www.sciencemag.org/cgi/content/full/science.1239028/DC1

Supplementary Materials for

The Gene Sr33, an Ortholog of Barley Mla Genes, Encodes Resistance to Wheat Stem Rust Race Ug99

Sambasivam Periyannan, John Moore, Michael Ayliffe, Urmil Bansal, Xiaojing Wang, Li Huang, Karin Deal, Mingcheng Luo, Xiuying Kong, Harbans Bariana, Rohit Mago,

Robert McIntosh, Peter Dodds, Jan Dvorak, Evans Lagudah*

*Corresponding author. E-mail: evans.lagudah@csiro.au

Published 27 June 2013 on Science Express DOI: 10.1126/science.1239028

This PDF file includes:

Materials and Methods Figs. S1 to S7 Tables S1 to S6 References

2

Materials and Methods Plant Materials Mapping populations consist of 85 recombinant inbred lines (RIL) and 1150 F2 lines derived from the cross between CS1D5405 (5) and Chinese Spring. Mutant lines were generated from CS1D5405 seeds using ethyl methane sulphonate (EMS) treatment. Using an initial kill-curve analysis, an EMS concentration of 0.2-0.4% with 12 hours exposure was identified as the optimal dose to render 50% mortality and reduced growth. Accordingly, 850 single heads harvested from the cultivation of 2000 CS1D5405 seeds treated with EMS were used for further analysis. Rust screening of the mapping populations and mutants was done using Puccinia graminis f. sp. tritici race 34-1,2,3,4,5,6,7,11 (Plant Breeding Institute culture no. 171) in accordance with Bariana and McIntosh (19). Along with CS1D5405, Aegilops tauschii accession CPI110799 (the original donor of Sr33; same as RL5288) was also used as a positive control. Haplotypes of Sr33 were identified using 368 Ae. tauschii accessions collected from different geographical locations and maintained at the Australian Winter Cereals Collection in Tamworth, Australia, the Commonwealth Plant Introduction collection (CPI) at CSIRO Plant Industry, Australia and UC Davis, USA Marker Analysis Simple sequence repeat (SSR) markers specific to Chromosome 1D (20) were screened on the 85 RILs using the method of Hayden et al. (21) and the polymorphic markers identified were mapped on the RIL populations using MAP MANAGER Version QTXb20 (22) and Kosambi (23) map function. Closely linked flanking markers were used to identify recombinants from the large F2 population of over 1200 lines using the method described in Kota et al. (24). For additional markers, wheat expressed sequence tags (wEST) specific to chromosome group 1 (25) were screened following the method of Lagudah et al. (26). Finally, AFLP analysis was done using 408 primer combinations derived from 17 PstI and 24 MseI selective amplification primers and methods as described in Mago et al. (16) BAC Screening and Sequence Analysis Screening of the D genome specific BAC library made from Ae. tauschii accession AL8/78 (7) and AUS18913 (8) were performed according to Lagudah et al. (26). Contigs of the positive clones of AL8/78 were identified from Ae. tauschii Physical Map (27). BACs were mapped using the isolated low copy sequences as described in Lagudah et al. (26). BAC clones from figure 1 (1. HI134N19, 2. HD512N18, 3. RI353E24, 4. HI328O18, 5. HD071G23, 6. HD147M18, 7. HD036N08, 8. RI074M08, 9. HI085F18, 10. 69I06, 11. 172J10 and 12.86D17) were sequenced at the Chinese Agricultural Academy of Sciences, Beijijng, China and at Integrated Genomics facility, Kansas State University, USA. Repeat sequences present in the assembled short contigs within the BACs were masked using the wheat repeats database (http://wheat.pw.usda.gov/ITMI/Repeats/blastrepeats3.html) and the non-repeat sequences were analysed for genes using the gene prediction software of Massachusetts Institute of Technology (http://genes.mit.edu). Comparative Genomics

3

The genomic sequence of Bpm (encoding a Pum/Mpt5/FBF-like protein) from wheat was used to identify syntenic regions in rice, barley and Brachypodium genomic sequences using the Phytozome database (www.phytozome.net). Wheat ESTs with more than 80% sequence identity with any of the genomic sequences of rice, barley and Brachypodium were targeted for mapping. The orthologous region of Bpm in barley chromosome 1H has the three classes of RGH families (associated with mildew resistance) and the CI2 genes (associated with insect resistance). The sequence of these gene members (9) from the barley cultivar Morex were used as a RFLP probe and mapped on the critical recombinants of the large F2 population as described in Lagudah et al. (26). Candidate Gene Identification Overlapping primer pairs (Table S5) designed along the entire length of the predicted genes were used to amplify the sequence from CPI110799 (Sr33 donor line) and various susceptible M2 mutants following the PCR method described by Lagudah et al. (28). Amplified sequences were compared for nucleotide variations using multiple sequence alignment (CLUSTAL-European Bioinformatics Institute-http://www.edi.ac.uk/Tools/sequence.html). C-DNA Analysis RNA extraction, cDNA synthesis, 5' and 3' RACE (rapid amplification of cDNA ends) were done using the method described in Krattinger et al. (29). Primers (5'-GTCCCCTCACGGATCTGGCC-3') and (5'-AGCACATCACACAACCTCTCGG-3') designed at the predicted 5' and 3' terminus were used for the RT-PCR analysis. For RACE, primers (5'-CCCTTGTGCAGTTTGTACTCCC-3') and (5'-CCTTCGGCGGGTGGTCTGGG-3') designed at the 5' and 3' coding regions were used as the gene specific primers. Wheat Transformation Complementation test was performed using 8 kb length of genomic fragment comprising of all the exons and introns and the 2.4 kb upstream and 1.5 kb downstream regions of AetRGA1e. The fragment was amplified using primers (5'-TTCAAGATGTCAAATTTTAAAAGGGC-3'), (5'-CTACTCATTAGGAACTCGAGCGG-3') and the Phusion High-Fidelity DNA Polymerase (New England Biolabs Inc.) under the manufacturer’s recommended conditions. The Sr33 gene fragment was cloned into the binary vector pVecNeo, a derivative of pWBvec8 (30) in which the 35S hygromycin gene has been replaced with a 35S NPTII selectable marker gene derived from pCMneoSTL2 (31). The Sr33 gene fragment in the binary vector was introduced into the stem rust susceptible wheat cultivar Fielder by Agrobacterium tumefaciens mediated transformation using a licensed procedure (Japan Tobacco, Japan, http://www.jti.co.jp/biotech/en/plantbiotech/index.html). T0 and T1 transformants were tested for Pgt resistance response. VIGS The original BSMV vectors utilized in these experiments were obtained from Dr. Andrew O. Jackson at UC Berkeley (32). The BSMV γ vector was reconstructed to include a

