PLANT SCIENCE

Variation in the AvrSr35 genedetermines Sr35 resistance againstwheat stem rust race Ug99Andres Salcedo,1* William Rutter,1*† Shichen Wang,1‡ Alina Akhunova,2

Stephen Bolus,3 Shiaoman Chao,4 Nickolas Anderson,1 Monica Fernandez De Soto,2

Matthew Rouse,5,6 Les Szabo,5,6 Robert L. Bowden,7

Jorge Dubcovsky,3,8 Eduard Akhunov1§

Puccinia graminis f. sp. tritici (Pgt) causes wheat stem rust, a devastating fungaldisease. The Sr35 resistance gene confers immunity against this pathogen’s mostvirulent races, including Ug99. We used comparative whole-genome sequencing ofchemically mutagenized and natural Pgt isolates to identify a fungal gene namedAvrSr35 that is required for Sr35 avirulence. The AvrSr35 gene encodes a secretedprotein capable of interacting with Sr35 and triggering the immune response.We show that the origin of Pgt isolates virulent on Sr35 is associated with thenonfunctionalization of the AvrSr35 gene by the insertion of a mobile element. Thediscovery of AvrSr35 provides a new tool for Pgt surveillance, identification of hostsusceptibility targets, and characterization of the molecular determinants of immunityin wheat.

The emergence of new virulent races ofpathogens that can overcome the resistanceof existing crop cultivars poses a threat toglobal food security. A prime example isthe outbreak of wheat stem rust in Africa

that was caused by a broadly virulent Pucciniagraminis f. sp. tritici (Pgt) race, Ug99, detectedin Uganda in 1999 (1). Ug99 was virulent onmost of the wheat varieties grown in Europe,Asia, and the United States, prompting researchinto the discovery of Ug99-effective resistancegenes. Since the discovery of Ug99, Pgt surveil-lance identifiednewUg99-derived strains virulentagainst additional wheat resistance genes (2).Plant resistance genes (R) defend against an

invading pathogen by detecting the correspondingpathogen avirulence factors (Avr), which are oftensecreted effector proteins. R genes encode recep-tors that trigger an immune response upon per-ception of pathogen Avr factors. This response

results in localized cell death at the site of infection[hypersensitive response (HR)] (3–5). A pathogenlacking an Avr gene renders the correspondingplant R gene ineffective. Here, we identified thefungalAvr gene recognized by theUg99-effectivewheat stem rust resistance gene Sr35 (6) andinvestigated the origin of Sr35-virulent fungalisolates.Using confocal microscopy of Pgt-infected leaf

tissues from resistant (Sr35+) and susceptible(Sr35–) wheat lines, we demonstrated that Sr35triggers a resistance response at the early stagesof infection (Fig. 1) (7). Inwheat lineU6169 (Sr35+),the development of fungal infection hyphae

stopped even before the formation of a hausto-rium, the structurewithwhich the fungus extractsnutrients from its host plant. This early immuneresponse is consistent with the lack of pronouncedHR symptoms in Triticum monococcum accessionG2919 used to identify the Sr35 gene (6) andsuggested early expression of a fungal gene rec-ognized by Sr35. To identify this Avr gene, wemutagenized the spores of the Pgt race RKQQC(Sr35-avirulent isolate 99KS76A-1) with ethylme-thane sulfonate (EMS). We isolated 15 Pgtmutantsvirulent to theSr35gene, suggesting that they carrymutations affecting the Sr35-specific Avr factor(tables S1 and S2) (7). Both microscopy and time-courseRNA-sequencing (RNA-seq) analyses showedno obvious effects of these mutations on the Pgtmutants’ interaction with a wheat host comparedto the wild-type Pgt isolate (figs. S1 and S2, andtables S3 to S5) (7), perhaps due to the functionalredundancy of virulence factors that can compen-satemutations in theAvrSr35 gene (8). The genomeof the wild-type Pgt isolate was assembled, anno-tated using RNA-seq data (tables S4 and S6), andcompared with Illumina reads generated for eachof 15 independent Pgt mutants (table S7), result-ing in the detection of 30,429 EMS-inducedmutations (table S8 and data S1).Only one gene (MF474174) carried mutations

in each Pgt mutant; 12 mutants had nonsensemutations, one mutant carried a splice-site dis-rupting mutation, and twomutants had the samenonsynonymous mutation producing valine toisoleucine (V128I) substitution (Fig. 2A and table S9).This AvrSr35 candidate gene encoded a 578–aminoacid protein with a predicted secretion signalpeptide (fig. S3). The protein was larger thanmany previously identified effectors (9); it showedno similarity to proteins fromother specieswithinthe protein databases, nor did it contain anydetectable protein domains (fig. S4) (7). Geneexpression analysis of a Pgt-infected susceptiblewheat line showed increased amounts of the

