolfsii in the stems and leaves of chickpea plants above the infection zone is therefore feasible. Siranidou et al. (2002) showed that the amount of p-coumaric acid increased significantly after inoculation of wheat by Fusarium culmorum. But the ferulic acid amount did not differ.

Specialized cells of plants synthesize phenolics and store them in their vacuoles during the normal processes of differentiation. Such phenolic-storing cells are distributed within most tissues. In some tissues they occur uniformly in all of the cells, whereas in other tissues they occur randomly scattered, and in still others they appear to be strategically located at potential points of entry. It is proposed that these cells can, first, by decompartmentation, rapid oxidation of their phenolic content, and the ensuing lignification and suberization of cells, and cell death, seal off infections or injuries at the immediate site of cellular penetration and, secondly, if this defence should fail and the stress persist, these same processes promote the prolonged build-up of IAA and ethylene that cause a further metabolic cascade in outlying cells that includes secondary metabolism and growth responses to produce a peridermal defence in depth (Beckman, 2002).

GENERAL CONCLUSIONS

13 different decamer primers have been used for studying the gene expression in the pathosystem P. infestans-Solanum spp. However not all the tested primers have amplified the cDNA.

More than 60 cDNA fragments have been cloned successfully.

In the case of compatible interaction, the polymorphism obtained by the amplification of cDNA is more important than in the case of incompatible interaction.

This induction/suppression of the expression of different genes which are not not the resistance genes (compatible interaction) represents the ability of P. infestans to manipulate the plant metabolsim in the case of susceptibility.

The suppression/induction of gene expression differs in time in function of the gene in question. It could be induced/suppressed after 24, 48, or 72h, or even earlier.

In the case of incompatible interaction, the number of induced/suppressed genes is minimum. When the same plant could be resistant and susceptible in function of the pathogen inoculum, the susceptibility is manifested by the suppression of an important number of gene products.

In this study we see the important role of many proteins in the potato defense against P. infestans. Many proteins with high molecular weight could not been identified, but they have a very important role in resistance, especially in the hypersensitive reaction. Also, the important role played by the pathogenesis-related protein in resistance: glucanases, lipoxygenases, glucosidases, endochitinase, serine protease inhibitor and others. In the susceptible plants, only a few numbers of proteins have been detected.

The resistance reaction is cooperation between the R proteins and PR proteins. Alone the PR proteins could not confer resistance to late blight.

The high molecular weight protein of 229 kDa detected in the case of inoculated R4, and the 230-233 kDa protein detected in the case of 21 could be the resistance protein coded by the R gene R4.

In the case of 21 where the resistance has manifested as a HR, many proteins with enzymatic activity have been induced, and many unknown others, with high molecular weight.

26  

In the case of wild relative species, the most part of proteins are putative proteins. They belong to the NBS-LRR family as the majority of potato R proteins. Also in this case, the resistance was a combination of possible R proteins and PR proteins.

We agree with Ivanova and Singh (2003), who thought that the FTIR might be useful for in evaluating the pathological changes in plant leaves after implementation of elicitins. So we extend the application to study the reaction of potato to infection of late blight agent. In the two most important cases: resistance and susceptibility.

One the most important conclusions from this study, is that infection by late blight agent induces a huge perturbation of the metabolism of the plant. This is expressed by the high number of peaks induced and/or suppressed in the case of compatible interaction. In the case of incompatible interaction, the most important volume of peaks is in common between the healthy and inoculated plants.

Many bands have been identified as a marker of resistance or susceptibility of potato to late blight. The spectra of different potato plant have the same aspect, but in the case of susceptible plant we noticed a delay of bands, especially in the zone of fingerprint (1800-600 cm-1).

Potato plant, in case of susceptibility does not react in the same way. As demonstrated by the case of R2, after infection with different late blight agent isolates. Also the composition of inoculums affects the reaction of potato to infection; we have seen that in the case of free R potato: bintje and desiree. The potato plant has reacted differently in function of the inoculums composition. The same thing could be said in the case of an incompatible interaction. The case of 21 with the three R genes, and it reaction to A2.2 and A2.3 has not been the same in the metabolomic level, even the final reaction has been the same (HR). Theoretically, A2.3 could infest 21, because in the test done individually in the laboratory, A2.3 was virulent on all potato plant carrying each gene separately. This demonstrates that there is a synergy between the genes. And the question of resistance in the case of P. infestans-Solanum spp. pathosystem is more than as suggested by the theory of Flor for gene-for-gene interaction. This is also demonstrated by the residual effect of the R2 gene, when the plant carrying it is infested by another isolate than A2.2, by causing a delay of infection.

The quantity of total phenolic compounds is not an important criterion for resistance. In this study we found that also in the case of resistant plant, inoculation by the P. infestans, has induced a decrease of total phenol compounds quantities, only in the case of Bintje where it has decreased. However, the types of phenolic compound could play an important role in resistance. Many phenolic compounds could be implicated in resistance: chlorogenic acid, ferulic acid, protocatecuic acid, caffeic acid and sinapic are the most encountered in the resistant potato plants. More investigations on the role of chlorogenic acid, ferulic and galic acid in late blight resistance are highly recommended.