4

PCR-ready cloning site. In details, the γ vector was digested with two restriction enzymes NotI and PacI, and ligated with a sequence of GCCCCACTCATGACATGGCGTTAGCCATGGGAAGCTTGGAT, which includes two XcmI restriction sites. The modified γ vector (named as γPCR vector) was linearized with restriction enzyme XcmI to produce a TA cloning site for direct cloning of PCR products. For simplicity, the BSMV-derived construct with no insert was named as γ00, and each BSMV silencing construct was named as γtarget. For example, a BSMV silencing construct carried a 185-bp fragment of the wheat PDS gene was named as γPDS. The BSMV construct utilized to silence the Sr33 gene carries a 190-bp Sr33 gene specific fragment. Two constructs were prepared for silencing the AetRGA2a gene; each construct carries a 190-bp gene specific fragment from either the N or C terminus of the gene, named as γAetRGA2aN and γAetRGA2aC. In contrast, RAR1, SGT1 and HSP90 each have three homeologs on A, B and D genomes of wheat. To silence all three homeologs in the genome, constructs were designed to carry a 190-bp conserved fragment among three homeologs of each gene.Infectious RNA transcripts were synthesized by in vitro transcription using T7 RNA polymerase (New England Biolabs, Ipswich, MA) from linearized α, β, and γ plasmids (14). The BSMV inoculum was prepared with an equimolar ratio of α, β, and γ transcripts plus inoculation buffer containing a wounding agent. The inoculum was rub-inoculated onto the second leaf of each nine day old wheat seedling. Stem rust assessments were conducted under a greenhouse conditions with American stem rust race QFCSC. The urediniospores were suspended in Soltrol 170 Isoparaffin (Chempoint, Bellevue, WA). The spore-inoculum density was calculated at 227,500 spores/ml using a Brightline hemocytometer as per the manufacturer’s recommendations (Hausser Scientific, Horsham, PA). The inoculum was applied at a rate of 0.05 mg spores /10 ml Soltrol / plant using a Badger 350-3 airbrush gun (Badger Air-Brush Co., Franklin Park, IL). Spore germination rate was assessed on an inoculated microscope slide using a light microscope. A dew chamber with lighting was pre-conditioned to an air temperature of 19-22°C and incubated for 24 h, followed by incubation under high humidity and light intensity conditions for at least 3 h before being transferred to a greenhouse. Assessments were made when Chinese Spring showed full susceptibility at 14 days post inoculation following the scale described in Bariana and McIntosh (19). Expression of the genes targeted for silencing was quantified by comparative quantitative real-time PCR (qRT-PCR). Transcript abundance was quantified via the iScript One-Step RT-PCR Kit with SYBR Green real time-PCR and quantified using the CFX96 real-time PCR detection system operated with the CFX Manager software (Bio-Rad, Hercules, CA). Transcript abundance was normalized to 18s and Actin transcript abundance and relative transcript abundance was calculated using the ∆∆Ct method as described in the CFX96 manual (Bio-Rad, Hercules, CA), where fold change = 2-∆∆Ct and percent transcript abundance = fold change x 100. Each reaction was conducted in a triplicate and data were used only if the Ct ≤ 30 and the Ct standard deviation between replicates was ≤0.3. The cycling conditions were as follows: 10 min at 50°C, 5 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at 55°C and 1min at 95°C, 1 min at 55°C, melt curve 55°C to 95°C, increment 0.5°C. In all the cases, relative expression of the targeted gene

5

was presented as the expression level of this gene in silenced plants relative to that of the same gene in plants infected with γ00, and the values of gene expression were the averages of three plants. For each PCR, the specificity of the amplifications was validated and the threshold cycle above background was calculated using Bio-Rad iCycler software. PCR efficiency was close to 100%. Relative quantification of the gene transcript abundances was calculated as described in Scofield et al. (14). Error bars in all figures showing qRT-PCR data indicated the standard deviations calculated from the original CT (cycle threshold) values. The primer sequences used to detect each gene were as follows: SR33-F：5' GCAGGAGGACGTGGAAATC 3' SR33-R: 5' AAAGTCTACCATACAGCGGAAC 3' RGA2a-F: 5' ATGGAGCAATGCCCAAAGT 3' RGA2a-R: 5' GGCATCAGCAAACACCAACT 3' HSP90-F：5' CGACCAGCACGCTCACGAT 3' HSP90-R：5' GCGATGGTCCCGAGGTTGT3' SGT1-F: 5' CAAGCTGGGCAGTTAC 3' SGT1-R: 5'TCCTTCGATGCATAAAGC 3' RAR1-F: 5'ATGCGGTGCCAGCGAATA 3' RAR1-R: 5'GGGTTGTCGTCGTCGGTG 3' Actin-F: 5’ AAATCTGGCATCACACTTTCTAC 3’ Actin-R: 5’ GTCTCAAACATAATCTGGGTCATC 3’ 18SF: 5’ GTGACGGGTGACGGAGAATT 3’ 18SR: 5’ GACACTAATGCGCCCGGTAT 3’ PDS-F: 5’ TGTCTTTAGCGTGCAAG 3’ PDS-R: 5’ GATGATTTCGGTGTCACT 3’ Yeast two-hybrid analysis All experiments were performed in Saccharomyces cerevisiae reporter strain Hf7c. The cDNA encoding full length or truncated Sr33 were cloned at EcoRI-SalI sites of pGBKT7 (Clontech) using primers at table S6. The cDNA of HSP90, SGT1, RAR1, WRKY1 and WRKY2 were amplified from wheat line CPI110799 and H. vulgare L. variety Golden Promise. Target cDNA were obtained by PCR amplification using primers designed with specific restriction enzyme sites (Table S6) and cloned into pGADT7(Clontech) at the corresponding sites. The yeast transformation was performed in accordance with Gietz et al. (33) and co-transformants were selected on SD media lacking leucine and tryptophan. The interaction analysis was performed on media lacking leucine, tryptophan and histidine with yeast grown at 30˚C for 3-4 days. As a positive control, the Flax L6 TIR domain, which has been shown to homodimerize in yeast or the MLA10 CC1-46-HvWRKY1260–353 combination were used (12, 34). Immunoblot Analysis Total yeast protein was extracted in accordance with Kushnirov (35). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Pall). Membranes were blocked in 5% skimmed milk and probed with anti- HA or anti-Myc mouse monoclonal antibodies (Roche), followed by goat anti-mouse antibodies conjugated with horseradish peroxidase (Pierce). Labelling was detected using the SuperSignal West Pico