RESEARCH

Salcedo et al., Science 358, 1604–1606 (2017) 22 December 2017 1 of 3

1Department of Plant Pathology, Kansas State University,Manhattan, KS 66506, USA. 2Integrated Genomics Facility,Kansas State University, Manhattan, KS 66506, USA.3Department of Plant Sciences, University of California,Davis, CA 95616, USA. 4U.S. Department of Agriculture,Agricultural Research Service (USDA-ARS) Cereal CropsResearch Unit, Fargo, ND 58102, USA. 5USDA-ARS, CerealDisease Laboratory, St. Paul, MN 55108, USA.6Department of Plant Pathology, University of Minnesota,St. Paul, MN 55108, USA. 7USDA-ARS, Hard Winter WheatGenetics Research Unit, Manhattan, KS 66506, USA.8Howard Hughes Medical Institute (HHMI), Chevy Chase,MD 20815, USA.*These authors contributed equally to this work. †Presentaddress: USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC29414, USA. ‡Present address: Texas A&M Engineering Exper-iment Station-AgriLife Center for Bioinformatics and GenomicSystems Engineering, Texas A&M University, College Station, TX77845, USA.§Corresponding author. Email: eakhunov@ksu.edu

Fig. 1. Sr35 provides prehaustorialresistance against Pgt. Infected leavesof susceptible cultivar Morocco (Sr35–)and resistant line U6169 (Sr35+) werecollected 28 and 48 hours after infec-tion (HAI). (A and B) Fungal infectionhyphae (IH) (stained blue) entered theleaf mesophyll tissue (stained red)through the plant stoma (S) in bothwheat lines at 24 HAI. Fungal haustoria(H) developed only in susceptibleMorocco (B). (C and D) Imaging at48 HAI revealed fungal growth in sus-ceptible Morocco (D) but no furtherfungal growth in U6169 (C). (E andF) Using different dyes, imaging at48 HAI revealed two presumably deadhost cells (DC) with increased fluores-cence in close proximity of the HM inU6169 (E); no dead cells were revealed in Morocco (F). Staining of nuclei with propidiumiodide (red) was indicative of cell death [insert in (E)]. SSV, fungal substomatal vesicle; MC,mesophyll cells; HM, haustorial mother cells.

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AvrSr35 transcripts in the leaf tissues over thecourse of infection (fig. S3).To understand the origin of virulence to Sr35

in the field, we resequencedAvrSr35 from 12 Sr35-virulent and 15 Sr35-avirulent natural isolates(tables S10 and S11 and data S2). Phylogeneticanalysis revealed twomajor clades (Fig. 2B). CladeA sequences had intact coding sequences andwere found only in the Sr35-avirulent isolates,including 99KS76A-1 and Ug99, indicating thatfunctional AvrSr35 is required for triggering Sr35

resistance against Ug99. Clade V sequences werepreferentially found in the Sr35-virulent isolates,except for five Sr35-avirulent isolates (77ND82A,72CA1A, 75-36-700-3, 69SD657C, and 74MN1049)that carry at least twoAvrSr35gene copies frombothclades (7). Clade V had the miniature invertedtransposable element (MITE) in exon 6, resultingin the premature stop codon. Because even lesssevere AvrSr35 truncations detected in the Pgtmutants (Fig. 2A) caused Sr35-avirulence functionloss, this MITE insertion is predicted to produce

a nonfunctional protein. These results suggestthat transposon-mediated disruption of AvrSr35resulted in the origin of natural Sr35-virulent Pgtisolates. It is likely that the loss of Avr factors isfacilitated by transposon proliferation in the rustgenomes, which display the higher abundanceof mobile elements compared with other fungi(9, 10), contributing to the erosion of plant R genesconferring resistance to rusts.The ability of thewheat Sr35 gene to recognize

the fungalAvrSr35 and triggerHRwas confirmed

Salcedo et al., Science 358, 1604–1606 (2017) 22 December 2017 2 of 3

Stop codon gainSplice site disruptionNon-synonymous mutationMiniature Inverted Transposable Element (MITE)Secretion signal peptide

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Fig. 2. Identification of the candidateAvrSr35 gene. (A) EMS-induced mutationsand MITE insertion site in the AvrSr35 gene.(B) Phylogeny of the AvrSr35 gene in diversePgt isolates. The colored tree tips correspondto alleles originating from the Sr35-avirulent(red) and Sr35-virulent (blue) isolates.The sequences from the Pgt isolates hetero-zygous at the AvrSr35 locus are markedwith stars. The different AvrSr35 alleles fromthese isolates have A or V appended to thesequence name. The sequences with MITEinsertion are marked by checkmarks. TheAvrSr35 gene sequences form two majorclades, A and V. Clade V includes the virulentallele with the MITE insertion. Bootstrapvalues above 0.7 are shown on the tree nodes.The second AvrSr35 allele (accession numberMF596174) from isolate 99KS76A-1 with anonsense mutation in coding sequence wasused as an outgroup.