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UNIVERSITATEA DE STIINTE AGRICOLE SI MEDICINA VETERINARA

CLUJ-NAPOCA

SCOALA DOCTORALA FACULTATEA DE ZOOTEHNIE SI BIOTEHNOLOGII

Ing. TAOUTAOU Abdelmoumen  

DIFFERENTIAL GENE EXPRESSION WITH DIFFERENT TYPES OF RESISTANCE IN THE

PATHOSYSTEM P. INFESTANS-SOLANUM SPP.

REZUMAT AL TEZEI DE DOCTORAT  

                          

CONDUCATORI STIINTIFICI:

PROF. DR. SOCACIU CARMEN

PROF. DR.PAMFIL DORU

CLUJ-NAPOCA, OCT. 2010

CUPRINS

Introducere…………………………………………………………………………………….. 33 Capitolul 1: Introducere generala……………………………………………………………. 33 1.1. Istoricul cultivarii cartofului…………………………………………………………….. 33 1.2. Daunatorii cartofului…………………………………………………………………….. 33 Capitolul 2: Interactiunea Gazda-Patogen………………………………………………….. 33 2.1 Transductia semnalului si reactii de aparare……………………………………………. 34 2.2. Cazul P. infestans-Solanum spp…………………………………………………………. 34 2.3. Mecanisme de patogeneitate……………………………………………………………. 34 2.3 Detectia efectorilor lui P. infestans……………………………………………………………… 34 2.4. Genele de rezistenta la cartof……………………………………………………………. 34 2.5. Reactia de rezistenta……………………………………………………………………….. 35 Capitolul 3: Differential display……………………………………………………………… 35 3.1. Metoda originala…………………………………………………………………………. 35 Capitolul 4:Transcriptomica…………………………………………………………………. 36 4.1. Intorducere ……………………………………………………………………………… 36 4.2. Materialul vegetal……………………………………………………………………….. 36 4.3. Rezultate si discutii…………………………………………………………………….. 36 4.4. Expresia genetica………………………………………………………………………… 36 4.4.1. Bintje si R2……………………………………………………………………………. 36 4.4.2. Cazul R4………………………………………………………………………………. 37 4.4.3. Cazul 21 (R2R3R4)……………………………………………………………………. 37 4.4.4.Solanum demissum si Solanum bulbocastanum…………………………………………… 37 Capitolul 7: Proteomica……………………………………………………………………… 38 7.1. Introducere……………………………………………………………………………… 38 7.2. Materialul vegetal……………………………………………………………………….. 38 7.3. Rezultate si Discutii……… ………………………………………………………………. 39 7.3.1. Cazul Bintje…………………………………………………………………………… 39 7.3.2. Cazul R2 ……………………………………………………………………………….. 39 7.3.3.Interactiunea incompatibila……………………………………………………………. 39 7.3.3.1. Cazul R4……………………………………………………………………………. 39 7.3.3.1. Cazul 21…………………………………………………………………………….. 39 7.3.3.2. Cazul S. demissum…………………………………………………………………………….. 40 7.3.3.3. Cazul S. bulbocastanum……………………………………………………………………… 40 Capitol 9: Metabolomică……………………………………………………………………. 40 9.1. Materialul vegetal………………………………………………………………………… 40 9.2. Rezultate si discutii……………………………………………………………………… 41 9.3. Interactiunea compatibila……………………………………………………………….. 41 9.3.1. Efectul asupra metabolismului plantei…………………………………………………. 41 9.3.4. Benzile marker de rezistenta si de sensibilitate………………………………………. 41 9.3.5. Rezistent sau sensibil, care e diferenta?................................................................... 41 Capitolul 9: Rezultatele preliminarii referitoare la rolul compusilor fenolici in rezistenta cartofului la mana……………………………………………………………………………..

42

9.1. Introducere………………………………………………………………………………. 42 9.2. Material si metoda……………………………………………………………………….. 42 9.3. Rezultate si discutii ……………………………………………………………………… 43 Concluzii generale…………………………………………………………………………….. 43 Bibliografia selective………………………………………………………………………… 45  

Introducere

Cartoful este una dintre cele mai importanta culture din lume. Productia modiala depaseste 300 de milioane tone. Cea mai importanta boala care afecteaza cartoful este mana, cauzata de P. infestans. Amelioarea rezistentei cartofului la mana este una daca nu cea mai promitoare cale de a rezolva aceasta problema. Dar, pentru a reusi acest obiectiv, intelegerea mecanismelor de rezistenta/patogeneitate este obligatoria. Aceasta lucrare, se inscrie in acest domeniu. Scopul acestei lucrarii a fost evedentiere diferentelor intre diferite tipuri de sensibilitate (cu sau fara gene de rezistenta) si diferite tipuri de rezistenta (cu o singura gena sau cu mai multe), si evidentiere unor gene implicate in ambele cazuri. Au fost folosite si 2 specii salbatice: S. demissum si S. bulbocastanum. In acest scop, am lucrat la 3 nivele diferite dar complimentare: transcriptomic, proteomic si metabolomic.

Capitolul 1

Introducere generala

1.1. Istoricul cultivarii cartofului

Cartoful (Solanum tuberosum) este originar din America de Sud, din regiunea Muntiilor Anzi. A fost introdus in Europa la sfarsitul secolului 16. Cartoful a fost introdus in Transilvania din Germania, in anii 1700. In Africa, a ajuns in jurul secolului 20. Dupa introducerea lui in Algeria, la mijlocul aniilor 1800, cartoful a fost cultivat doar pentru a fi exportat in Franta. Dupa obtinerea independentei, recolta era in jur de 250 000 tone/an, din care o treime se exporta.