6

or Femto chemiluminescence kit (Pierce). Membranes were stained with Ponceau S to confirm equal loading. Isolation of Ae. tauschii HSP90, RAR1, SGT1 and WRKY1, WRKY2 Public databases were scrutinized using H. vulgare L. HSP90, RAR1, SGT1, WRKY1/2 as queries to isolate related expressed sequence tags (ESTs) derived from wheat. Using the available literature and sequence data, ESTs CK208966.1 and CJ619316.1 for SGT1, CJ684577.1 for RAR1, GQ240780.1 for HSP90, DR741433.1, BQ578389.1for WRKY1and DR740124.1, DR741886.1 for WRKY2 (36, 37) were selected and primer pairs (Table S6) were developed for the isolation of the full-length cDNA of HSP90, RAR1, SGT1 and WRKY1/2 from Ae. tauschii line CPI110799. Microscopic Analysis Rust infected leaf tissues were cleared and stained with wheat germ agglutinin (WGA) conjugated to FITC as described in Ayliffe et al. (38). The WGA lectin specifically binds to chitin oligomers present in fungal cells walls (38). To visualise autofluroscent cells, the same leaf samples were cleared in a saturated chloral hydrate solution and observed under UV light.

7

Fig. S1. Structure of AetRGA2a. The amino acid sequence predicted through RT-PCR analysis comprised of coiled coil (CC) nucleotide binding site (NBS) and leucine rich repeat (LRR) domains related to RGA2 class of barley Mla locus and an unusual C terminal domain related to Exo70 subunit of exocyst complex

8

Fig. S2. Structure of Sr33 and the nucleotide changes of SNP mutants. (A) Structure of AetRGA1e. Coloured and uncoloured rectangle bars represent Exons and UTRs, respectively while the black lines in-between indicate the introns. (B) Details of the nucleotide and the corresponding amino acid change in the four point mutants. E9 and E7 have the changes in P-loop (Walker A) while E6 and E8 have in RNBS-B and GLPL motifs of NBS domain, respectively.

9

Fig. S3. Neighbor-joining tree analysis of RGA from Ae. tauschii (AetRGA), functional MLA members of barley (HvMLA) and T. monococcum (TmMLA) and leaf rust resistance of CNL type (LR1, LR10 and LR21). The sequence alignment and bootstrap confidence values were calculated using MEGA v5.05 (39)

10

Fig. S4. Virus induced gene-silencing of Sr33, AetRGA2a, RAR1, SGT1 and HSP90 genes in CS1D5405 and their resistance response against the American stem rust race QFCSC. Stem rust infection on (A) Chinese Spring and (D) CS1D5405 seedlings without inoculation with BSMV constructs. Rust response on Chinese Spring seedlings inoculated with BSMV constructs containing; (B) no insert (C) with PDS gene fragment. Rust response on CS1D5405 seedlings inoculated with BSMV constructs containing; (E) no insert, (F) PDS gene fragment, (G) Sr33 gene fragment, (H) HSP90 gene fragment, (I) SGT1 gene fragment, (J) RAR1 gene fragment, (K) N terminus and (L) C terminus fragment of AetRGA2a gene.

11

Fig. S5

Fig. S5. Functional characterization of SR33 using yeast two-hybrid assessment. SR33 was tested for an ability to interact with both Ae. tauschii and H. vulgare equivalents of (A) the chaperonins HSP90, RAR1, SGT1 and (C) the transcription factors WRKY1 and WRKY2. SR33 CC domain self-association was tested (E) using three CC domain variants representative of a truncated portion of the CC domain (CC1-125), the entire CC domain (CC1-160) and a truncated CC-NB domain (CC1-225). Expression of each GAL4-AD and GAL4-BD fusion protein was analysed in corresponding yeast cells (B, D, F) by protein immunoblot using α-HA and α-cMyc antibodies, respectively.

12

Fig. S6. Leaves of CS1D5405 (panels A,B,C,D) and CS1D5406 (carrying Sr45) (panels E,F,G,H) lines collected on 5th day after post infection with an Australian stem rust race 98-1, 2, 3, 5, 6. Panels labelled A, C, E, G show WGA-FITC staining of fungal infection structures within the leaf segments while panels labelled B, D, F, H show autoflorescent cell death present on the same leaf segments. On average much larger infection sites are present on Sr33 tissue when compared with Sr45 tissue (A verses E). Autoflorescence indicative of plant cell death is very limited on Sr33 material (B) while obvious autoflorescence is associated with Sr45 resistance (F). Panels G and H demonstrate that on Sr45 material autoflorescence is usually associated with small infection sites only (G, infection site marked with and arrow). In this same panel the significantly larger infection sites (upper two) show little evidence of autoflorescent cell death.

