Peter Dodds, CSIRO Effectors from flax rust and Puccinia graminis

1. University of Minnesota
Effectors from flax rust and Puccinia graminis

2. Gene-for-gene resistance in flax:
Basic Research Questions
1. What are rust resistance genes and
how do they work?
2. How do rusts overcome resistance?
3. Can we apply knowledge from the model
system to cereal rust diseases?

3. Gene-for-gene resistance in flax
R
R genes encode recognition
components of plant immune system
S
~30 Rust resistance genes in flax
Cloned 20 R genes
R Locus Cloned
L L, L1-L11
TIR NB LRR
M M, M1, M3
N N, N1, N2
P P, P2 Signalling Activation Recognition

4. Flax rust Avirulence Genes:
Haustorially expressed secreted proteins
Identified Avr genes from four loci
- encode small secreted proteins
- expressed in haustoria HR
R AVR
- recognised inside plant cells cell death
L5 Bison
Avr Locus Matching R genes
AvrL567 L5, L6, L7
AvrM M
AvrP123 P, P1, P2, P3
35S
promoter AvrL567 Nos
AvrP4 P4
No signal peptide
(cytoplasmic)

5. R-Avr recognition occurs by direct interaction
Receptor ligand model Avr proteins escape recognition
R by altering surface residues
Avr
AvrL567-A
Yeast-2-hybrid assays
AvrL567
A B D
L5
AvrL567-D
AvrM avrM
M

6. Flax rust AvrM protein secreted and
translocated to host cell
Anti-haustoria Anti-AvrM
Plant
Cytoplasm
Haustorium
0.5 μm
Rafiqi et al Plant Cell 2010 22:2017

7. AvrM protein is taken up in host vesicles
Haustorium Plant
cytoplasm
Plant Haustorium
cytoplasm
Pamela Gan and Adrienne Hardham et al unpublished

8. AvrM in the plant endomembrane system
Plant
cytoplasm
Haustorium
Pamela Gan and Adrienne Hardham et al unpublished

9. Effector delivery is pathogen-independent and
requires N-terminal uptake signals
Plasmolysed cells
SP-GFP
SP-AvrM-GFP
SP GFP
Uptake signal
MKFLKPDQVQKLSTDDLITYMAEKDKNVRDL
FL V Ls LI Yqaekd
Rafiqi et al Plant Cell 2010 22:2017

10. Effector delivery from transgenic rust also
requires the N-terminal uptake signal
M
CH5F2-96 AvrM-YFP AvrM-∆106-153-YFP

11. Secreted effectors in rust infection and host
immunity
R
Immune
Host
Recognition
Manipulation

12. Stem rust effectoromics
• What is the set of stem rust effector proteins?
> Secreted (have signal peptide)
> Expressed in haustoria
• Which of these are recognised by wheat R genes?
> Genetic association
> Functional assays

13. Wheat stem rust genomic resources
Reference genome – strain CRL75-36-700 (7a)
Broad Institute (Cuomo, Szabo)
- 81.5 Mbp; ~92% coverage
- 20,567 genes annotated
- 1342 have signal peptides for secretion
Four Australian isolates – represent founder strains
- 21-0
- 126
- 194
- 326

14. Identifying stem rust effector genes
Isolated haustoria
• Haustorial isolation (21-0) haustoria
> 13K ESTs
> Illumina RNAseq
• Genome sequence
- 21-0 454/Illumina
- 126 Illumina
- 194 Illumina
- 326 Illumina
• Illumina RNAseq – infected tissue
- 21-0 - 194
- 126 - 326

15. Identifying stem rust effector genes
21-0 Haustorial ESTs 7a reference genome
212 with SP - 84 predicted SP
56 no SP (incorrect annotation)
77 absent
105 no SP - predicted SP
(incomplete clones)
- Manually curate using genome and RNAseq data
369 candidates

16. p7a genome-assisted building of 21-0 reference
genome
Assembled on 7a ref (79 Mbp)
De novo assembly of non-aligned reads (18 Mbp)
Mbp
Gene calling based on Illumina RNAseq data
Gene models 13,921 5370
Candidate effectors 339 30