1.2. Daunatorii cartofului

Cartoful este o planta gazda pentru o multime de patogeni (mai mult de 60), printre care se afla fitoplasma, viroizi, virusi, bacterii, nematozi, ciuperci si oomicete. Cei mai importanti patogeni ai cartofului sunt Phytophthora infestans, Streptomycesscabies, Ralstonia solanacearum, Alternaria solani, PVY, PVX, PLRV. In cazul insectelor, gandacul de Colorado este cel mai important daunator.

P. infestans este cel mai important patogen cauzand mana. Este si foarte devastator si in cazul rosiilor, si celorlalte plante din familia Solanaceae (Agrios, 1997). In anii 1840, a cauzat marea foamete Irlandeza. Costurile si pagubele au fost estimate la mai mult de 3 miliarde $/an (CIP, 1996 in Fry, 2008). Cu toate ca, in zilele noastre, folosirea fungicidelor ajuta la combaterea manei, pagubele si costurile de tratament depasesc 5 miliarde $/an (Duncan, 1999).

Capitolul 2

Interactiunea Gazda-Patogen

In general plantele sunt rezistante la marea majoritate a patogenilor. Plantele au dezvoltat multe mecanisme sofisticate pentru a detecta prezenta patogenului si a raspunde cu efecienta (van der Hoorn & Kamoun, 2008). Protectia pasiva impotriva patogenilor este asigurata de ceara cuticulara si de compusii antimicrobieni (Dangl &Jones, 2001). Rezistenta activa include o serie de procese interconectate, care

33  

sunt induse dupa recunoasterea patogenului in celula gazda, care au ca si rezultat final excluderea, inhibarea sau eliminarea patogenului potential (Heitefuss, 1982). Plantele se pot apara in mod activ cu neosinteza compusilor antimicrobieni, intarirea peretilor celulari, formarea unui strat de abscisie, tiloze, depozitarea de rasina, si intr-un final reactia de hipersensibilitate.

Recunoasterea rapida a patogenului este foarte importanta pentru a activa sisteme de aparare. Plantele par ca ar primi semnale care indica prezenta patogenului in cel mai scurt timp de la intrarea acestuia in contact fizic cu planta. (Agrios, 1997).

2.1 Transductia semnalului si reactii de aparare

Dupa detectatea prezentei patogenului, semnalul trebuie sa fie transmis pana la genele de rezistenta. Reactiile de aparare sunt induse de schimbari la nivelul membranelor, fluxulului ionilor, productia moleculelor de oxigen active (active oxygen species), peroxidarea lipidelor, fosforilarea proteinelor, care altereaza fiziologia celulelor si genereaza multe molecule cu rol de mesager, care sunt implicate in activarea reactiei de aparare la nivel local sau sistemic (Dixon et al., 2002). Multe molecule sunt implicate in transmisie: acidul salicylic (Bennett & Wallsgrove, 1994), acid iasmonic (Bennett & Wallsgrove, 1994; Wasternack, 2007) etilena (Nimchuck et al., 2003), oxidul nitric (Neil et al., 2003), tipurile active de oxigen (Grant & Loake, 2000; Garcia-Olmedo, 2001), si calciu (Lecourieux et al., 2006).

2.2. Cazul P. infestans-Solanum spp.

2.3. Mecanisme de patogeneitate

Patogenii au dezvoltat si ei diferite strategii pentru a evita si a manipula sistemul imunitar al plantei (Qutob et al., 2006). P. infestans fiind un hemibiotrof, infesteaza planta in 2 etape: in prima etapa, are nevoie de celule viabile, urmata de o necroza extinsa care rezulta in colonizare si sporulare (Kamoun and Smart, 2005). Pentru a reusi infectia si colonizarea, o serie de procese sunt necesare, care include aderare, penetrare si apoi colonizare (Huitema et al., 2004). Aceasta implica secretia proteinelor si a altor molecule (Huitema et al., 2004).

Cateva din aceste molecule participa la atasarea patogenului pe suprafata plantei, altele la distrugerea obstacolelor fizice ale infectiei (peretele celular si membranele), si o mare parte dintre acestea intervin in schimbarea fiziologiei plantei prin suprimarea sistemului de aparare (Kamoun and Smart, 2005). Suprimarea sistemul de aparare poate fi efectuata prin productia proteinelor inhibitoare care actioneaza asupra enzimelor (Kamoun, 2003). Efectorii (Effectors) sunt molecule care manipuleaza structura si functia celulei gazdei (factori de virulent sau toxine) si/sau inducerea reactiilor de apare (factori de avirulenta sau elicitori) (Kamoun, 2006).

2.3 Detectia efectorilor lui P. infestans

Multi efectori sunt descrisi in literatura. Cativa dintre acesti efectori nu sunt detectabili pentru planta, si prin urmare, planta se imbolnaveste. Daca efectorul a fost detectat, planta activeaza reactii de aparare. In cazul lui P. infestans, pana acum, singurul efector caracterizat, si care induce reactii de aparare este Avr3a. Rasele care au gena Avr3a sunt avirulente in cazul cultivarelor de cartof cu gena R3a.