13

Fig. S7 HaplotypeI RSMLQPIYSDMGNVYACRVHDMVLDLICNLSHEAKFVNVFDGTGNIMSSQSNVRRLSLQN 540 HaplotypeII RSMLQPIYSDMGNVYACRVHDMVLDLICNLSHEAKFVNVFDGTGNIMSSQSNVRRLSLQN 540 HaplotypeIII RSMLQPIYSDMGNVYACRVHDMVLDLICNLSHEAKFVNVFDGTGNIMSSQSNVRRLSLQN 540 HaplotypeIV RSMLQPIYSDMGNVYACRVHDMVLDLICNLSHEAKFVNVLDGTGNIMSSQSNVRRLSLQN 540 HaplotypeV RSMLQPIYSDMGNVYACRVHDMVLDLICNLSHEAKFVNVFDGTGNIMSSQSNVRRLSLQN 540 HaplotypeI KNEDHQAKPLTNIMSISQVRSITIFPPAVSIMPALSRFEVLRVLDLSDCNLGESSSLQPN 600 HaplotypeII KNEDHQAKPLTNIMSISQVRSITIFPPAVSIMPALSRFEVLRVLDLSNCNLGESSSLQPN 600 HaplotypeIII KNEDHQAKPLTNIMSISQVRSITIFPPAVSIMPALSRFEVLRVLDLSDCNLGESSSLQPN 600 HaplotypeIV KNEDHQAKPLTNIMSISQVRSITIFPPAVSIMPALSRFEVLRVLDLSDCNLGESSSLQPN 600 HaplotypeV KNEDHQAKPLTNIMSISQVRSITIFPPAVSIMPALSRFEVLRVLDLSDCNLGESSSLQPN 600 HaplotypeI LKGVGHLIHLRYLGLSGTRISKLPAEIGTLQFLEVLDLGYNHELDELPSTLFKLRRLIYL 660 HaplotypeII LKGVGHLIHLRYLGLSGTRISKLPAEIGTLQFLEVLDLGYNHELDELPSTLFKLRRLIYL 660 HaplotypeIII LKGVGHLIHLRYLGLSGTRISKLPAEIGTLQFLEVLDLGYNHELDELPSTLFKLRRLIYL 660 HaplotypeIV LKGVGHLIHLRYLGLSGTRISKLPAEIGTLQFLEVLDLGYNHELDELPSTLFKLRRLIYL 660 HaplotypeV LKGVGHLIHLRYLGLSGTRISKLPAEIGTLQFLEVLDLGYNHELDELPSTLFKLRRLIYL 660 HaplotypeI NVSPYKVVPTPGVLQNMTSIEVLRGIFVSLNIIAQELGKLARLRELQIYFKDGSLDLYEG 720 HaplotypeII NVSPYKVVPTPGVLQNMTSIEVLRGIFVSLNIIAQELGKLARLRELQIYFKDGSLDLYEG 720 HaplotypeIII NVSPYKVVPTPGVLQNMTSIEVLRGIFVSLNIIAQELGKLARLRELQIYFKDGSLDLYEG 720 HaplotypeIV NVSPYKVVRTLGVLQNMTSIEVLRGIFVSLNIIAQELGKLARLRELQIRFKDGSLDLYEG 720 HaplotypeV NVSPYEVVPTPGVLQNMTSIEVLRGIFVSEHYCT---------RAWQTGKAEGASDLLQG 711 HaplotypeI FVKSLCNLHHIESLIVSCNSGETSFELMDLLGEQWVPPVHLREFVSEMPSQLSALRGWIK 780 HaplotypeII FVKSLCNLHHIESLIVSCNSGETSFELMDLLGEQWVPPVHLREFVSEMPSQLSALRGWIK 780 HaplotypeIII FVKSLCNLHHIESLIVSCNSGETSFELMDLLGEQWVPPVHLREFVSHMPSQLSALRGWIK 780 HaplotypeIV FVKSLCNLHQIESLIIDCNSEEASFELMDLLGERWVPPVHLREFVSYMPSQLSALRGWIK 780 HaplotypeV W----------------------------------------------------------- 712 HaplotypeI RDPSHLSNLSELILPTVKEVQQEDVEIIGGLLSLRRLLIESTHQTQRLLVIRADGFRCMV 840 HaplotypeII RDPSHLSNLSELILPTVKEVQQEDVEIIGGLLSLRRLLIESTHQTQRLLVIRADGFRCMV 840 HaplotypeIII RDPSHLSNLSELILPTVKEVQQEDVEIIGGLLSLRRLWIKTTHQTQRLLVIRADGFRCMV 840 HaplotypeIV RDPSHLSNLSELILTSVKEVQQEDVEIIGGLLSLRRLRIWSTHQTQRLLVIRADGFRCMV 840 HaplotypeV ------------------------------------------------------------ HaplotypeI DFYLNCGSATQIMFESGALPRAEEVCFSLGVRVAKEDGNRGFDLGLQGNLLSLRRVVWVK 900 HaplotypeII DFYLNCGSATQIMFESGALPRAEEVCFSLGVRVAKEDGNRGFDLGLQGNLLSLRRVVWVK 900 HaplotypeIII DFHLNCGSATQIMFESGALPRAEEVCFSLGVRVAKEDGNRGFDLGLQGNLLSLRRVVWVK 900 HaplotypeIV DFYLNCRSAAQIKFEPGALPRAEGVVFSLGVRVAKEDGNRGFDLGLQGNLLSLRRVVLVK 900 HaplotypeV ------------------------------------------------------------ HaplotypeI MYCGGARVGEAKEAKAAVRHALEDHPNHPPIQINMFPRIAEGAQDDDLMCYPVGGPISDAE 961 HaplotypeII MYCGGARVGEAKEAKAAVRHALEDHPNHPPIQINMFPRIAEGAQDDDLMCYPVGGPISDAE 961 HaplotypeIII MYCGGARVGEAKEAKAAVRHALEDHPNHPPIQINMFPRIAEGAQDDDLMCYPAGGPISDAE 961 HaplotypeIV MYCGGARVGEAKEAEAAVRHALEDHPNHPPIEINMLPHIAK-------------------- 941 HaplotypeV -------------------------------------------------------------

Fig. S7. Amino acid sequence alignment of the sequence variants (haplotypes) identified for SR33 in Ae. tauschii. Amino acid from position 481 is shown, as the difference between the haplotypes starts only from position 520. Polymorphic changes are indicated in red and the dotted lines represent deletion variation. Haplotype I through V are equivalent to sequence variants from I through V in the main text.