17. Sequence Variations Among Different Pgt Isolates
21-0
anchored
100
pg7a
% Similarity Plot
80
126 100
80
326 100
80
100
194
80
SuperContig1
Pgt-p7a genome assisted assembly of Pgt-21-0 genome and subsequent assembly of genomes of other Australian strains
assisted by derived Pgt21-0 genome (Illumina GAII/HiSeq 100 bp paired-end sequencing)

18. Strain variation in effector genes
Only 13 effector candidates show no differences between five strains
~40 have < 5 SNPs across five strains
SNPs in secreted effector genes
reference Protein
strain length
gene 7a 126 194 21-0 326 (aa)
PGTT_02220 15 18 20 10 5 189
PGTT_19738 7 18 17 7 4 209
PGTT_01618 23 30 27 12 8 354
PGTT_07595 17 19 29 15 10 339
PGTT_09048 17 14 4 3 177
PGTT_05791 9 6 5 2 103
PGTT_13278 21 26 31 20 12 425
PGTT_10668 9 9 6 1 123
PGTT_12848 7 7 17 2 2 138
PGTT_00796 9 11 2 1 115
PGTT_02680 14 10 2 134
PGTT_06969 5 9 23 5 4 217
PGTT_08533 10 11 9 1 1 117

19. Expression variation of candidate effectors
Relative
18.00 absent
Expression ~100fold absent
(log2) 16.00
14.00
12.00
10.00
8.00
6.00
4.00 HS_210
2.00 210
0.00 126
194.00
326.00

20. Functional Assay: bacterial type 3 delivery
Pseudomonas fluorescens ‘Ethan’ (Pfo):
Express as fusion to AvrRPM1 bacterial effector
Adenylate cyclase AvrM delivery
delivery to wheat to tobacco (M)
cAMP mg/protein
7000
6000
5000 Agro:
4000 mock avrM
3000
2000 Agro:
1000 Pfo AvrM
0
Pfo:
Pfo: avrM
AvrRPM1
Pfo:
AvrM

21. Wheat accessions with different R genes
Pfo inoculation
Differentials R genes
BW56 Sr31
Acme Sr9g, SrX
Agent Sr24
Comb X Sr5b, Sr7b, Sr9b
Festiguay Sr30
Gatcher L06-139 Sr2, Sr5, Sr6, Sr8a, Sr12
Kite Sr26
Banks L09-94 Sr5, Sr8a, Sr9b, Sr12
Line S L09_10 Sr13, Sr17 DAB stain
Mendos L09_89 Sr11,Sr17, Sr36
Norka L09_95 Sr15
Trident L09_91 Sr38
W3534 Sr22

22. Screening Effector Candidates
13 Wheat accessions
1 2 3
4 5 6
7 8 9
10 11 12
Treatments: 5. Lcontig10 al4
1. Mock 6. Lcontig625_al1
2. Pfo Ethan/AvrRPM1:Cya 7. Lcontig75_al1
13
3. Lcontig10 _al1 8. Lcontig10 al6
4. Lcontig10 _al3 9. Lcontig10 al7

23. Two potential effector-R gene recognition
responses
Kite Sr26 Norka Sr15
Treatments
Mock
G1LContig1_al1_G1#1-1
G1LContig1_al5_G1#1-9
G1LContig7_al1-al2_G1#2-4
G1LContig7_al3_G1#2-7
G1LContig7_al4_G1#2-3

24. Acknowledgements
CSIRO-Plant Industry U. Sydney ANU
Narayana Upadhyaya Robert Park Adrienne Hardham
Robyn East Colin Wellings David Jones
Rohit Mago Pamela Gan
U. Minnesota Maryam Rafiqi
Jeff Ellis Les Szabo
Xiaodi Xia Jerry Johnson
Kim Newell Jane Glazebrook
Jen Taylor Fumi Katagiri
Andrew Spriggs
Maud Bernoux Broad Institute
Greg Lawrence Christina Cuomo
Jane Wilkinson
Two Blades
Foundation