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. 2017 Nov 7;114(45):E9483-E9492.
doi: 10.1073/pnas.1706277114. Epub 2017 Oct 23.

Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group

Wenjun Zhang  1 , Shisheng Chen  1 , Zewdie Abate  1 , Jayaveeramuthu Nirmala  2 , Matthew N Rouse  3   4 , Jorge Dubcovsky  5   6
Affiliations

Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group

Wenjun Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

The Puccinia graminis f. sp. tritici (Pgt) Ug99 race group is virulent to most stem rust resistance genes currently deployed in wheat and poses a threat to global wheat production. The durum wheat (Triticum turgidum ssp. durum) gene Sr13 confers resistance to Ug99 and other virulent races, and is more effective at high temperatures. Using map-based cloning, we delimited a candidate region including two linked genes encoding coiled-coil nucleotide-binding leucine-rich repeat proteins designated CNL3 and CNL13. Three independent truncation mutations identified in each of these genes demonstrated that only CNL13 was required for Ug99 resistance. Transformation of an 8-kb genomic sequence including CNL13 into the susceptible wheat variety Fielder was sufficient to confer resistance to Ug99, confirming that CNL13 is Sr13CNL13 transcripts were slightly down-regulated 2-6 days after Pgt inoculation and were not affected by temperature. By contrast, six pathogenesis-related (PR) genes were up-regulated at high temperatures only when both Sr13 and Pgt were present, suggesting that they may contribute to the high temperature resistance mechanism. We identified three Sr13-resistant haplotypes, which were present in one-third of cultivated emmer and durum wheats but absent in most tested common wheats (Triticum aestivum). These results suggest that Sr13 can be used to improve Ug99 resistance in a large proportion of modern wheat cultivars. To accelerate its deployment, we developed a diagnostic marker for Sr13 The identification of Sr13 expands the number of Pgt-resistance genes that can be incorporated into multigene transgenic cassettes to control this devastating disease.

Keywords: CC-NBS-LRR; Ug99; durum wheat; resistance genes; stem rust.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genetic and physical maps of Sr13. (A) B. distachyon chromosome 3 region colinear with the wheat Sr13-candidate region. Rectangles in red indicate NLR genes (likely nonfunctional since they carry frame-shift mutations and premature stop codons). (B) High-density genetic map of Sr13 on chromosome arm 6AL. (C) Physical map of the Sr13 region constructed with overlapping BACs from the Sr13-resistant durum wheat variety Langdon. (D) Diagrammatic representation of the annotated sequence of the Sr13 region (GenBank accession no. KY924305). Genes are indicated by arrows (uppercase names indicate complete genes and lowercase names 5′ or 3′ truncated genes). Red lines below this graph indicate regions where no CS orthologs were found.
Fig. 2.
Fig. 2.
Sr13 mutants. (A) Schematic representation of NLR candidate genes CNL3 and CNL13. Dotted lines indicate introns, yellow rectangles coding exons, and gray rectangles 5′ and 3′ UTRs. The positions of the selected truncation mutations are indicated by blue arrows. Mutations in Kronos mutant lines T4-403, T4-1065, T4-3715, T4-771, and T4-3102 are expected to result in premature stop codons, whereas the mutation in mutant line T4-476 is expected to eliminate the donor splice site of the third intron. (B) CNL3 and CNL13 mutant lines inoculated with race TTKSK. Kronos is the Sr13-resistant control and Rusty is the susceptible control. CNL3 mutants are all resistant, whereas CNL13 mutants are all susceptible to TTKSK (Ug99). Numbers below the leaves are average pustule sizes (n = 3). Superscripts indicate significance of the differences with Kronos using Dunnett’s test (ns, not significantly larger than Kronos, *P < 0.05). Results for races TKTTF, JRCQC, and TRTTF are presented in SI Appendix, Fig. S1.
Fig. 3.
Fig. 3.
Sr13 transgenic plants. (A) Transcript levels of CNL13 in the susceptible control Fielder and in transgenic plants from four independent events (Fielder background). qRT-PCR primers amplify both the susceptible CNL13 allele in Fielder (S1) and the resistant allele (R3) in the transgenic plants. Transcript levels are expressed as fold-ACTIN using the 2−ΔCT method. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant (Dunnett’s test). (B) Semiquantitative PCR products from dCAPS marker Sr13F/R digested with restriction enzyme HhaI. The lower band corresponds to the transcript from the resistant allele (R = LDN) and the upper band to the transcript from the susceptible allele (S = Fielder). Six independent T1 plants from every transgenic event were evaluated. (C) Reaction to Pgt race TTKSK (Ug99) in Fielder and transgenic families T1Sr13-2 and T1Sr13-3. R, Resistant; S, Susceptible; −, no resistant CNL13 allele; +, resistant CNL13 allele present. Numbers below leaves are average pustule sizes (n = 8). Superscripts indicate significance of the differences between Fielder and each of the transgenic families using Dunnett’s test (***P < 0.0001). Results for the four transgenic families for races TTKSK, TKTTF, and TRTTF are presented in SI Appendix, Fig. S4.
Fig. 4.
Fig. 4.
Pathogen growth in different genotypes at different temperatures. Interaction graphs for pathogen growth at two temperatures (low = 18 °C day/15 °C night and high = 25 °C day/22 °C night) and in two genotypes (Sr13 gene present or absent). Resistant genotypes are indicated in orange (LMPG-Sr13, transgenic-Sr13, and Kronos) and susceptible genotypes in blue (LMPG, Fielder, and Kronos sr13-mutant). Pathogen growth was estimated using average sporulation area 13 dpi (A–C), ratio between fungal and host DNA 5 dpi (D–F), and average size of individual fungal infection areas estimated by fluorescence microscopy 5 dpi (G–I). All plants were inoculated with TTKSK. Statistical analyses (including replication numbers) are detailed in SI Appendix, Table S4.
Fig. 5.
Fig. 5.
Infection areas visualized by fluorescent staining. Visualization of TTKSK growth at different temperatures in resistant genotypes carrying Sr13 (LMPG-Sr13, transgenic-Sr13, and Kronos) and in susceptible genotypes without Sr13 (LMPG, Fielder, and Kronos sr13-mutant T4-476) 5 dpi. Infected leaves were cleared with KOH and stained with WGA-FITC.
Fig. 6.
Fig. 6.
Transcript levels of PR genes. Transcript levels of PR genes PR1, PR2, PR3, PR4, PR5, and PR9 are presented in interaction graphs including two genotypes (Kronos vs. sr13-mutant T4-476) and two inoculation treatments (TTKSK vs. mock). Values relative to ACTIN endogenous control calculated using the ΔCT method. Error bars indicate SEMs. All interactions between genotype and inoculation were highly significant (P < 0.001). n = 6. Samples were collected 5 dpi. Statistical analyses are presented in SI Appendix, Table S6.
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References

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