14

Table S1. Stem rust response of Sr33 mutants against Ug99 and its derivative races * Ug99 and derivative races. Scores and methods as described in Rouse et al. (6). Scores from 2 to 2+ indicate resistant to moderately resistant host response while 3+ represent the susceptible response of the wheat lines to a rust race. All mutants (E1 to E9; E1 to E4 contain very large deletions) were susceptible to the Australian Pgt race 34-1,2,3,4,5,6,7,11 and subsequent tests with the Ug99 stem rust race group (TTKSK, TTKST, TTTSK) were conducted on mutants E5 to E9.

EMS mutant Races and rust response scores* TTKSK TTKST TTTSK

E5 3+ 3+ 3+ E6 3+ 3+ 3+ E7 3+ 3+ 3+ E8 3+ 3+ 3+ E9 3+ 3+ 3+ CS1D5405 22+ 22+ 2 Chinese Spring 3+ 3+ 3+

15

Table S2. QRT-PCR analysis of Sr33, AetRGA2a, RAR1, SGT1 and HSP90 expression during silencing by BSMV: VIGS

*Relative expression was calculated by dividing the expression value determined for the target gene in silenced plants by the expression value of the same gene measured in plants infected with Bsmv:00. Each number is an average of triplicates.

Genes Relative expression* Average

Relative Expression

SD Exp.1 Exp.2 Exp.3

Sr33 0.47 0.59 0.45 0.50 0.08 AetRGA2aN 0.47 0.34 0.04 0.28 0.22 AetRGA2aC 0.06 0.26 0.62 0.31 0.29 RAR1 0.12 0.22 0.31 0.22 0.09 HSP90 0.34 0.32 0.43 0.36 0.06 SGT1 0.09 0.12 0.27 0.16 0.10

16

Table S3. Haplotypes of Sr33 and the details of Ae. tauschii accessions in each type.

*Ae. tauschii accession used as the original Sr33 source which is the same as RL5288

Haplotype Ae. tauschii accession number

I *CPI110799, CPI110659, CPI110801, CPI110855, CPI110818, AUS18905, AUS18955, AUS18986

II PI603225

III

CPI110661, CPI110667, CPI110691, CPI110787, CPI110800, CPI110810, CPI110811, CPI110815, AUS18836, AUS18880, AUS18899, AUS18906, AUS18909, AUS18912, AUS18913, AUS18988, AUS18990, AUS18993, AUS19013, AUS22981

IV CPI110629, CPI110633, CPI110908

V CPI110716, CPI110934, CPI110936, AUS18911, AUS18994

17

Table S4. Stem rust response of Sr33 haplotype I and II

*34-0 and 17-1,2,3,4 are Australian stem rust isolates. Rust resistance response against TTKSK (Ug99 isolate from Kenya), QTHJC, RKQQC and TPMKC (isolates from United States) are reproduced from Rouse et al. (6) and Olson et al. (15) - data not available. All these stem rust isolates were avirulent for Sr33.

Haplotype Ae. tauschii accession

Stem rust isolates and rust response scores* 34-0 17-1,2,3,4 TTKSK QTHJC RKQQC TPMKC

I CPI110799 ;1 ;1 2- 2 ;2- 2

CPI110659 ; 0; ; ;,1 ;,1- ;,1

CPI110801 ;1 ;1 - - - -

CPI110855 12- 12- 2- 1 ;,1= 1-

CPI110818 ;1- ;1 1,2- 0 0 0;

AUS18905 ;1- ;1 - - - -

AUS18955 - - - - - -

AUS18986 - - - - - -

II PI603225 - - 2- 2 2- 2

18

Table S5. List of primers used to isolate gene specific sequence.

Gene Primer Pair Primer Sequence 5’-3’ (Forward) Primer Sequence 5’-3’ (Reverse)

