High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis.

Wheat is one of the most important staple food crops. Leaf rust caused by Puccinia triticina Eriks. (Pt) is a destructive foliar disease that threatens world wheat production. Complementary wheat genes Necrosis 1 (Ne1) and Necrosis 2 (Ne2) on chromosome arms 5BL and 2BS, respectively, cause hybrid necrosis (Caldwell and Compton, 1943Caldwell R.M. Compton L.E. Complementary lethal genes in wheat.J. Hered. 1943; 34: 67-70Crossref Scopus (34) Google Scholar; Chu et al., 2006Chu C. Faris J. Friesen T. Xu S. Molecular mapping of hybrid necrosis genes Ne1 and Ne2 in hexaploid wheat using microsatellite markers.Theor. Appl. Genet. 2006; 112: 1374-1381Crossref PubMed Scopus (60) Google Scholar). Ne2 shows extremely tight genetic linkage with leaf rust resistance gene Lr13 (Zhang et al., 2016Zhang P. Hiebert C. McIntosh R. McCallum B. Thomas J. Hoxha S. Singh D. Bansal U. The relationship of leaf rust resistance gene Lr13 and hybrid necrosis gene Ne2m on wheat chromosome 2BS.Theor. Appl. Genet. 2016; 129: 485-493Crossref PubMed Scopus (26) Google Scholar). We previously identified a temperature-sensitive leaf rust resistance gene LrZH22 in Chinese wheat cultivar Zhoumai 22 (ZM, Figure 1A) that was effective against most Chinese Pt pathotypes (Wang et al., 2016Wang C. Yin G. Xia X. He Z. Zhang P. Yao Z. Qin J. Li Z. Liu D. Molecular mapping of a new temperature-sensitive gene LrZH22 for leaf rust resistance in Chinese wheat cultivar Zhoumai 22.Mol. Breed. 2016; 36: 18Crossref Scopus (19) Google Scholar) and shared an overlapping genetic interval with Lr13. In a separate study, a recessive early leaf senescence 1 (els1) gene (Figure 1B) shared an overlapping interval with LrZH22/Lr13/Ne2 (Chu et al., 2006Chu C. Faris J. Friesen T. Xu S. Molecular mapping of hybrid necrosis genes Ne1 and Ne2 in hexaploid wheat using microsatellite markers.Theor. Appl. Genet. 2006; 112: 1374-1381Crossref PubMed Scopus (60) Google Scholar; Wang et al., 2016Wang C. Yin G. Xia X. He Z. Zhang P. Yao Z. Qin J. Li Z. Liu D. Molecular mapping of a new temperature-sensitive gene LrZH22 for leaf rust resistance in Chinese wheat cultivar Zhoumai 22.Mol. Breed. 2016; 36: 18Crossref Scopus (19) Google Scholar; Zhang et al., 2016Zhang P. Hiebert C. McIntosh R. McCallum B. Thomas J. Hoxha S. Singh D. Bansal U. The relationship of leaf rust resistance gene Lr13 and hybrid necrosis gene Ne2m on wheat chromosome 2BS.Theor. Appl. Genet. 2016; 129: 485-493Crossref PubMed Scopus (26) Google Scholar; Li et al., 2018Li M. Li B. Guo G. Chen Y. Xie J. Lu P. Wu Q. Zhang D. Zhang H. Yang J. et al.Mapping a leaf senescence gene els1 by BSR-Seq in common wheat.Crop J. 2018; 6: 236-243Crossref Scopus (18) Google Scholar). However, the function and relationship of LrZH22/Lr13/Ne2/els1 remained unknown for decades. Here, we report the map-based cloning of LrZH22 and els1 and characterize their relationship with Lr13 and Ne2 in conjunction with a companion paper (Hewitt et al., 2021Hewitt T. Zhang J. Huang L. Upadhyaya N. Li J. Park R. Hoxha S. Luo M. McIntosh R. Lagudah E. et al.Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.05.010Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar) studying the same gene. Seedling leaf rust reactions of ZM, susceptible wheat cultivar Zhengzhou 5389 (ZZ), early leaf senescence line M114, and its normal senescent sib line W301 to Pt pathotype FHDS were investigated at 18°C, 22°C, and 25°C, respectively. In each situation, ZM and M114 were resistant with infection types (IT) 1–2, whereas ZZ and W301 were susceptible with IT 3–4 (Figure 1A). ZM and M114 were more resistant at 25°C than at 22°C and 18°C. There was significantly accumulated H2O2 and reduced mycelial growth in ZM and M114 seedlings compared with ZZ and W301 at 48 h post inoculation (hpi) (p < 0.01; Figure 1A and Supplemental Figure 1). To fine-map LrZH22, we used flanking markers Xbarc55 and Xgwm374 (Wang et al., 2016Wang C. Yin G. Xia X. He Z. Zhang P. Yao Z. Qin J. Li Z. Liu D. Molecular mapping of a new temperature-sensitive gene LrZH22 for leaf rust resistance in Chinese wheat cultivar Zhoumai 22.Mol. Breed. 