2.4. Genele de rezistenta la cartof

Sunt 2 tipuri de rezistenta la mana: prima calitativa, controlata de genele R dominante si a doua cantitativa (Gebhardt &Valkonen, 2001). 20 de gene R care confera rezistenta la mana au fost localizate

34  

in harta genetica. 11 dintre ele (R1-R11) provin de la S. demissum, 5 (RB/Rpi-blb1, Rpi-blb2, Rpi-blb3, Rpi-abpt), de la S. bulbocastanum, si cate una de la S. berthauli (Rber/Rpi-ber), S. pinnatisectum (Rpi1), si S. mochiquense (Rpi-moc1) (Simko et al., 2007).

Rezistanta cantitativa cartofului la mana este de altfel cunoscuta. Factorii care controleaza acest tip de rezistanta au fost localizati in aproape fiecare cromozom (Gebhardt & Valkonen, 2001). Wang et al. (2005) au elucidat unele mecanizme ale rezistantei cantitative si au evidentiat implicarea a 348 de gene. Majoritatea acestor gene joaca un rol in metabolism, reactii de aparare si semnalizare, si regularizeaza transcriptia. Pe de alta parte, 114 gene cu rol neidentificat au fost detectate (Wang et al., 2005). Abu-Nada et al. (2007) au pus in evidenta ca aceste metabolite denumite metabolite legate de patogeneitate ( pathogenesis related (PR) metabolites) faptul ca indeplinesc mai multe roluri: 1- homeostazie, 2- reactii primare de aparare, 3- aparare secundara, 4- colapsul apararii primare si secundare. Modul de expresie difera in timp, anumite gene sunt activate mai repede decat celelalte (Tian et al., 2006).

2.5. Reactia de rezistenta

Reactia plantelor la atacul patogenului este complexa (Gobel et al., 2002). “Elicitation” induce activarea unei serii de raspunsuri, care includ: reintarirea peretilor celulari prin depozitarea calozei si ligninei, productia de enzime litice ca si chitinaza si glucanaza, si biosinteza fitoalexinelor si proteinelor legate de patogeneitate, si apoptoza (Desender et al., 2007).

Compusii fenolici si cu caloza reprezinta componente majore ale papilei, care este o depunere pe perete, care reprezinta primul obstacul al invaziei (Schmelzer, 2002). Un rol crucial in sistemul de aparare este reorganizarea citoplasmei si a citoscheletului.

Capitolul 3

Differential display

A fost prima data descrisa de catre Liang & Pardee (1992) ca si o metoda de “fingerprinting” pentru identificare si comparare a ARN-ului mesager in timpul diferitelor procese celulare. Cerintele acestui protocol au fost: reproductibilitatea, compararea tuturor tipurilor de ARN mesager si posibilitatea de a izola AND- ul complementar de interes.

3.1. Metoda originala:

Procedura consta in 2 etape:

• Revers transcriptia • Amplificarea PCR

Seturile de primeri sunt construite pentru a amplifica intre 50 si 100 de ARNm-uri (Sturtevant, 2000). Setul original era constituit din 12 oligo (dT): 11 sunt T-uri care se hibrideaza cu capatul poli(A) si 2 baze suplimentare.

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Capitolul 4

Transcriptomica

4.1. Intorducere

Expresia genetica este procesul prin care informatia stocata in genom este transformata in fenotip. Intr-un organism, exprimarea spatiala si temporala a diferitelor gene este un process foarte regulat, este determinata atat in cazul normal cat si cel pathologic. Intelegerea acestor mecanisme ale acestor procese normale sau patologice necesita identificarea, izolarea si caracterizarea genelor exprimate diferential (Sturtevant, 2000).

4.2. Materialul vegetal

Materialul vegetal folosit a fost reprezentat de plante de cartof cu vârsta de 5 săptămâni crescuti ȋn seră.. Doua cultivare cu grade diferite de rezistenţă la mană au fost utilizate pentru experimente (Bintje si Desiree). Au mai fost folosite si alte genotipuri de cartof precum plante cu cate o gena de rezistenta (R2, R4), plante cu mai multe gene de rezistenta “21” (R2R3R4) si inca 2 specii salbatice de cartof (S. demissum si S. bulbocastanum).

Agentul patogen

Au fost utilizate 3 izolate de P. infestans: A2.2, A2.3 si A1.3 ca si inoculums. Un alt tip de inoculums a fost preparat prin amestecul a tuturor isolatelor de P. infestans (5 izolate). Inoculumul a fost preparat prin amestecarea ȋn părţi egale a celor 5 izolate şi incubarea lor la 4° ȋn vederea eliberării zoosporilor.

Procesul de infectare

S-au utilizat plante sănătoase de cartof care au fost inoculate pe nervurile mediane cu o cantitate de 30 µl de suspensie P. Infestans. Martorul a fost inoculat cu apă bidistilată sterilă. Au fost incubate ȋn fitotron la 20°C, la o fotoperioadă de 16 h/ziuă, 8 h /noapte şi umiditate relativă de 70 %.

t0= congelare imediată dupa infecţie, t1 după 24 ore de la infecţie, t2 după 48 ore de la infecţie şi t3 după 72 ore.

4.3. Rezultate si discutii

Analizele RAPD au fost realizate in scopul selectarii primerilor care urmeaza sa fie folosite in DD. Din 22 de primeri testati 16 au amplificat AND-ul genomic: OPA-13, OPA-17, OPA-18, OPC-20, 70-03, 70-04, 70-08, Mic-07, Mic-13, Mic-14, 595, 270, 534, 563, 594, si 570. 13 din acesti primeri au fost selectati pentru studiul expresiei genetice: OPC-13, OPC-20, 70-03, 70-04, 70-08, Mic-07, Mic-13, Mic-14, 595, 270, 534, 563, 594.