AetRGA1a AtM1 F1 R1 CTGCGCGCGTGGTTGGC GATCGATAACAACTGCTTCCC

AtM1 F2 R2 GATCGGAATCGGATAGGGC AATGGTTAGGTAGATCTATTGG

AtM1 F3 R3 AGCAGAATATACTCGAAAGGG CTCCCTCAGCCTTGCCAG

AtM1 F4 R4 TTAATCTACCTAAATGTTTCTCC CAGTGAAATTAGCGTGCAGC

AetRGA1b AtM2 F1 R1 TCTTCTTCTTCCACACTGGG CCAAATCCAACAATGGAGACC

AtM2 F2 R2 AGCTTTGTACGCAGAAGCAAC ATGAATGAAACAAGAAGTACTTC

AtM2 F3 R3 CCTAGAGAACAAAAGGTATGC CAAAACTCAGAGCTATATGAAC

AtM2 F4 R4 TTTATTCAGATTGTTTATCATCTG AAGCATGTACCTGGCCTAGATC

AtM2 F5 R5 TCCAGAAGATAGCATGATTGC AGGAGTTGGAACCACCTTAG

AtM2 F6 R6 TGTTGGATCTTGGAGACAATTA CAATACATATAAACGCAGACATC

AtM2 F7 R7 GAAGTAGTTAGGTTCAGCCTG GCCAGCCGGTTGTGGCG

AtM3 F1 R1 CATATGGATGTGAAGGAGGC TCTTGTTAGAGGCATCGTCG

AetRGA1c AtM3F2 R2 GGCTTTGTACACAGAAGCTAC TAAAACTGTGTGGATAGAACAG

AtM3 F3 R3 ATCCAAACATTTTACATTTCACC AAGGTCTACACACATCACATAT

AtM3 F4 R4 ATTTATTCTTTTTTTGGAGGGCA AAGCATATACCTGGCCTTTATA

AtM3 F5 R5 ATCCAGAAGATAGCAAGATTGA AGATTCTGCAACACACCAGC

AtM3 F6 R6 GGAGGTGTTGGATATTGGAAG CAATACAACCAAACCTTGACATA

AtM3 F7 R7 GGAAAAAGTTGATTTCAGCCTT CTAAAAGCCATTCACATTAACC

AetRGA1d AtM4F1R1 GGGCTTGGTCCAGATCCC CACCCGCTGGCCACTAGTT

AtM4F2R2 CCATAAGAGAATATTTCCTGACGC GAAAACACCAGCATGCCATGGG

AetRGA1e AtM5 F1 R1 CTTGCCAACTCAGTTCCACC TTGCATTATCATTCCGTGCAC

AtM5 F2 R2 CATATCGTACAATACATGCACC TATTCTGAAGGGACAAGCGG

AtM5 F3 R3 ATGCTCCAGCCAATATATTCG AGCACATCACACAACCTCTCGG

AetRGA1f AtM6 F1 R1 CTTGGATCAATGTTATTACTTCTCC ACAAGCTGAGCTCTAGAAGATGG

AetRGA2a AtM7F1R1 GTTGAACTATCTTTCGAACTCG TAAACAAACAACCTATCTGCGC

AetRGA3a AtM8F1R1 GGGTCCTGTACATTCCCTCGC CTGGTTTATCCATCCGATCCACC

19

Table S6. List of primers used to isolate cDNA of Sr33 from CPI110799 (Ae. tauschii) and cDNA of HSP90, RAR1, SGT1 and WRKY1/2 of CPI110799 and Golden Promise (H. Vulgare L.)

Species Primers Primer Sequence (5’-3’) Ae. tauschii Sr33 FL Fwd EcoRI ATGAATTCATGGATATTGTCACGGGTGCCATTG

Ae. tauschii Sr33 FL Rev SalI ATGTCGACTCACTCTGCGTCAGAAATCGGTCCTC

Ae. tauschii Sr33 CC46 Rev SalI ATGTCGACTCACGCAGCGTTCATGGTCTTGAG

Ae. tauschii Sr33 CC125 Rev SalI ATGTCGACTCAGTCCTTGATCGCGTGAGCTATTCC

Ae. tauschii Sr33 CC160 Rev SalI ATGTCGACTCATAGAGCACGGAGACGAGGATCAATTGC

Ae. tauschii Sr33 CC225 Rev SalI ATGTCGACTCAGTGACAATCAAAATCACCTTTAATCTTCTCGTA

Ae. tauschii Sr33 LRR550 Fwd EcoRI ATGAATTCATGCTCACAAATATCATGAGTATCTCACAAGTGAGGT

H. vulgare L. RAR1 FL Fwd NdeI ATCATATGTCGGCGGAGACGGAGAGG

H. vulgare L. RAR1 FL Rev ClaI GCATCGATTCACACAGCATCAGCATTGTGCCA

Ae. tauschii RAR1 FL Fwd NdeI ATGTCGGCGGAGACGGAGACG

Ae. tauschii RAR1 FL Rev ClaI TAATCGATTCATACGGCATCAGCATTGTGCCA

Ae. tauschii/H. vulgare L. SGT1 FL Fwd EcoRI ATGAATTCATGGCCGCCGCCGCC

Ae. tauschii/H. vulgare L. SGT1 FL Rev ClaI ATATCGATTTAATACTCCCACTTCTTGAGCTCCATTCCA

Ae. tauschii/H. vulgare L. HSP90 Fwd EcoRI GCTATGAATTCATGGCGACGGAGACCGAG

Ae. tauschii/H. vulgare L. HSP90 Rev ClaI GCATAATCGATTTAGTCGACCTCCTCCATCTTGC

H. vulgare L. WRKY1 FL Fwd EcoRI ATGAATTCATGGATCCATGGATGGGCAGCC

H. vulgare L. WRKY1 FL Rev ClaI ATATCGATTTAATTGATGTCCCTGGTCGGCGA

Ae. tauschii WRKY1 FL Fwd EcoRI ATGAATTCATGGATCCATGGGTCAGCAGCCA

Ae. tauschii WRKY1 FL Rev ClaI ATATCGATTTAATTGATGTCCCTGGTCGGCGATA

Ae. tauschii/H. vulgare L. WRKY1260 Fwd EcoRI ATGAATTCATGCCGCAGCAGCAGAACGACGG

H. vulgare L. WRKY2 FL Fwd EcoRI ATGAATTCATGGAGGAGCAGTGGATGATCGGG

H. vulgare L. WRKY2 FL Rev ClaI ATATCGATTCAAGCAACAGGGATCCGACCAGA

H. vulgare L. WRKY2242 Fwd EcoRI ATGAATTCATGCCGCCGCCCAAGCATCAAG

Ae. tauschii WRKY2 FL Fwd EcoRI ATGAATTCATGGACGAGCAGTGGATGATCGGG

Ae. tauschii WRKY2 FL Rev ClaI ATATCGATTCAAGCAACAGGGATCCGACCAGAG

Ae. tauschii WRKY2246 Fwd EcoRI ATGAATTCATGCCGCCGCCCAAGCAACAAG

20

References 1. P. G. Pardey, J. M. Beddow, D. J. Kriticos, T. M. Hurley, R. F. Park, E. Duveiller, R. W.