2016; 36: 18Crossref Scopus (19) Google Scholar) to genotype an F2 population of 7213 plants from the cross ZM × ZZ. Six simple sequence repeat markers and four single-nucleotide polymorphism (SNP) markers (Supplemental Table 1) were developed and used to narrow down the LrZH22 locus to a 0.15 cM genetic interval between markers HBAU3 and HBAU46, corresponding to a 68.95 kb genomic sequence on Chinese Spring chromosome arm 2BS (IWGSC, 2018IWGSC, International Wheat Genome Sequencing ConsortiumShifting the limits in wheat research and breeding using a fully annotated reference genome.Science. 2018; 361: eaar7191Crossref PubMed Scopus (1386) Google Scholar). Three markers, HBAU5, HBAU12, and HBAU458, co-segregated with LrZH22 (Figure 1C). Two annotated genes, TraesCS2B02G182800 and TraesCS2B02G182900, which encoded a nucleotide-binding site and leucine-rich repeat protein (NLR) and a ribonuclease (RP), respectively, were identified (IWGSC, 2018IWGSC, International Wheat Genome Sequencing ConsortiumShifting the limits in wheat research and breeding using a fully annotated reference genome.Science. 2018; 361: eaar7191Crossref PubMed Scopus (1386) Google Scholar) (Figure 1C). Sequence comparison revealed a number of SNPs and insertions and deletions (InDels) between the contrasting alleles of TraesCS2B02G182800 (NLR), but not TraesCS2B02G182900, in ZM and ZZ (Supplemental Figure 2 and Supplemental Table 2). Thus the NLRZM was the most likely candidate gene for LrZH22. The cDNA of NLRZM were isolated from uninfected seedling leaves of ZM grown at 22°C in the greenhouse and three splicing isoforms—IF1, IF2, and IF3—were detected (Figure 1D). IF1 is the majority isoform and accounts for over 90% of the transcripts resulting from the splicing of four exons and three introns. Both IF1 and IF2 encode the complete NLR protein containing 1072 amino acids with typical Rx-CC-like, nucleotide-binding site, and leucine-rich repeat (LRR) domains (Figure 1D and 1F; Supplemental Figure 3), whereas IF3 encodes a truncated protein with 564 amino acids (Figure 1D). qRT–PCR analyses showed that the NLR alleles were expressed in seedling tissues of ZM and ZZ and upregulated following infection with Pt pathotype FHDS (Supplemental Figure 4). Eight leaf rust susceptible mutants (EM1 to EM8) were identified from an ethyl methanesulfonate (EMS)-treated ZM population (Figure 1E) and used in MutRenSeq analysis (Steuernagel et al., 2016Steuernagel B. Periyannan S. Hernández-Pinzón I. Witek K. Rouse M. Yu G. Hatta A. Ayliffe M. Bariana H. Jones J. et al.Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture.Nat. Biotechnol. 2016; 34: 652-655Crossref PubMed Scopus (213) Google Scholar). Compared with the ZM de novo assembly, six contigs showing SNPs were filtered out from five mutants. Priority was given to Contig_13 835 as it had a high score hit in the NLR (Supplemental Figure 5). The genomic DNAs and full-length cDNAs of the NLR were amplified from ZM and the eight susceptible mutants to allow validation of the SNPs. Sequence alignment revealed that one or two nonsynonymous SNPs were present in the coding region (CDS) of the NLR from six mutants (EM1 to EM6), resulting in missense or nonsense mutations, respectively (Figure 1F). The other two susceptible mutants (EM7 and EM8) had no SNP in the NLR compared with NLRZM, suggesting that mutations might have occurred in other regulators in the LrZH22-mediated defense pathway. Mutations in the NLR provided additional evidence that NLRZM is LrZH22. To determine whether NLRZM was