Cracking the codes: Genetic basis of nonhost resistance of barley to heterologous rust fungi

Rients Niks, Hossein Jafary, Thierry MarcelLaboratory of Plant Breeding, Wageningen University

P. triticina

Barley

Wheat

Barley is host to Puccinia hordei, but nonhost to leaf rusts of rye, wheat and grasses.

Host status:

Can we make wheat a “quasi-nonhost” to P. triticina ?

What do we know about Nonhost resistance?

Non-host resistance:

• constitutive• morphological

• physiological

• induced

cell wall PAMP

PAMP = Pathogen associated molecular pattern

RLKperception of non-self

RLK = Receptor-like kinase, recognizing the PAMP

signal transduction

Unadapted pathogen

defence reaction

cell wall PAMP

PAMP = Pathogen associated molecular pattern

RLKperception of non-self

RLK = Receptor-like kinase, recognizing the PAMP

signal transduction

Adapted pathogen

defence reaction

effectors

Suppression of defence

cell wall PAMP

PAMP = Pathogen associated molecular pattern

RLKperception of non-self

RLK = Receptor-like kinase, recognizing the PAMP

signal transduction

Unadapted pathogen

defence reaction No suppression of defence

Plant cell

Pathogen

The players

The defencePerception

Signal transduction

Defence

PAMP

PRR

SA, JA

PR proteins,Chitinases, Callose deposition, Silicium deposition

Switch off the alarm

1 0 6 71 0 6 7Effectors

Host-status: degree of match between effectors of pathogens and targets in the plant

Which are the targets?

Relevant effectors are presumably transcription factors or specific proteinases

Barley

P. hordei

host

host

non-host

non-host

P. triticina

Wheat

Barley

P. hordei P. triticina

host

host

non-host

non-host

++

--

Monogenic or polygenic?

Wheat

Barley

P. hordei

host

host

non-host

non-host

+ + + + + +

+ + + + + + +- - - - - -

- - - - - - -

Monogenic or polygenic?

Wheat

P. triticina

Are the same genes responsible for resistance to other unadapted rusts?

Barley

P. hordei

host

host

non-host

non-host

+ + + + + +

+ + + + + + +- - - - - -

- - - - - - -

Wheat

P. triticina

- - - - - - -- - - - - - -- - - - - - -

+ + + + + ++ + + + + ++ + + + + +

Do all barley accessions share the same genes?

- - - - + + -- - - + - - +Barley

P. hordei

host

host

non-host

non-host

+ + + + + +

+ + + + + + +Wheat

P. triticina

- + - - - - -+ + + - - - -

+ + + + + ++ + + + + ++ + + + + +

Do all barley accessions share the same genes?Are such nonhost genes present also for the adapted pathogen?

- - - - - -

+ - + + + ++ + + + - +

- + + + + + - - - + - - +Barley

P. hordei

host

host

non-host

non-host

+ + + + + + +Wheat

P. triticina

- - - - + + -- + - - - - -+ + + - - - -

+ + + + + +

Are such nonhost genes present also for the adapted pathogen?

- - - - - -

- + + + + + - - - + - - +Barley

P. hordei

host

host

non-host

non-host

+ + + + + + +Wheat

P. triticina

- - - - + + -- + - - - - -+ + + - - - -

+ + + + + ++ - + + + ++ + + + - +

- - - - - -

Are such nonhost genes present also for the adapted pathogen? Quantitative basal host resistance?

Basal resistance (partial resistance)

“what makes the plant less than extremely susceptible”

non-hypersensitive resistance

L94

Vada

Puccinia hordei

Vada

Barley-Puccinia is an excellent system to study those questions

P. triticina

Barley

Wheat

rare barley accession, L94

Quantification of host status of barley to rusts of grasses

• 110 barley accessions:

landraces, modern cultivars, Asian, American, African, …; Hordeum spontaneum

• Rust species collected from other Hordeum species, or from Triticum, Agropyron, etc

• Inoculation with 3x as much inoculum than “normal” (= in P. hordei studies)

• All tests in seedling stage

Atienza et al (2004)Atienza et al (2004)