Sutherst, J. J. Burdon, D. Hodson, Right-sizing stem-rust research. Science 340, 147–148 (2013). doi:10.1126/science.122970 Medline

2. Y. Jin, R. P. Singh, Resistance in U.S. wheat to recent eastern African isolates of Puccinia graminis f. sp. tritici with virulence to resistance gene Sr31. Plant Dis. 90, 476–480 (2006). doi:10.1094/PD-90-0476

3. R. P. Singh, D. P. Hodson, J. Huerta-Espino, Y. Jin, S. Bhavani, P. Njau, S. Herrera-Foessel, P. K. Singh, S. Singh, V. Govindan, The emergence of Ug99 races of the stem rust fungus is a threat to world wheat production. Annu. Rev. Phytopathol. 49, 465–481 (2011). doi:10.1146/annurev-phyto-072910-095423 Medline

4. E. R. Kerber, Resistance to leaf rust in hexaploid wheat: Lr32 a third gene derived from Triticum tauschii. Crop Sci. 27, 204–206 (1987). doi:10.2135/cropsci1987.0011183X002700020013x

5. S. S. Jones, J. Dvorak, D. R. Knott, C. O. Qualset, Use of double-ditelosomic and normal chromosome 1D recombinant substitution lines to map Sr33 on chromosome arm 1DS in wheat. Genome 34, 505–508 (1991). doi:10.1139/g91-077

6. M. N. Rouse, E. L. Olson, B. S. Gill, M. O. Pumphrey, Y. Jin, Stem rust resistance in germplasm. Crop Sci. 51, 2074–2078 (2011). doi:10.2135/cropsci2010.12.0719

7. M. C. Luo, C. Thomas, F. M. You, J. Hsiao, S. Ouyang, C. R. Buell, M. Malandro, P. E. McGuire, O. D. Anderson, J. Dvorak, High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis. Genomics 82, 378–389 (2003). doi:10.1016/S0888-7543(03)00128-9 Medline

8. O. Moullet, H. B. Zhang, E. S. Lagudah, Construction and characterisation of a large DNA insert library from the D genome of wheat. Theor. Appl. Genet. 99, 305–313 (1999). doi:10.1007/s001220051237

9. F. Wei, R. A. Wing, R. P. Wise, Genome dynamics and evolution of the Mla (powdery mildew) resistance locus in barley. Plant Cell 14, 1903–1917 (2002). doi:10.1105/tpc.002238 Medline

10. S. Seeholzer, T. Tsuchimatsu, T. Jordan, S. Bieri, S. Pajonk, W. Yang, A. Jahoor, K. K. Shimizu, B. Keller, P. Schulze-Lefert, Diversity at the Mla powdery mildew resistance locus from cultivated barley reveals sites of positive selection. Mol. Plant Microbe Interact. 23, 497–509 (2010). doi:10.1094/MPMI-23-4-0497 Medline

11. H. Q. Ling, S. Zhao, D. Liu, J. Wang, H. Sun, C. Zhang, H. Fan, D. Li, L. Dong, Y. Tao, C. Gao, H. Wu, Y. Li, Y. Cui, X. Guo, S. Zheng, B. Wang, K. Yu, Q. Liang, W. Yang, X. Lou, J. Chen, M. Feng, J. Jian, X. Zhang, G. Luo, Y. Jiang, J. Liu, Z. Wang, Y. Sha, B. Zhang, H. Wu, D. Tang, Q. Shen, P. Xue, S. Zou, X. Wang, X. Liu, F. Wang, Y. Yang, X. An, Z. Dong, K. Zhang, X. Zhang, M. C. Luo, J. Dvorak, Y. Tong, J. Wang, H. Yang, Z. Li, D. Wang, A. Zhang, J. Wang, Draft genome of the wheat A-genome progenitor Triticum urartu. Nature 496, 87–90 (2013). doi:10.1038/nature11997 Medline

21

12. T. Jordan, S. Seeholzer, S. Schwizer, A. Töller, I. E. Somssich, B. Keller, The wheat Mla homologue TmMla1 exhibits an evolutionarily conserved function against powdery mildew in both wheat and barley. Plant J. 65, 610–621 (2011). doi:10.1111/j.1365-313X.2010.04445.x Medline

13. K. Shirasu, The HSP90-SGT1 chaperone complex for NLR immune sensors. Annu. Rev. Plant Biol. 60, 139–164 (2009). doi:10.1146/annurev.arplant.59.032607.092906 Medline

14. S. R. Scofield, L. Huang, A. S. Brandt, B. S. Gill, Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol. 138, 2165–2173 (2005). doi:10.1104/pp.105.061861 Medline

15. E. L. Olson, M. N. Rouse, M. O. Pumphrey, R. L. Bowden, B. S. Gill, J. A. Poland, Simultaneous transfer, introgression, and genomic localization of genes for resistance to stem rust race TTKSK (Ug99) from Aegilops tauschii to wheat. Theor. Appl. Genet. 126, 1179–1188 (2013). doi:10.1007/s00122-013-2045-5 Medline

16. R. Mago, W. Spielmeyer, J. Lawrence, S. Lagudah, G. Ellis, A. Pryor, Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines. Theor. Appl. Genet. 104, 1317–1324 (2002). doi:10.1007/s00122-002-0879-3 Medline

17. M. Ayliffe, S. K. Periyannan, A. Feechan, I. Dry, U. Schumann, M. B. Wang, A. Pryor, E. Lagudah, A simple method for comparing fungal biomass in infected plant tissues. Mol. Plant Microbe Interact. 26, 658–667 (2013). doi:10.1094/MPMI-12-12-0291-R Medline

18. C. Saintenac et al., Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group. Science 340, 1239022 (2013); 10.1126/science.1239022

19. H. S. Bariana, R. A. McIntosh, Cytogenetic studies in wheat. XV. Location of rust resistance genes in VPM1 and their genetic linkage with other disease resistance genes in chromosome 2A. Genome 36, 476–482 (1993). doi:10.1139/g93-065 Medline

20. D. J. Somers, P. Isaac, K. Edwards, A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 109, 1105–1114 (2004). doi:10.1007/s00122-004-1740-7 Medline