sufficient to confer leaf rust resistance, we generated an overexpression construct pUbi:NLRZM and transformed it into susceptible hexaploid wheat cultivar Fielder by Agrobacterium-mediated transformation (Supplemental Figure 6). Seven positive transgenic events were identified from 12 regenerated transgenic plants. NLRZM-positive plants in T0 and seven T1 families were resistant to Pt pathotype FHDS (Figure 1G and Supplemental Table 3), which indicated that NLRZM conferred resistance to leaf rust pathogen. Wheat lines Manitou and RL4031 with leaf rust resistance gene Lr13 shared an identical NLRZM gDNA sequence, indicating that LrZH22 is Lr13. Seedling leaf rust tests of ZM, Manitou, RL4031, and ZZ to eight Pt pathotypes revealed that ZM was consistently more resistant than Manitou and RL4031 (Supplemental Table 4). Seedling responses of ZM, Manitou, and RL4031 to Pt pathotype FHDS at different temperatures indicated that ZM was moderately resistant, whereas Manitou and RL4301 were scored susceptible at 18°C (Figure 1H). However, at the higher temperatures of 22°C and 25°C, Manitou and RL4301 were moderately to highly resistant and ZM was consistently more resistant. This is quite similar to the temperature-responsive stripe rust resistance gene Yr36 that with shifting temperatures affects P. striiformis f. sp. tritici development (Bryant et al., 2014Bryant R.R.M. McGrann G.R.D. Mitchell A.R. Schoonbeek H.J. Boyd L.A. Uauy C. Dorling S. Rodout C.J. A change in temperature modulates defence to yellow (stripe) rust in wheat line UC1041 independently of resistance gene Yr36.BMC Plant Biol. 2014; 14: 10Crossref PubMed Scopus (22) Google Scholar). Although ZM additionally carries a 1BL.1RS translocation containing Lr26 and partial resistance gene Lr27/Sr2, we have no evidence that these genes affected the Lr13 response. In addition, the FHDS pathotype was virulent to Lr26. The distinctly lower response of ZM is also noted in the companion paper (Hewitt et al., 2021Hewitt T. Zhang J. Huang L. Upadhyaya N. Li J. Park R. Hoxha S. Luo M. McIntosh R. Lagudah E. et al.Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.05.010Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). These results indicated that LrZH22/Lr13 is a high-temperature leaf rust resistance gene likely influenced by genetic background. Another possibility is the presence of an enhancer in ZM. The els1 locus was independently fine-mapped in cross M114 (predicted genotype Ne1Ne1Ne2Ne2) × W301 (Ne1Ne1ne2ne2) using 266 recombinants between markers WGGB306 and WGGB307 identified from 3453 F2 plants evaluated for hybrid necrosis in the field (Figure 1B and 1C). els1 was narrowed down to a 0.07 cM genetic interval flanked by markers WGGB618 and WGGB476, and co-segregated with markers WGGB302 and WGGB466 (Figure 1C). The corresponding 1.7 Mb sequence in Chinese Spring chromosome arm 2BS contained 17 annotated genes, a region that overlapped with the interval containing LrZH22. RNA-sequencing results based on lines M114 and W301 indicated that nine of the 17 annotated genes were expressed in the mapped interval (Supplemental Table 5). No expression was observed for six of the eight non-expressed genes in the expVIP database (www.wheat-expression.com), the exceptions being TraesCS2B01G181600 and TraesCS2B01G181800 (Supplemental Figure 7). Genomic DNA sequence variations between lines M114 and W301 were identified in four of the nine expressed genes (Supplemental Tables 5 and 6). Based on the fact that NLR protein usually triggers R-gene-mediated pathogen recognition with programmed cell death (Jia et al., 2021Jia H.Y. Xue S.L. Lei L. Fan M. Peng S.X. Li T. Nagarajan R. Carver B. Ma Z.Q. Deng J.P. et al.A semi-dominant NLR allele causes whole-seedling necrosis in wheat.Plant Physiol. 