Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99

1The emergence of new virulent races of pathogens that can overcome the resistance of existing crop cultivars poses a threat to global food security. A prime example is the outbreak of wheat stem rust in Africa that was caused by a broadly virulent Puccinia graminis f. sp. tritici (Pgt) race, Ug99, detected in Uganda in 1999. Ug99 was virulent on most of the wheat varieties grown in Europe, Asia, and the United States, prompting research into the discovery of Ug99-effective resistance genes. Since the discovery of Ug99, Pgt surveillance identified new Ug99-derived strains virulent against additional wheat resistance genes.

2Plant resistance genes (R) defend against an invading pathogen by detecting the corresponding pathogen avirulence factors (Avr), which are often secreted effector proteins. R genes encode receptors that trigger an immune response upon perception of pathogen Avr factors. This response results in localized cell death at the site of infection [hypersensitive response (HR)]. A pathogen lacking an Avr gene renders the corresponding plant R gene ineffective. Here, we identified the fungal Avr gene recognized by the Ug99-effective wheat stem rust resistance gene Sr35 and investigated the origin of Sr35-virulent fungal isolates.

3Using confocal microscopy of Pgt-infected leaf tissues from resistant (Sr35+) and susceptible (Sr35–) wheat lines, we demonstrated that Sr35 triggers a resistance response at the early stages of infection (Fig. 1). In wheat lineU6169 (Sr35+), the development of fungal infection hyphae stopped even before the formation of a haustorium, the structurewithwhich the fungus extracts nutrients from its host plant. This early immune response is consistent with the lack of pronounced HR symptoms in Triticum monococcum accession G2919 used to identify the Sr35 gene and suggested early expression of a fungal gene recognized by Sr35. To identify this Avr gene, we mutagenized the spores of the Pgt race RKQQC (Sr35-avirulent isolate 99KS76A-1) with ethylmethane sulfonate (EMS). We isolated 15 Pgt mutants virulent to the Sr35 gene, suggesting that they carry mutations affecting the Sr35-specific Avr factor (tables S1 and S2). Both microscopy and timecourseRNA- sequencing (RNA-seq) analyses showed no obvious effects of these mutations on the Pgt mutants’ interaction with a wheat host compared to the wild-type Pgt isolate (figs. S1 and S2, and tables S3 to S5), perhaps due to the functional redundancy of virulence factors that can compensatemutations in the AvrSr35 gene. The genome of the wild-type Pgt isolate was assembled, annotated using RNA-seq data (tables S4 and S6), and compared with Illumina reads generated for each of 15 independent Pgt mutants (table S7), resulting in the detection of 30,429 EMS-induced mutations (table S8 and data S1).

4Only one gene (MF474174) carried mutations in each Pgt mutant; 12 mutants had nonsense mutations, one mutant carried a splice-site disruptingmutation, and two mutants had the same nonsynonymous mutation producing valine to isoleucine (V128I) substitution (Fig. 2A and table S9). This AvrSr35 candidate gene encoded a 578–amino acid protein with a predicted secretion signal peptide (fig. S3). The protein was larger than many previously identified effectors; it showed no similarity to proteins from other species within the protein databases, nor did it contain any detectable protein domains (fig. S4). Gene expression analysis of a Pgt-infected susceptible wheat line showed increased amounts of the AvrSr35 transcripts in the leaf tissues over the course of infection (fig. S3).

5To understand the origin of virulence to Sr35 in the field, we resequenced AvrSr35 from 12 Sr35- virulent and 15 Sr35-avirulent natural isolates (tables S10 and S11 and data S2). Phylogenetic analysis revealed twomajor clades (Fig. 2B). Clade A sequences had intact coding sequences and were found only in the Sr35-avirulent isolates, including 99KS76A-1 and Ug99, indicating that functional AvrSr35 is required for triggering Sr35 resistance against Ug99. Clade V sequences were preferentially 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 two AvrSr35 gene copies fromboth clades. Clade V had the miniature inverted transposable element (MITE) in exon 6, resulting in the premature stop codon. Because even less severe AvrSr35 truncations detected in the Pgt mutants (Fig. 2A) caused Sr35-avirulence function loss, this MITE insertion is predicted to produce a nonfunctional protein. These results suggest that transposon-mediated disruption of AvrSr35 resulted in the origin of natural Sr35-virulent Pgt isolates. It is likely that the loss of Avr factors is facilitated by transposon proliferation in the rust genomes, which display the higher abundance of mobile elements compared with other fungi, contributing to the erosion of plant R genes conferring resistance to rusts.

6The ability of the wheat Sr35 gene to recognize the fungal AvrSr35 and trigger HR was confirmed by coexpressing thewild-type andmutated AvrSr35 and Sr35 gene constructs (table S12) in Nicotiana benthamiana leaves (Fig. 3, fig. S5, andmovie S1). Even though the coexpressed wild-type constructs induced HR, the coexpression of truncated AvrSr35 with wild-type Sr35 failed to induce HR. No HR was evident in the combination of wild-type AvrSr35 with either of the two loss-of-function sr35 alleles, one with mutation (K206L) in the Ploop domain required forHR andanotherwith three mutations found in the leucine-rich repeat (LRR) domain of the Ug99-susceptibleM1120mutant of diploid wheat accession G2919. The expression of individual wild-type constructs also failed to elicit HR (fig. S6). Because loss-offunction mutations in either AvrSr35 or Sr35 resulted in both HR loss in N. benthamiana and a compatible interaction between Pgt and wheat, direct or indirect recognition of AvrSr35 by Sr35 appears to induce a resistance response in wheat. Consistent with this conclusion, the infiltration of the AvrSr35 protein caused HR in the leaves of a Sr35+ but not in the leaves of a Sr35– wheat lines (Fig. 3E).

7In N. benthamiana cells, fluorescently tagged Sr35 and AvrSr35 proteins, either coexpressed together or expressed individually, colocalized in the same subcellular compartment (Fig. 4, A to C). Colocalization of coexpressed Sr35:GFP protein fusion and the endoplasmic reticulum (ER) marker suggest that Sr35 and AvrSr35 expressed in N. benthamiana are likely associated with the ER (Figs. 4C and fig. S7). To investigate whether colocalized Sr35 and AvrSr35 interact in planta, we used bimolecular fluorescent complementation (BiFC) (Fig. 4D and figs. S8 and S9). The complementary AvrSr35 and Sr35 fusion proteins coexpressed in N. benthamiana produced a fluorescence signal that is consistent with a protein-protein interaction. The nonsynonymous mutations (sr35 M1120 allele) affecting the LRR domain reduced the fluorescent signal intensity implicating the LRR domain in the Sr35- AvrSr35 interaction. These results suggest that the Ug99-susceptibility of wheat mutant M1120 is associated with the inability of sr35M1120 to interact effectively with AvrSr35. The coimmunoprecipitation of epitope-tagged Sr35 and AvrSr35 expressed in N. benthamiana leaves supported the BiFC results, indicating that these proteins are capable of interacting in plant cells (fig. S10).

8The identification of AvrSr35 and AvrSr50 provides valuable tools for molecular surveillance and early detection of virulent fungal pathogen races, which can inform the deployment of resistance genes to prevent epidemics. AvrSr35 can also be used to confirm the expression of the functional Sr35 protein in the resistance gene cassettes, allowing for Sr35 to be quickly pyramided alongside other R genes. As more corresponding R-Avr gene pairs are identified, this information can guide the selection of complementary R genes targeting multiple avirulence factors to increase the durability of the deployed resistance gene pyramids and reduce the probability of spontaneous virulent Pgt strain origin.