4.4. Expresia genetica

4.4.1. Bintje si R2

Cultivarul bintje (Fig.1) si plantele R2 au fost sensibili la infectie cu P. infestans. Polimorfismul obtinut in urma amplificarii ADNc a fost foarte mare, la diferiti primeri folositi. Multe benzi au fost induse sau suprimate dupa caz. Asta corespunde cu gene care au fost activate sau carora expresia a fost suprimata.

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Fig. 1. Expresie genetica in cazul bintje. Expresia unor gene a fost suprimata iar altora a fost indusa in comparative cu controlul. C: control, I: infestat cu Izolat A2.2, M: infestat cu amesctul de isolate ,0,1,2,3 timp dupa inoculare: 0, 24,48,72 h

4.4.2. Cazul R4

Plantele R4 s-au dovedit a fi rezistente dupa inoculare cu P. infestans. Polimorfismul obtinut din ADNc a fost foarte slab.

4.4.3. Cazul 21 (R2R3R4)

21 a fost rezistent prin reactia de hipersensibilitate la ambele izolate A2.2 si A2.3, dar sensibil la amestecul de isolate. La nivel transcriptomic asta s-a tradus prin suprimarea mai multor benzi dupa inoculare cu amestec.

Fig. 2. Expresie genetica in cazul plantei rezistente cu 3 gene de rezistenta. Expresia unor gene a fost suprimata iar altora a fost indusa in comparative cu controlul. C: control, I: infestat cu Izolat A2.2, M: infestat cu amesctul de isolate ,0,1,2,3 timp dupa inoculare: 0, 24,48,72 h

4.4.4.Solanum demissum si Solanum bulbocastanum

Ambele specii au fost rezistente la inoculare cu izolatul A2.2. La nivel transcriptomic la Solanum demissum polimorfismul a fost foarte slab, dar la S. bulbocastanum mai multe benzi au fost suprimate.

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Capitolul 7

Proteomica

7.1. Introducere

Sunt 2 tipuri de proteine implicate in rezistenta. Proteine de rezistenta (R) si proteine legate de patogeneitate (PR). Cele de rezistenta sunt codate de gene rezistenta, si sunt caracterizate de situsuri de legare a nucleotidelor (NBS) si fragmente bogate in leucina repetate (LRR) ca si de altfel de domenii amino si carboxi- terminale variabile (McHale et al., 2006).

Cea mai raspandita definitie a proteinelor PR este ca ele sunt polippeptide cu o greutate moleculara scazuta (10-40 kDa) si se acumuleaza in spatiul extracellular in tesuturile infectat, prezinta o rezistenta ridicata la degradarea proteolitica si adesea, dar nu intotdeauna prezenta puncte izoelectrice extreme (van Loon, 1985).

7.2. Materialul vegetal

Materialul vegetal folosit a fost reprezentat de plante de cartof cu vârsta de 5 săptămâni crescuti ȋn seră.. Doua cultivare cu grade diferite de rezistenţă la mană au fost utilizate pentru experimente (Bintje si Desiree). Au mai fost folosite si alte genotipuri de cartof precum plante cu cate o gena de rezistenta (R2, R4), plante cu mai multe gene de rezistenta “21” (R2R3R4) si inca 2 specii salbatice de cartof (S. demissum si S. bulbocastanum).

Agentul patogen

Au fost utilizate 3 izolate de P. infestans: A2.2, A2.3 si A1.3 ca si inoculums. Un alt tip de inoculums a fost preparat prin amestecul a tuturor isolatelor de P. infestans (5 izolate). Inoculumul a fost preparat prin amestecarea ȋn părţi egale a celor 5 izolate şi incubarea lor la 4° ȋn vederea eliberării zoosporilor.

Procesul de infectare

S-au utilizat plante sănătoase de cartof care au fost inoculate pe nervurile mediane cu o cantitate de 30 µl de suspensie P. Infestans. Martorul a fost inoculat cu apă bidistilată sterilă. Au fost incubate ȋn fitotron la 20°C, la o fotoperioadă de 16 h/ziuă, 8 h /noapte şi umiditate relativă de 70 %.

t0= congelare imediată dupa infecţie, t1 după 24 ore de la infecţie, t2 după 48 ore de la infecţie şi t3 după 72 ore.

Extracţia proteinelor

Extracţia proteinelor s-a realizat conform protocolului lui Wang şi colab. (2006).

Separaţia proteinelor a fost realizată prin metoda SDS page. Pentru identificarea proteinelor, am facut recurs la baza de date Swissprot (http://expasy.org/sprot/).

38  

7.3. Rezultate si Discutii

7.3.1. Cazul Bintje:

Proteina cu masa moleculara de 10 kDa a fost identificata ca si o proteina inhibatoare a metalocarboxipeptidazei, cu activitate carboxipeptidaza si hidrolaza si proteaza (http://www.uniprot.org/uniprot/Q41432). Aceasta proteina a fost detectata in toate cazurile bintje, in afara de BP T3 (bintje infestat dupa 72 h). aceasta protein n-a fost detectata, dar a aparut in toate tratamentele R4. In cazul 21, a fost detectata in toate tratamentele control, si in cazul 21A2.2 T0, 21 A2.2 T3 si 21A2.3 T3.