21. M. J. Hayden, T. M. Nguyen, A. Waterman, G. L. McMichael, K. J. Chalmers, Application of multiplex-ready PCR for fluorescence-based SSR genotyping in barley and wheat. Mol. Breed. 21, 271–281 (2008). doi:10.1007/s11032-007-9127-5

22. K. F. Manly, R. H. Cudmore, Jr., J. M. Meer, Map Manager QTX, cross-platform software for genetic mapping. Mamm. Genome 12, 930–932 (2001). doi:10.1007/s00335-001-1016-3 Medline

23. D. D. Kosambi, The estimation of map distance from recombination values. Ann. Eugen. 12, 172–175 (1944).

24. R. Kota, W. Spielmeyer, R. A. McIntosh, E. S. Lagudah, Fine genetic mapping fails to dissociate durable stem rust resistance gene Sr2 from pseudo-black chaff in common wheat (Triticum aestivum L.). Theor. Appl. Genet. 112, 492–499 (2006). doi:10.1007/s00122-005-0151-8 Medline

22

25. E. D. Akhunov, A. R. Akhunova, O. D. Anderson, J. A. Anderson, N. Blake, M. T. Clegg, D. Coleman-Derr, E. J. Conley, C. C. Crossman, K. R. Deal, J. Dubcovsky, B. S. Gill, Y. Q. Gu, J. Hadam, H. Heo, N. Huo, G. R. Lazo, M. C. Luo, Y. Q. Ma, D. E. Matthews, P. E. McGuire, P. L. Morrell, C. O. Qualset, J. Renfro, D. Tabanao, L. E. Talbert, C. Tian, D. M. Toleno, M. L. Warburton, F. M. You, W. Zhang, J. Dvorak, Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes. BMC Genomics 11, 702 (2010). doi:10.1186/1471-2164-11-702 Medline

26. E. S. Lagudah, H. McFadden, R. P. Singh, J. Huerta-Espino, H. S. Bariana, W. Spielmeyer, Molecular genetic characterization of the Lr34/Yr18 slow rusting resistance gene region in wheat. Theor. Appl. Genet. 114, 21–30 (2006). doi:10.1007/s00122-006-0406-z Medline

27. M. C. Luo, Y. Q. Gu, F. M. You, K. R. Deal, Y. Ma, Y. Hu, N. Huo, Y. Wang, J. Wang, S. Chen, C. M. Jorgensen, Y. Zhang, P. E. McGuire, S. Pasternak, J. C. Stein, D. Ware, M. Kramer, W. R. McCombie, S. F. Kianian, M. M. Martis, K. F. Mayer, S. K. Sehgal, W. Li, B. S. Gill, M. W. Bevan, H. Simková, J. Dolezel, S. Weining, G. R. Lazo, O. D. Anderson, J. Dvorak, A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor. Proc. Natl. Acad. Sci. U.S.A. 110, 7940–7945 (2013). doi:10.1073/pnas.1219082110 Medline

28. E. S. Lagudah, S. G. Krattinger, S. Herrera-Foessel, R. P. Singh, J. Huerta-Espino, W. Spielmeyer, G. Brown-Guedira, L. L. Selter, B. Keller, Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which confers resistance to multiple fungal pathogens. Theor. Appl. Genet. 119, 889–898 (2009). doi:10.1007/s00122-009-1097-z Medline

29. S. G. Krattinger, E. S. Lagudah, W. Spielmeyer, R. P. Singh, J. Huerta-Espino, H. McFadden, E. Bossolini, L. L. Selter, B. Keller, A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science 323, 1360–1363 (2009). doi:10.1126/science.1166453

30. M. B. Wang, P. R. Matthews, N. M. Upadhyaya, P. M. Waterhouse, Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hortic. 461, 401–405 (1998).

31. C. Maas, C. G. Simpson, P. Eckes, H. Schickler, J. W. S. Brown, B. Reiss, K. Salchert, I. Chet, J. Schell, C. Reichel, Mol. Breed. 3, 15–28 (1997). doi:10.1023/A:1009602923403

32. I. T. Petty, B. G. Hunter, N. Wei, A. O. Jackson, Infectious barley stripe mosaic virus RNA transcribed in vitro from full-length genomic cDNA clones. Virology 171, 342–349 (1989). doi:10.1016/0042-6822(89)90601-6 Medline

33. R. D. Gietz, R. A. Woods, Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96 (2002). doi:10.1016/S0076-6879(02)50957-5 Medline

34. M. Bernoux, T. Ve, S. Williams, C. Warren, D. Hatters, E. Valkov, X. Zhang, J. G. Ellis, B. Kobe, P. N. Dodds, Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation. Cell Host Microbe 9, 200–211 (2011). doi:10.1016/j.chom.2011.02.009 Medline

23

35. V. V. Kushnirov, Rapid and reliable protein extraction from yeast. Yeast 16, 857–860 (2000). doi:10.1002/1097-0061(20000630)16:9<857::AID-YEA561>3.0.CO;2-B Medline

36. Y. S. Tai, Interactome of signaling networks in wheat: the protein-protein interaction between TaRAR1 and TaSGT1. Mol. Biol. Rep. 35, 337–343 (2008). doi:10.1007/s11033-007-9091-5 Medline

37. G. F. Wang, X. Wei, R. Fan, H. Zhou, X. Wang, C. Yu, L. Dong, Z. Dong, X. Wang, Z. Kang, H. Ling, Q. H. Shen, D. Wang, X. Zhang, Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): Functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance. New Phytol. 191, 418–431 (2011). doi:10.1111/j.1469-8137.2011.03715.x Medline

38. M. Ayliffe, R. Devilla, R. Mago, R. White, M. Talbot, A. Pryor, H. Leung, Nonhost resistance of rice to rust pathogens. Mol. Plant Microbe Interact. 24, 1143–1155 (2011). doi:10.1094/MPMI-04-11-0100 Medline

39. K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011). doi:10.1093/molbev/msr121 Medline