Rients Niks, Wageningen University

1. Cracking the codes:
Genetic basis of nonhost resistance of
barley to heterologous rust fungi
Rients Niks, Hossein Jafary, Thierry Marcel
Laboratory of Plant Breeding, Wageningen University

2. Host status:
Barley is host to Puccinia hordei, but nonhost to leaf
rusts of rye, wheat and grasses.
Can we make wheat a “quasi-nonhost” to P. triticina ?
What do we know about Nonhost resistance?
P. triticina
Barley
Wheat

3. Non-host resistance:
• constitutive
• morphological
• physiological
• induced

4. Unadapted pathogen
cell wall PAMP
perception of RLK
non-self
signal
transduction
defence
reaction
PAMP = Pathogen associated molecular pattern
RLK = Receptor-like kinase, recognizing the PAMP

5. Adapted pathogen
cell wall PAMP effectors
perception of RLK
non-self
signal
transduction Suppression of defence
defence
reaction
PAMP = Pathogen associated molecular pattern
RLK = Receptor-like kinase, recognizing the PAMP

6. Unadapted pathogen
cell wall PAMP
perception of RLK
non-self
signal
transduction
defence
reaction No suppression of defence
PAMP = Pathogen associated molecular pattern
RLK = Receptor-like kinase, recognizing the PAMP

7. The players
Plant cell
Pathogen

8. The defence
Perception
PRR
SignalSA, JA
transduction
PAMP
PR proteins,Chitinases,
Defence
Callose deposition,
Silicium deposition

9. Effectors 1 0 6 7
1 0 6 7
Switch off
the alarm

10. Host-status: degree of match
between effectors of pathogens
and targets in the plant
Relevant effectors are presumably transcription
factors or specific proteinases
Which are the targets?

11. P. hordei P. triticina
host non-host
Barley
non-host host
Wheat

12. P. hordei P. triticina
host non-host
Barley
+ -
non-host host
Wheat
- +
Monogenic or polygenic?

13. P. hordei P. triticina
host non-host
++++++ -------
Barley
non-host host
Wheat
------ +++++++
Are the same genes responsible for resistance to other unadapted rusts?
Monogenic or polygenic?

14. P. hordei P. triticina
++++++ -------
host non-host
Barley ++++++ -------
++++++ -------
++++++ -------
non-host host
Wheat
------ +++++++
Do all barley accessions share the same genes?

15. P. hordei P. triticina
++ ++ ++ ---+--+
host non-host
Barley ++ ++ ++ ----++-
++ ++ ++ -+-----
++ ++ ++ +++----
non-host host
Wheat
------ +++++++
Are suchall barley accessions share the sameadapted pathogen?
Do nonhost genes present also for the genes?

16. P. hordei P. triticina
-+++++ ---+--+
host non-host
Barley ++++++ ----++-
+-++++ -+-----
++++-+ +++----
non-host host
Wheat
------ +++++++
Are such nonhost genes present also for the adapted pathogen?

17. P. hordei P. triticina
-+++++
host
---+--+
non-host
Barley ++++++ ----++-
+-++++ -+-----
++++-+ +++----
non-host host
------ +++++++
Wheat
Are such nonhost genes present also for the adapted pathogen?
Quantitative basal host resistance?

18. Basal resistance (partial resistance)
“what makes the plant less than extremely susceptible”
non-hypersensitive resistance
L94
Puccinia hordei
Vada
Vada

19. Barley-Puccinia is an excellent system to study those questions
P. triticina
rare barley accession,
L94
Barley
Wheat

20. 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)

21. <3 pustules, more flecks
< 3 pustules, few flecks
Atienza et al (2004)
11-100 pustules
> 500 pustules
> 100 pustules
3-10 pustules
(Near)-non-host status of barley to Puccinia rusts
i
tic
tri
) p.
.1 f. s
.2
(1 inis
ei
rd am
ho gr ri
P. eria ecu
p
um lo
Bl a c. a
Full nonhost-status
re n
P. lpia a
vu dit
P. con
re ina
P. om s
br lidi
P. ty
ac
.d .
U lci spp
.
ho ff
P. r. 5 ini
co ur ii
P. r.-m lol
i
ho inis os
P. am ulb
Near-nonhost-status
gr i-b i
P. rde alin
ho sec
P. r.-
ho a
P. icin ns
t
tri ste
P. rsi
pe
P.
0%
90%
80%
70%
60%
50%
40%
30%
20%
10%
100%