7.3.2. Cazul R2:

Ambele izolate A2.2 si A1.3 au reusit sa infesteze frunzele plantei R2. 3 proteine au fost induse ca si raspuns la infectie cu diferite mase moleculare: 50-51, 47-49 si 27-28 kDa. Alta cu masa moleculara de 30-31 a fost suprimata. Proteina cu 50-51 kDa masa moleculara a fost identificata ca si beta-glucozidaza 08, si fac parte din familia 17 glicozil hidrolaza si care are o activitate hidrolaza. Proteina cu masa moleculara 27-28 kDa a fost identificate ca si o protein legata de patogeneitate, si un activator transcriptional, gasita la tomate. Activeaza genele de rezistenta la plante. Este si implicate in reactii de aparare, transcriptie si reglarea transcritptiei (http://www.uniprot.org/uniprot/O04682). A fost detectata numai in cazul R2 infestat si 21 inoculat, care a fost extreme de rezistent la infectie cu A2.2. cu o greutate moleculara de 101 kDa, protein gasita in cazul R2A1.3 poate fi o proteina de rezistenta PSH-RGH6.

7.3.3.Interactiunea incompatibila

7.3.3.1. Cazul R4

R4 a fost imune la izolat A2.2, dar n-a prezentat HR. multe proteine pot fi implicate in rezistenta plantei R4 la P. infestans: 112, 97, 65-67, 41-42, si 30 kDa.

Lipoxigenazele sunt dioxigenaze care catalizeaza hidroperoxidarea acizilor grasi polinesaturati (Kolomiets et al., 2000). Aceasta protein a fost detectata in cazul R4 inoculat, si bintje. In R4, a fost identificata in R4P T0 si R4P T3, in timp ce in cazul lui Bintje, numai in BP T3. Proteina cu masa moleculara de 37 kDa a fost identificata ca si glucan endo-1,3-beta glucozidaza. Joaca un rol important in apararea impotriva ciupercilor patogene, detectata in cazul R4 in toate tratamentele. Proteina cu 65-67 kDa ca si masa moleculara a fost identificata ca si Endo-1,4-beta-glucanaza. A fost gasita in cazurile R4P T0, R4P T1, R4P T3.

7.3.3.1. Cazul 21

Planta de cartof “21” a fost rezistenta la infectie cu izolatele A2.2 si A2.3 prin reactia de HR. proteina cu masa moleculara 144 kDa detectata in cazul 21A2.3 T3 a fost identificata ca protein CC-NBS-LRR. Alta proteina cu masa moleculara de 34-36 kDa a fost identificata ca si endochitinaza, care este implicata in reactiile de aparare impotriva patogenilor care contin chitin. O alta cu masa de 24 kDa a fost detectata in cazul 21 inoculat cu A2.2 si A2.3. a fost identificata ca si o protein inhibatoare a proteazei cu serina. 3 proteine cu masa moleculara mare, de 243, 169 si 156 kDa.

39  

7.3.3.2. Cazul S. demissum

In reactia de rezistenta 3 proteine poate fi implicate in cazul lui S. demissum: 40-43, 24-25, 20-21 kDa. Proteina cu masa moleculara de 149 kDa a fost identificata ca si o proteina CC-NBS-LRR, implicata in apoptoza si in reactiile de aparare. A fost detectata in cazul DMSP T0. Proteina cu 17 kDa a fost identificata ca si proteina de rezistenta la mana (putativa), identificat in cazul DMSP T3. Alta proteina detectata in cazul DMSP T1 cu masa moleculara de 45 kDa. Proteina cu 33-34 kDa care a fost detectata in toate cazurile. A fost idenfiticata ca si o kinaza. In cazurile DMSP T1 si DMSP T2 a fost identificata ca si proteina de rezistenta la mana (putativa). O alta proteina CC-NBS-LRR a fost identificata in cazul DMSP T1.

7.3.3.3. Cazul S. bulbocastanum

Proteina cu masa molculara de 146 kDa corespunde proteinei de rezistenta la mana Rpi-blb2. O alta cu masa moleculara de 38 kDa si care a fost suprimata in toate cazurile S. bulbocastanum inoculat. Proteina cu 24-25 kDa a fost detectata in cazurile S. bulbocastanum neinoculat, dar si inoculate in T0. Ea corespunde proteinei 30S ribozomala S3.

Capitol 9

Metabolomică

9.1. Materialul vegetal

S-au utilizat 3 categorii de material vegetal: 1. fără gene de rezistenţă - cutivarurile Bintje şi Desiree ; 2. Care prezintă gene de rezistenţă divizate ȋn două subgrupe: Care prezintă o singură genă de rezistenţă: R1, R2, R3, R4, R5 (R4 rezistant). Care prezintă mai multe gene de rezistenţă: 21 (R2R3R4) 3. reprezentate de celelalte specii : S. demissum şi S. bulbocastanum Plantele au fost crescute ȋn seră ȋn condiţii controlate de mediu.

Agentul patogen A fost folosit un amestec de 6 rase de mană precum şi 4 suspensii care conţin o singură rasă.

Infecţia

Au fost aplicate 2 metode de infectare: una cu acelaşi izolat şi diferite genotipuri gazdă şi a doua cu acelaşi genotip gazdă şi diferite izolate. Cultivarele Bintje şi Desiree au fost inoculate cu izolatele A 2.2 şi un amestec de 6 izolate.