2021; https://doi.org/10.1093/plphys/kiab058Crossref Scopus (2) Google Scholar), NLRM114 was proposed as the candidate gene for els1. Genomic DNA sequences of the NLR alleles were isolated from lines M114 and W301. Sequence alignment revealed that NLRM114 shared an identical sequence with NLRZM and varied from NLRZZ and NLRM301 by a number of SNPs and InDels (Supplemental Figure 2). Since the same NLR was the candidate for both LrZH22 and els1, homozygous NLRZM overexpression transgenic line OE-T1-1-1 was used for genetic analysis of LrZH22 and els1 by crossing lines M114 and W301 with Fielder and OE-T1-1-1, respectively. The Fielder × M114 and Fielder × W301 F1 hybrids were normal, and OE-T1-1-1 × M114 and OE-T1-1-1 × W301 F1 hybrids showed necrosis. When line M114 was separately crossed with ZM and leaf rust susceptible mutant EM6, respectively, EM6 × M114 F1 hybrids showed normal growth whereas ZM × M114 F1 plants exhibited hybrid necrosis (Figure 1I and Supplemental Table 7). These results indicated that the els1 encoded NLR is the Ne2 gene responsible for hybrid necrosis in wheat. The 34 CDS of NLR from a panel of wheat accessions and another 15 CDS of NLR in wheat accessions from the companion paper (Hewitt et al., 2021Hewitt T. Zhang J. Huang L. Upadhyaya N. Li J. Park R. Hoxha S. Luo M. McIntosh R. Lagudah E. et al.Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.05.010Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar) were selected to determine allelic variations of the NLR (Supplemental Table 8). Among the 49 accessions tested, 24 had identical NLRZM sequences, whereas the other 25 accessions had highly variable sequences (Figure 1J and Supplemental Figure 8). Sequence alignment of the NLR alleles revealed two key single-nucleotide variations (SNVs), T1308G and A1684C, between the leaf rust resistant allele (TA) and all the susceptible alleles (GC) (Figure 1J). However, no sequence variation was observed at the two SNVs among the EMS mutants (Figure 1F and 1J; Supplemental Figure 3). A cleaved amplified polymorphism sequences marker, HBAU-Lr13, was developed for LrZH22 based on SNV A1684C (Supplemental Figure 9) and tested on a panel of 390 diverse wheat accessions and a 262-member Mini-Core Collection. Twenty-three and eight accessions carried the LrZH22 allele, respectively (Supplemental Tables 9 and 10) and were resistant to Pt pathotype FHDS at the seedling stage (Supplemental Table 11). The mean adult plant rust responses of accessions with the LrZH22 were significantly lower than that of accessions without LrZH22 in the field trials at Baoding in Hebei province and Zhoukou in Henan province during the 2015/2016, 2016/2017, and 2017/2018 cropping seasons (p < 0.01, Supplemental Table 10). These results indicated that LrZH22 conferred effective leaf rust resistance at the adult plant stage. The cornerstone parental breeding line Zhou 8425B and several elite wheat cultivars widely grown in the Northern China Plain and the Yellow and Huai River Valley Winter Wheat Zone of China, such as Nongda 212, Yannong 15, Liangxing 99, and Zhoumai 22, carry LrZH22 and have made significant contributions to leaf rust control in China. In summary, we isolated the high-temperature leaf rust resistance gene LrZH22/Lr13 from the elite wheat cultivar Zhoumai 22 using map-based cloning, MutRenSeq, EMS mutagenesis, transgenic validation, and haplotype analysis. Independent cloning and genetic analysis revealed that els1/Ne2 is the same as LrZH22/Lr13. LrZH22/Lr13/els1/Ne2 encodes an NLR protein that triggers programmed cell death and exhibits pleiotropic effects on leaf rust resistance and hybrid necrosis. However, the presence of a second gene (Ne1) is necessary for expression of hybrid necrosis. This study was supported by the National Key Research and Development Program of China ( 2017YFD0300906-07 ) and National Natural Science Foundation of China ( 31361140367 and 31571662 ).