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Fig. 3. AvrSr35 triggers Sr35-dependent cell death in N. benthamiana and wheat leaves.(A) The AvrSr35 and Sr35 gene constructs were delivered into the N. benthamiana leavesby Agrobacterium tumefaciens infiltration. (B) Expression of gene constructs in N. benthamianawas validated by reverse transcription polymerase chain reaction (RT-PCR). The proteinphosphatase 2A (PP2A) gene was used as an internal control (7). (C) Coinfiltration ofN. benthamiana leaves with wild-type and mutant Sr35 and AvrSr35 constructs. The imageswere taken 48 to 72 HAI. (D) The accumulation of reactive oxygen species accompanying HRwas assessed by staining leaves with 3,3′-diaminobenzidine 20 to 24 HAI. Each leaf wasinfiltrated at four sections formed by a midvein and two secondary veins (dashed lines).(E) Infiltration of the AvrSr35 protein into the leaves of Sr35+ and Sr35– wheat lines. TheSDS–polyacrylamide gel electrophoresis analysis of AvrSr35 before (1) and after (2) smallubiquitin-related modifier (SUMO) protease treatment. Bovine serum albumin (BSA) was usedas a negative control.

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by coexpressing thewild-type andmutatedAvrSr35and Sr35 gene constructs (table S12) inNicotianabenthamiana leaves (Fig. 3, fig. S5, andmovie S1).Even though the coexpressedwild-type constructsinduced HR, the coexpression of truncatedAvrSr35 with wild-type Sr35 failed to induce HR.NoHRwas evident in the combination of wild-typeAvrSr35 with either of the two loss-of-functionsr35 alleles, one with mutation (K206L) in the P-loop domain required forHR (7) and anotherwiththree mutations found in the leucine-rich repeat(LRR) domain of the Ug99-susceptible M1120mu-tant of diploid wheat accession G2919 (6). The ex-pression of individual wild-type constructs alsofailed to elicit HR (fig. S6). Because loss-of-function mutations in either AvrSr35 or Sr35resulted in both HR loss in N. benthamiana anda compatible interaction between Pgt and wheat,direct or indirect recognition of AvrSr35 by Sr35appears to induce a resistance response in wheat.Consistent with this conclusion, the infiltration oftheAvrSr35 protein caused HR in the leaves of aSr35+ but not in the leaves of a Sr35– wheatlines (Fig. 3E).

In N. benthamiana cells, fluorescently taggedSr35 and AvrSr35 proteins, either coexpressedtogether or expressed individually, colocalized inthe same subcellular compartment (Fig. 4, A toC). Colocalization of coexpressed Sr35:GFP pro-tein fusion and the endoplasmic reticulum (ER)marker suggest that Sr35 and AvrSr35 expressedin N. benthamiana are likely associated with theER (Figs. 4C and fig. S7). To investigate whethercolocalized Sr35 and AvrSr35 interact in planta,we used bimolecular fluorescent complementa-tion (BiFC) (Fig. 4D and figs. S8 and S9). Thecomplementary AvrSr35 and Sr35 fusion pro-teins coexpressed inN. benthamiana produced afluorescence signal that is consistent with aprotein-protein interaction. The nonsynonymousmutations (sr35M1120 allele) affecting the LRRdomain reduced the fluorescent signal inten-sity implicating the LRR domain in the Sr35-AvrSr35 interaction. These results suggest thatthe Ug99-susceptibility of wheat mutant M1120(6) is associated with the inability of sr35M1120

to interact effectively with AvrSr35. The coim-munoprecipitation of epitope-tagged Sr35 and

AvrSr35 expressed in N. benthamiana leavessupported the BiFC results, indicating that theseproteins are capable of interacting in plant cells(fig. S10).The identification of AvrSr35 and AvrSr50

(11) provides valuable tools for molecular sur-veillance and early detection of virulent fungalpathogen races, which can inform the deploy-ment of resistance genes to prevent epidemics.AvrSr35 can also be used to confirm the ex-pression of the functional Sr35 protein in theresistance gene cassettes, allowing for Sr35 tobe quickly pyramided alongside other R genes.As more corresponding R-Avr gene pairs areidentified, this information can guide the selec-tion of complementary R genes targeting multipleavirulence factors to increase the durability of thedeployed resistance gene pyramids and reducethe probability of spontaneous virulent Pgt strainorigin.