FTIR

Frunzele au fost mojarate ȋn azot lichid şi apoi transferate ȋn tuburi eppendorf. S-a adăugat 1ml de metanol 70% , au fost omogenizate sonic 15 minute apoi centrifugate. 100 µl de supernatant au fost utilizaţi pentru analiză. Profilul infraroşu a fost realizat utilizând Shimatzu Prestige 2 şi spectofotometrul Happ-Genzel.

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9.2. Rezultate si discutii

9.3. Interactiunea compatibila

9.3.1. Efectul asupra metabolismului plantei

Numarul total de peakuri n-a fost afectat de infectia plantei R2. In cazul R1, infectia a scazut numarul de peakuri semnificativ din 43 in cazul controlului pana la 12 cand a fost infestat cu A1.1, si pana la 21, 19 si 15 cand a fost inoculate cu A1.3, A2.2 si A2.3. in cazul plantei R5. Infectia cu A2.3 n-a afectat semnificativ numarul total a peakurilor. Utilizarea izolatelor A1.1, A1.3 si A2.2 a crescut numarul total de peakuri. Un alt criteriu foarte important este numarul de peakuri indus/suprimat. Infectia cu P. infestans a perturbat metabolismul plantei. Peakuri communi intre controlul si inoculat R1 au fost numai 7, 11 in cazul R2, si 9 in cazul R5. Restul peakurilor a fost suprimat.

9.3.4. Benzile marker de rezistenta si de sensibilitate

In aceasta parte, am incercat sa gasim benzi specifice plantelor rezistente si alte benzi specifice celor sensibile. Pentru cele rezistente, 3 zone au fost identificate: 941-1180 cm-1, 1336-1483 cm-1, 1483-1703 cm-1. Pentru celor sensibile: 1056-1294 cm-1, 1442-1585 cm-1, 1585-1832 cm-1.

9.3.5. Rezistent sau sensibil, care e diferenta?

a de unda de 600 cm-1a fost identificat ca si amind V. a fost detectat in cazul R2, R4, 21 A2,2. Peak-ul la 1722 cm-1 a fost detectat numai in cazul speciei S. bulbocastanum a fost identific

4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

1 . 2

1 . 4

1 . 6

1 . 8

2 . 0

Proteinele si aminoacizii

Peak-ul la lungime

at ca si acid aspartic (Stuart, 1997). Peakul la 1560 cm-1, detectat in cazul R2 si desiree, a fost identificat ca si acidul glutamic. La 1558 cm-1 (argenina), multe tratamente au absorbit: bintje, R5A2.2, DA2.2, si ambele cazuri inoculat si neinoculat a speciei S. bulbocastanum. Tot argenina a fost detectata in

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A b s o r b a n c e B a n d 1 / c m

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Fig.48. Spectru FTIR cu benzi caracteristice cartofului rezistent/sensibil la mană.toate spectrele au același comportament, dar pentru plantele sensibile a fost decalat.

41  

cazul 21A2.2, S. demissum inoculate. Tirozina a fost detectata in cazul 21 A2.3, S. demissum (inoculate si neinoculat) , R1A2.2 si R5. Alanina a fost detectata in cazul R4A2.2, R2, R5A2.2. serina a fost detectata in urmatoarele cazuri: R1, R2, R3A2.2, R5A2., desiree si R4. Prolina a fost detectata in cazul 21A2.2.

Metionina a fost detectata in cazuri R2A2.2, BA2.2, si blbA2.2. cisteina a fost detectata in cazul 21 si blbA2.2.

a 1015 cm-1 a fost identificat ca si riboza din AND/ARN (Stuart, 1997), detectat in cazul S. bulbocastanum neinoculat. Inocularea a indus o exprimare a acizilor nucleic in cazul R4A2.2.

ectat in cazul blbA2.2. fructoza in cazul R4 (inoculate si neincolat) si in cazuri R2A2.2, 21A2.2 si R1. Manoza in cazul R1A2.2, bintje, 21 si blb. Galctoza in cazul R5A2.2, desiree,

595 cm-1, a fost identificat ca si nucleu aromatic, detectata in cazul R1, R4, Desiree si Desiree infestat, dar intensitatea a fost mai mare in cazului R4, si care corespunde de fapt cu lignina.

Capitolul 9

Rezultatele preliminarii referitoare la rol r fenolici in rezistenta cartofului la mana

9.1. Introducere

fenolici sunt molecule care contin unul sau mai multe grupuri hidroxil atasate direct la un carboxil aromatic. Grupul compusilor fenolici include metabolite derivate din condensare unitati de acetat (

l utilizat in teste a fost constituit din 3 genotipuri de cartof: Bintje ca si martor si alte doua genotipuri fiecare detinand gene diferite, R2 respectiv R5. Toate acestea sunt sensibile la isolate testate d

Acizii nucleici

Peakul l

Glucidele si polizaharidele

Trehaloza a fost det

si R4. Amidonul in cazul R4A2.2, dar glucoza in cazul blb. Pectina in cazurile R4, 21A2.2, blbA2.2, dmsA2.2

Compusi fenolici

Peakul la 1

ul compusilo

Compusii

ex. terpenoizi). Acesti compusi pot fi implicati in reactiile de rezistenta de la formarea obstacolele fizice si chimice impotriva patogenului pana la molecule semnal implicate in reactia de rezistenta locala sau sistematica si inducerea genelor de rezistenta (Dixon et al., 2002).