22. Development of hyper-susceptible experimental lines
P. triticina
several barley
accessions , like L94
SusPtrit
Atienza et al (2004)

23. P. triticina P. recondita
P. hordei-bulbosi P. holci
P. hordei-murini P. bromi
P. gram. lolii etc
P. persistens
SusPtrit susceptible immune
Vada immune immune
C. Capa immune immune
Atienza et al 2004

24. Vada X SusPtrit X C. Capa Oregon Wolfe
barleys
3 mapping populations x 4 heterologous rusts
Quantitative inheritance! Jafary et al, MPMI 2006
Jafary et al, Genetics 2008

25. Vada x SusPtrit
Immunity of Vada to each heterologous
rust depends on a different set of QTLs!
Jafary et al, MPMI 2006
Jafary et al, Genetics 2008

26. 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!

27. Host range genes to Puccinia
Jafary et al, MPMI 2006
Jafary et al, Genetics 2008

28. Hypothesis: L94
• These QTLs may contain genes (or encode proteins) that are
targets to the effectors of rust fungal effectors
Vada
• In that case, each rust has different targets to suppress
PAMP triggered defence!
•What kind of genes are these targets? Are they defence
genes? Same genes as genes for basal resistance to P.
hordei?

29. Host range genes to Puccinia
BINs of 5 cM
each
Whole 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
Jafary et al, MPMI 2006
Jafary et al, Genetics 2008
Gonzalez et al PLoS-ONE 2010

30. Possible role for peroxidase genes
Aminoacid sequences of 119 barley peroxidases
http:/ / peroxidase.isb-sib.ch
150 bp
FHDCFV and VSCADI conserved motifs
30
Gonzalez et al PLoS-ONE 2010

31. Motif-directed profiling for prx-motifs
• RILs from SusPtrit x Vada (118 RILs) • RILs from L94 x Vada (96 RILs)
MseIFH2Y-680L/ 676V
MseIFH1C-660S/ 657V
MseIFH2Y-650L/ 640V
MseIFH1C-650S/ 590V
MseIFH2Y-678L
MseI/ FH1C
31
MseI/FH2Y
Gonzalez et al PLoS-ONE 2010

32. 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. Significant association of QTLs for basal resistance
with location of PRX genes
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
22 15
O (E) 11 (5.2) 14 (6.7) 3 (2.6) 12 (8.3) 5 (3.2) 5 (3.8) 9 (6.8)
(13.4) (11.5)
χ2 9.9 * 12.6** 9.9* 1.8 0.2 2.8 1.4 0.6 1.1
PRX not associated with QTLs for
agronomic traits
33
Gonzalez et al PLoS-ONE 2010

34. P. hordei P. triticina
-+++++ ---++-+
host non-host
Barley ++++++ ----++-
+-++++ ++---++
++++-+ ++++---
non-host host
Wheat
------ +++++++
• 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

35. How to proceed?

36. Mapping and fine-mapping QTLs
QTL-NILs developed:
L94 background: SusPtrit background:
Rphq2 Rphq2 (Vada)
Rphq3 Rphq3 (Vada)
Rphq4
Rphq2 + Rphq3 Rphq11 (Steptoe)
Rnhq Rphq16 (Dom)
All from Vada
Rnhq (L94)
Rhnq (Vada)
Yeo et al, unpubl.

37. Charactirization of QTLs in NILs
P. hordei.1.2.1
P. hordei.Co-4
P. trtiticina. Flamingo
P. trtiticina.Swiz
P. hor murini
P. gram. Lolii
P. persistens
P. graqm trit
P. hor sec W
P. hor sec G
P. hor sec. F
P. hor b
NILs
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
LP IF

38. What may be the targets of the effectors?
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?

39. 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

40. Concluding remarks
 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

41. Acknowledgements
Wageningen University- IPK
Dr. Rajeev K. Varshney
Lab of Plant Breeding Prof. Dr. Andreas Graner
Richard G.F. Visser Dr. Nil Steins
Anton Vels Dr. Marion Röder
Reza Aghnoum Dr. Patrick Schweizer
Ana Maria Gonzalez
Zuzana Kohutova URGV-INRA
Students Dr. Boulos Chalhoub
Cécile Huneau
Jerome Durand
Thijs van Dijk
Alice Loriaux SCRI
Freddy Yeo Kuok San
Dr. Ingo Hein
Dr. Robbie Waugh