REFERENCES AND NOTES

1. Z. A. Pretorius, R. P. Singh, W. W. Wagoire, T. S. Payne, PlantDis. 84, 203 (2000).

2. R. P. Singh et al., Phytopathology 105, 872–884(2015).

3. J. D. G. Jones, J. L. Dangl, Nature 444, 323–329(2006).

4. J. G. Ellis, M. Rafiqi, P. Gan, A. Chakrabarti, P. N. Dodds, Curr.Opin. Plant Biol. 12, 399–405 (2009).

5. J. M. Elmore, Z.-J. D. Lin, G. Coaker, Curr. Opin. Plant Biol. 14,365–371 (2011).

6. C. Saintenac et al., Science 341, 783–786 (2013).7. Materials and methods are available as supplementary

materials.8. W. B. Rutter et al., BMC Genomics 18, 291 (2017).9. S. Duplessis et al., Proc. Natl. Acad. Sci. U.S.A. 108, 9166–9171

(2011).10. D. Cantu et al., BMC Genomics 14, 270 (2013).11. J. Chen et al., Science 358, 1607–1610 (2017).

ACKNOWLEDGMENTS

Data are available from the National Center for BiotechnologyInformation database: MF474174, MF596174, PRJNA415853,PRJNA313186, SRX1619622-SRX1619626, and GEOGSE106397. The project is funded by U.S. Department ofAgriculture (USDA) National Institute of Food and Agriculturegrant 2012-67013-19401 and Bill and Melinda GatesFoundation grant BMGF:01511000146 to E.A., and USDAAgricultural Research Service funding 3020- 21000-010-00-Dto B.B. J.D. acknowledges support from the HowardHughes Medical Institute. We thank Y. Jin for providingstem rust cultures, H. Liang and Y. Lun for technicalassistance, R. Todd for valuable suggestions, and D. Cantu forinitial contribution to the sequencing of Pgt races. Thesupplementary materials contain additional data. This workis licensed under a Creative Commons Attribution 4.0International (CC BY 4.0) license, which permits unrestricteduse, distribution, and reproduction in any medium, providedthe original work is properly cited. To view a copy of thislicense, visit http://creativecommons.org/licenses/by/4.0/.This license does not apply to figures/photos/artworkor other content included in the article that is credited to athird party; obtain authorization from the rights holder beforeusing such material.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/358/6370/1604/suppl/DC1Materials and MethodsFigs. S1 to S10Tables S1 to S12Movie S1Data S1 and S2References (12–30)

21 August 2017; accepted 9 November 201710.1126/science.aao7294

Salcedo et al., Science 358, 1604–1606 (2017) 22 December 2017 3 of 3

AvrSr35 SP:mRFP

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Fig. 4. Sr35 and AvrSr35 proteins colocalize in plant cells and interact. (A) Coexpressedfluorescently tagged Sr35 and AvrSr35 colocalized in the N. benthamiana leaf epidermalcells. Scale bar, 20 mm. (B) In the N. benthamiana cells, the AvrSr35DDSP:mRFP proteinfusion accumulated in the ER strands (small arrows) and perinuclear space. Scale bar, 10 mm.(C) The Sr35(K206L):GFP protein fusion colocalized with the ER marker ER-mCherry inN. benthamiana cells (fig. S7). (D) Bimolecular fluorescent complementation showed interactionbetween Sr35 and AvrSr35 in the N. benthamiana cells. Compared with wild-type Sr35, thefluorescence intensity was significantly reduced in the cells expressing sr35M1120 (Tukey’s testadjusted P = 7.4 × 10−4) and negative control (Tukey’s test adjusted P = 8.7 × 10−5). Scale bar,50 mm. N, nucleus.

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resistance against wheat stem rust race Ug99Sr35 gene determines AvrSr35Variation in the

Fernandez De Soto, Matthew Rouse, Les Szabo, Robert L. Bowden, Jorge Dubcovsky and Eduard AkhunovAndres Salcedo, William Rutter, Shichen Wang, Alina Akhunova, Stephen Bolus, Shiaoman Chao, Nickolas Anderson, Monica

DOI: 10.1126/science.aao7294 (6370), 1604-1606.358Science 

, this issue p. 1607, p. 1604; see also p. 1541Scienceinactivating these Avr effectors leaves the plant defenseless and susceptible to disease.

show that fungal AvrSr35 binds to Sr35. Successful binding activates the plant's immune defenses. Removing oral.et show that fungal AvrSr50 binds to the plant's immune receptor Sr50, and Salcedo et al.Moscou and van Esse). Chen

wheat crops. Two papers describe two peptides secreted by the fungus as it attacks the wheat (see the Perspective byUg99 can kill entire fields of wheat and is undeterred by many of the disease-resistance genes that otherwise protect

The fungal pathogen Ug99 (named for its identification in Uganda in 1999) threatens wheat crops worldwide.Fungal effectors of wheat stem rust

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