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Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence.

ABSTRACT: Tetep is a rice cultivar known for broad-spectrum resistance to blast, a devastating fungal disease. The molecular basis for its broad-spectrum resistance is still poorly understood. Is it because Tetep has many more NLR genes than other cultivars? Or does Tetep possess multiple major NLR genes that can individually confer broad-spectrum resistance to blast? Moreover, are there many interacting NLR pairs in the Tetep genome? We sequenced its genome, obtained a high-quality assembly, and annotated 455 nucleotide-binding site leucine-rich repeat (NLR) genes. We cloned and tested 219 NLR genes as transgenes in 2 susceptible cultivars using 5 to 12 diversified pathogen strains; in many cases, fewer than 12 strains were successfully cultured for testing. Ninety cloned NLRs showed resistance to 1 or more pathogen strains and each strain was recognized by multiple NLRs. However, few NLRs showed resistance to >6 strains, so multiple NLRs are apparently required for Tetep's broad-spectrum resistance to blast. This was further supported by the pedigree analyses, which suggested a correlation between resistance and the number of Tetep-derived NLRs. In developing a method to identify NLR pairs each of which functions as a unit, we found that >20% of the NLRs in the Tetep and 3 other rice genomes are paired. Finally, we designed an extensive set of molecular markers for rapidly introducing clustered and paired NLRs in the Tetep genome for breeding new resistant cultivars. This study increased our understanding of the genetic basis of broad-spectrum blast resistance in rice.

SUBMITTER: Wang L

PROVIDER: S-EPMC6744916 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence.

Wang Long L Zhao Lina L Zhang Xiaohui X Zhang Qijun Q Jia Yanxiao Y Wang Guan G Li Simin S Tian Dacheng D Li Wen-Hsiung WH Yang Sihai S

Proceedings of the National Academy of Sciences of the United States of America 20190826 37

Tetep is a rice cultivar known for broad-spectrum resistance to blast, a devastating fungal disease. The molecular basis for its broad-spectrum resistance is still poorly understood. Is it because Tetep has many more NLR genes than other cultivars? Or does Tetep possess multiple major NLR genes that can individually confer broad-spectrum resistance to blast? Moreover, are there many interacting NLR pairs in the Tetep genome? We sequenced its genome, obtained a high-quality a ...[more]

PMID: 31451649

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Project description:BackgroundR gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure.ResultsTo identify novel factors required for R gene-mediated resistance in rice, we used rice insertional mutant lines, induced by the endogenous retrotransposon Tos17, in a genetic screening involving the rice blast fungus Magnaporthe oryzae. We inoculated 41,119 mutant lines with the fungus using a high throughput procedure, and identified 86 mutant lines with diminished resistance. A genome analysis revealed that 72 of the 86 lines contained mutations in a gene encoding a nucleotide binding site (NBS) and leucine rich repeat (LRR) domain-containing (NBS-LRR) protein. A genetic complementation analysis and a pathogenesis assay demonstrated that this NBS-LRR gene encodes Pish, which confers resistance against races of M. oryzae containing avrPish. The other 14 lines have intact copies of the Pish gene, suggesting that they may contain mutations in the signaling components downstream of Pish. The genome analysis indicated that Pish and its neighboring three NBS-LRR genes are high similar to one another and are tandemly located. An in silico analysis of a Tos17 flanking sequence database revealed that this region is a "hot spot" for insertion. Intriguingly, the insertion sites are not distributed evenly among these four NBS-LRR genes, despite their similarity at the sequence and expression levels.ConclusionsIn this work we isolated the R gene Pish, and identified several other mutants involved in the signal transduction required for Pish-mediated resistance. These results indicate that our genetic approach is efficient and useful for unveiling novel aspects of defense signaling in rice. Furthermore, our data provide experimental evidence that R gene clusters have the potential to be highly preferred targets for transposable element insertions in plant genomes. Based on this finding, a possible mechanism underlying the high variability of R genes is discussed.

Project description:BackgroundAs rice (Oryza sativa) is the staple food of more than half the world's population, rice production contributes greatly to global food security. Rice blast caused by the fungus Magnaporthe oryzae (M. oryzae) is a devastating disease that affects rice yields and grain quality, resulting in substantial economic losses annually. Because the fungus evolves rapidly, the resistance conferred by most the single blast-resistance genes is broken after a few years of intensive agricultural use. Therefore, effective resistance breeding in rice requires continual enrichment of the reservoir of resistance genes, alleles, or QTLs. Seed banks represent a rich source of genetic diversity; however, they have not been extensively used to identify novel genes and alleles.ResultsWe carried out a large-scale screen for novel blast-resistance alleles in 1883 rice varieties from major rice-producing areas across China. Of these, 361 varieties showed at least moderate resistance to natural infection by rice blast at rice blast nurseries in Enshi and Yichang, Hubei Province. We used sequence-based allele mining to amplify and sequence the allelic variants of the major rice blast-resistance genes at the Pi2/Pi9 locus of chromosome 6 from the 361 blast-resistant varieties, and the full-length coding region of this gene could be amplified from 107 varieties. Thirteen novel Pi9 alleles (named Pi9-Type1 to Pi9-Type13) were identified in these 107 varieties based on comparison to the Pi9 referenced sequence. Based on the sequencing results, the Pi2/Pi9 locus of the 107 varieties was divided into 15 genotypes (including three different genotypes of Pi9-Type5). Fifteen varieties, each representing one genotype, were evaluated for resistance to 34 M. oryzae isolates. The alleles from seven varieties with the highest resistance and widest resistance spectra were selected for transformation into the susceptible variety J23B to construct near-isogenic lines (NILs). These NILs showed resistance in a field test in Enshi and Yichang, indicating that the seven novel rice blast-resistance tandem-repeat regions at the Pi2/Pi9 locus of chromosome 6 could potentially serve as a genetic resource for molecular breeding of resistance to rice blast.ConclusionsThe thirteen novel Pi9 alleles identified in this study expand the list of available of blast-resistance alleles. Seven tandem-repeat regions of the Pi2/Pi9 locus from different donors were characterized as broad-spectrum rice blast-resistance fragments; these donors enrich the genetic resources available for rice blast-resistance breeding programs.

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Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence.

Publications

Large-scale identification and functional analysis of <i>NLR</i> genes in blast resistance in the Tetep rice genome sequence.

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