9.2. Material si metoda

Materialul vegeta

e mana. Ca si planta rezistenta din specia S. tuberosum , doua genotipuri: R4 si 21.

De asemenea, doua specii salbatice au fost testate: S. demissum si S. bulbucastanum.

Patogen: au fost utilizate 4 izolate A1.1, A1.3, A2.2, si A2.3.

42  

Extractia compusilor fenolici totali

zot lichid, apoi transferate in tuburi Eppendorf si a fost adaugat 1 ml de metanol 70%, apoi sonificat si centrifugat. 50 μl au fost utilizati pentru extractia prin metoda Folin Ciocalte

Pentru analizele HPLC au fost utilizate numai plante rezistente: R4, 21 si S. demissum. 0,2-0,5 g de frunze a fost utilizat pentru extractie. Extractul a fost filtrat si apoi 20 µl au fost introdusi in HPLC.

fenoli ai diferitelor plante de cartof sensibile a fost aproximativ acelasi. Totusi reactia la infectie a fost diferita. In cazul Bintje am inregist

enta existenta mai multor compusi fenolici implicati in rezisten

Concluzii generale

13 primeri au fost folositi pentru a studia expresia genetica. Dar nu cu toate primeri au dat polimorfism. Indicand ca nu toate genele au fost activate. 60 de banzi au fost clonate si urmeza sa fie secvent

. Cand planta a fost si rezistenta si sensibila (in functie de inoculum) sensibiliate a insemnat suprisie expresiei acestor gene.

fie exprimate si proteine de rezistenta si proteine legate de patogeneitate (chitinaze, glucanaze).

nocularea cu “elicitin”. Astfel noi am extins aplicabilitatea acestei metode pentru a studia reactia cartofului la infectie cu mana

stui studiu se desprinde din faptul ca infectia cu mana induce perturbari majore in metabolismul plantei. Aceasta se traduce prin numarul ridicat de

Frunzele au fost mojarate in a

u.

HPLC

9.3. Rezultate si discutii

Continutul total de

rat o crestere a continutului in compusi fenolici totali imediat dupa inoculare care a scazut dupa 5 zile. In cazul R5 si R2, dimpotriva, am inregistrat o scadere imediat dupa inoculare, dar care a crescut dupa 5 zile. In cazul plantelor rezistente am inregistrat o scadere a cantitatii de compusi fenolici totali imediat dupa inoculare.

Prin metoda HPLC s-a pus in evid

0200400600800

R4

R4P

Fig.3. Compusi fenolici detectat in cazul plantei R4 ta la mana, de exemplu acidul galic, acidul

ferulic si acidul clorogenic.

iate. In reactia de senstibilitate, mai multe gene au fost induse, dar si inca un numar mare a fost suprimate. Inducerea/suprimarea expresiei genetice, a fost in functia de timp. Cateva au fost la prima zi, si altele chiar mai devreme. Intarziere a fost mai ales in cazul inducerea expresiei.

In cazuri de rezistenta numarul genelor induse/suprimate a fost minim

Multe proteine au fost implicate in reactia de rezistenta. Dar sa fie planta rezistenta a trebuit sa

FTIR ar putea fi util in evaluarea modificariilor patologice la nivelul frunzelor dupa i

, in ambele cazuri: rezistenta si sensibilitate.

Una dintre cele mai relevante concluzii ale ace

43  

peakuri

ilitate la mana. Spectrul diferitelor plante de cartofi au avut acelasi aspect, dar in cazul plantelor sensibile un decalaj de denzi a fost obs

in acelasi mod, cum s-a si dovedit in cazul R2, dupa infectie cu diferite izolate ale manei. Pe de alta parte, compozitia inoculumului afecteaza reactia cartoful

ici totali nu reprezinta un criteriu de apreciere a rezistentei sau sensibilitatii la mana. Pe de alta parte se poate concluz

induse si/sau suprimate in cazul unei interactiuni compatibile. In celalalt caz (incompatibil) marea majoritatea a peakurilor este comuna intre plantele neinoculate si cele inoculate.

Multe benzi au fost identificate ca si markeri de rezistenta sau sensib

ervat, mai ales in zona de fingerprint (1800-600 cm-1).

In cazul sensibilitatii, plantele de cartof n-au reactionat

ui la infectie, cum a fost observat in cazul cultivarelor Bintje si Desiree. Acelasi lucru poate fi zis in cazul interactiunii incompatibile. In cazul 21 care continte 3 gene de rezistenta si reactia lui fata de A2.2 si A2.3 care a fost diferita la nivel metabolomic, chiar daca rezultatul final a fost la fel (Reactie de Hipersensibilitate). In mod teoretic A2.3 ar putea infesta 21, deoarece a reusit sa infesteze fiecare planta de cartof cu o singura gena dintre cele 3 pe care le detine 21. Aceasta demonstreaza o sinergie intre aceste gene. Astfel, problema de resistenta in cazul P. infestans-Solanum spp. nu se poate explica numai cu teorie gena-pentru-gena a lui Flor. Aceasta este de altfel demostrata si de efectul residual al genei R2, atunci cand planta este infectata cu un izolat diferit de A2.2, cauzand o intarziere a infectiei.

Din analizele efectuate se poate remarca faptul ca cantitatea compusilor fenol

iona ca compozitia compusilor fenolici are o importanta majora, mai ales acidul galic, acidul ferulic si acidul clorogenic.

44  

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