0%10%20%30%40%50%60%70%80%90%

100%

P. persistens

P. triticina

P. hor.-secalini

P. hordei-bulbosi

P. graminis lolii

P. hor.-murini

P. cor. 5 ff.spp.

P. holci

U. dactylidis

P. bromina

P. recondita

P. vulpiana

P.rec. alopecuri

Blumeria graminis f. sp. tritici

P. hordei (1.2.1)

> 500 pustules

> 100 pustules

11-100 pustules

3-10 pustules

<3 pustules, more flecks

< 3 pustules, few flecks

(Near)-non-host status of barley to Puccinia rustsFull nonhost-statusNear-nonhost-status

Atienza et al (2004)

Development of hyper-susceptible experimental lines

P. triticina

several barley accessions , like L94

SusPtrit

Atienza et al (2004)

SusPtritVada

C. Capa

P. tr iticina

P. hordei-bulbosi

P. hordei-murini

P. gram. lolii

P. persistens

susceptibleimmune

immune

P. recondita

P. holci

P. bromi

etc

immuneimmune

immune

Atienza et al 2004

SusPtritVada C. CapaXX

3 mapping populations x 4 heterologous rusts

Oregon Wolfe barleys

Jafary et al, MPMI 2006Jafary et al, Genetics 2008

Quantitative inheritance! Jafary et al, MPMI 2006Jafary et al, Genetics 2008

Vada x SusPtrit

Immunity of Vada to each heterologous rust depends on a different set of QTLs!

Jafary et al, MPMI 2006Jafary et al, Genetics 2008

Hypothesis:

• These QTLs may contain genes (or encode proteins) that are targets to the effectors of rust fungal effectors

• In that case, each rust has different targets to suppress PAMP triggered defence!

Host range genes to Puccinia

Jafary et al, MPMI 2006Jafary et al, Genetics 2008

Hypothesis:

• These QTLs may contain genes (or encode proteins) that are targets to the effectors of rust fungal effectors

• In that case, each rust has different targets to suppress PAMP triggered defence!

• What kind of genes are these targets? Are they defencegenes? Same genes as genes for basal resistance to P. hordei?

L94

Vada

Host range genes to Puccinia

Jafary et al, MPMI 2006Jafary et al, Genetics 2008

BINs of 5 cM eachWhole genome = 220 BINs

Some markers developed on PRX motif

Chi-square test on co-incidence of peak markers of nonhost QTLs with QTLs for partial resistance and PRX genes

Gonzalez et al PLoS-ONE 2010

30FHDCFV and VSCADI conserved motifs

Possible role for peroxidase genesAminoacid sequences of 119 barley peroxidases http:/ / peroxidase.isb-sib.ch

150 bp

Gonzalez et al PLoS-ONE 2010

31MseI/FH2Y

MseI/ FH1C

Motif-directed profiling for prx-motifs

• RILs from L94 x Vada (96 RILs)• RILs from SusPtrit x Vada (118 RILs)MseIFH2Y-680L/ 676V

MseIFH2Y-650L/ 640V

MseIFH2Y-678L

MseIFH1C-660S/ 657V

MseIFH1C-650S/ 590V

Gonzalez et al PLoS-ONE 2010

Prx markers and resistance QTLs placed on integrated linkage map

Prx markers occur in ~ 40 clusters

Significant tendency of resistance QTLs to map in prx-gene clusters

Gonzalez et al PLoS-ONE 2010

33

Prx QTLph1 QTLbg QTLnh QTLdh QTLdp QTLplh QTLkw QTLtw QTLyi

Marker no. 200 19 23 63 52 15 31 13 18 24

BIN no. 63 18 23 47 39 9 28 11 13 23

O (E) 11 (5.2) 14 (6.7) 22 (13.4)

15 (11.5) 3 (2.6) 12 (8.3) 5 (3.2) 5 (3.8) 9 (6.8)

χ2 9.9 * 12.6** 9.9* 1.8 0.2 2.8 1.4 0.6 1.1

Gonzalez et al PLoS-ONE 2010

Significant association of QTLs for basal resistance with location of PRX genes

PRX not associated with QTLs for agronomic traits

Barley

P. hordei

host

host

non-host

non-host

- + + + + +

+ + + + + + +- - - - - -

- - - + + - +

Wheat

P. triticina

- - - - + + -+ + - - - + ++ + + + - - -

+ + + + + ++ - + + + ++ + + + - +

• Quantitative inheritance

• Occasionally R-gene for HyR

• In each parental combination the resistance resides on different loci

• For each rust species a different set of genes

• Significant co-incidence with basal resistance to P. hordei

• Significant co-incidence with defence related ESTs, especially PRX

How to proceed?

Mapping and fine-mapping QTLs

QTL-NILs developed:

L94 background:

Rphq2Rphq3Rphq4Rphq2 + Rphq3RnhqAll from Vada

SusPtrit background:

Rphq2 (Vada)Rphq3 (Vada)

Rphq11 (Steptoe)Rphq16 (Dom)

Rnhq (L94)Rhnq (Vada)

Yeo et al, unpubl.

NILs

P. h

orde

i.1.2

.1

P. h

orde

i.Co-

4

P. h

or s

ec. F

P. h

or s

ec W

P. h

or s

ec G

P. g

ram

. Lol

ii

P. h

or m

urin

i

P. p

ersi

sten

s

P. tr

titic

ina.

Fla

min

go

P. tr

titic

ina.

Swiz

P. g

raqm

tri

t

P. h

or b

RLP (3 x) RLP (3 x) RIF (1 x) RIF (1 x) RIF (1 x) RIF (2 x) RIF (2 x) RIF (2 x) RIF (3 x) RIF (1 x) RFF (2 x) RIF (2 x)

SusPtrit 100 100 100 100 100 100 100 100 100 100 100 100

Su-Rphq2 106 100 98 70 81 108 103 140 110 144 102 126

Su-Rphq3 106 101 126 111 85 31 80 114 57 59 91 88

Su-Rphq11 105 103 27 33 26 32 44 54 87 25 66 2

Su-Rphq16 108 106 165 107 87 102 113 189 90 123 88 91

Su-Rnhq.L 100 100 96 53 61 57 93 75 85 131 116 87

Su-Rnhq.v 101 101 54 55 75 54 87 65 88 122 123 71

L94 100 100 100 100 100 100 100 HR 100 100 100 100

L94-Rphq2 106 108 14 123 76 115 72 HR 87 48 115 84

L94-Rphq3 105 106 9 42 36 31 80 HR 35 52 49 86

L94-Rnhq 100 99 2 12 16 3 28 HR 75 23 107 93

Charactirization of QTLs in NILs

LP IF

Defence genes: have general effectiveness: those effective to P. triticina should also be effective to P. hordei.

However, small sequence variations in coding region or promoter may determine whether effector can reprogramme the gene expression?

What may be the targets of the effectors?

Barley/ Puccinia offers an excellent opportunity to investigate the specificity aspect of non-host resistance:

- Diploid autogamous plant species, with great diversity

- Extensive knowledge on markers, micro-arrays, dense linkage maps

- Is near-nonhost to several different rust species and forms

- Related to other important cereal crops

Gonzalez et al PLoS-ONE 2010

Barley-Puccinia rust is effective system to understand the inheritance of host status of plants

Arabidopsis is not (yet) used to tackle this question

We uncover natural variation (no mutagenesis)

TILLING is no immediate option unknown which is the functional allele (susc or res?)

redundancy of QTLs for nonhost resistance

Most common mapping populations do not segregate (but OWB!)

LD mapping on cultivar collection is no option

BAC libraries available • Vada: resistance alleles of 28 QTLs • Cebada Capa: resistance allele of 13 QTLs• SusPtrit: QTL-genes can be susceptibility factors

Concluding remarks

IPKDr. Rajeev K. Varshney Prof. Dr. Andreas GranerDr. Nil SteinsDr. Marion Röder Dr. Patrick Schweizer

URGV-INRADr. Boulos Chalhoub Cécile Huneau

StudentsJerome DurandThijs van DijkAlice LoriauxFreddy Yeo Kuok San

Wageningen University-Lab of Plant BreedingRichard G.F. VisserAnton VelsReza Aghnoum Ana Maria GonzalezZuzana Kohutova

SCRIDr. Ingo Hein Dr. Robbie Waugh

Acknowledgements