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Identification of miRNAs Contributing to the Broad-Spectrum and Durable Blast Resistance in the Yunnan Local Rice Germplasm

ABSTRACT: MicroRNAs are 20–24 nucleotide non-coding RNAs and play important roles in plant-environment interactions. In recent years, many microRNAs (miRNAs) have been found to regulate rice immunity against rice blast fungus. However, there are limited studies about miRNAs that directly target resistance (R) genes to regulate rice immunity. In this study, by deep sequencing, small RNA libraries were constructed from four-leaf stage seedlings of the resistant variety Ziyu44 and susceptible variety Jiangnanxiangnuo (JNXN) upon Magnaporthe oryzae infection, we found that much more miRNAs were significantly differentially expressed in Ziyu44 than in JNXN. Among these miRNAs, we focused on miR9664, a newly identified rice miRNA in our sequencing, which was upregulated lightly in Ziyu44 and drastically in JNXN at 24–48 h post-inoculation (hpi). The transgenic plants overexpressing miR9664 (miR9664-oe) displayed reduced defense responses to M. oryzae, while those knocking down miR9664 (miR9664-m) displayed enhanced defense responses to M. oryzae. Most of the detected miR9664 predicted target genes were reduced in the miR9664-oe lines while increased in the miR9664-m lines. The cleavage site of LOC_Os08g07774 was confirmed by RLM-RACE. Meanwhile, after being inoculated with M. oryzae, the genes were expressed differently between Ziyu44 and JNXN. The results suggest that miR9664-mediated R gene turnover contributes to Ziyu44 broad-spectrum resistance to rice blast fungus. Taken together, our research identified a new rice miRNA that directly targets R genes to regulate rice immunity against rice blast fungus, adding significant information to the study of rice–M. oryzae interaction.

SUBMITTER: Li J

PROVIDER: S-EPMC8551726 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rice blast, one of the most destructive epidemic diseases, annually causes severe losses in grain yield worldwide. To manage blast disease, breeding resistant varieties is considered a more economic and environment-friendly strategy than chemical control. For breeding new resistant varieties, natural germplasms with broad-spectrum resistance are valuable resistant donors, but the number is limited. Therefore, artificially induced mutants are an important resource for identifying new broad-spectrum resistant (R) genes/loci. To pursue this approach, we focused on a broad-spectrum blast resistant rice mutant line SA0169, which was previously selected from a sodium azide induced mutation pool of TNG67, an elite japonica variety. We found that SA0169 was completely resistant against the 187 recently collected blast isolates and displayed durable resistance for almost 20 years. Linkage mapping and QTL-seq analysis indicated that a 1.16-Mb region on chromosome 6 (Pi169-6(t)) and a 2.37-Mb region on chromosome 11 (Pi169-11(t)) conferred the blast resistance in SA0169. Sequence analysis and genomic editing study revealed 2 and 7 candidate R genes in Pi169-6(t) and Pi169-11(t), respectively. With the assistance of mapping results, six blast and bacterial blight double resistant lines, which carried Pi169-6(t) and/or Pi169-11(t), were established. The complementation of Pi169-6(t) and Pi169-11(t), like SA0169, showed complete resistance to all tested isolates, suggesting that the combined effects of these two genomic regions largely confer the broad-spectrum resistance of SA0169. The sodium azide induced mutant SA0169 showed broad-spectrum and durable blast resistance. The broad resistance spectrum of SA0169 is contributed by the combined effects of two R regions, Pi169-6(t) and Pi169-11(t). Our study increases the understanding of the genetic basis of the broad-spectrum blast resistance induced by sodium azide mutagenesis, and lays a foundation for breeding new rice varieties with durable resistance against the blast pathogen.

Project description:BackgroundThe rice Pi2/9 locus harbors multiple resistance (R) genes each controlling broad-spectrum resistance against diverse isolates of Magnaporthe oryzae, a fungal pathogen causing devastating blast disease to rice. Identification of more resistance germplasm containing novel R genes at or tightly linked to the Pi2/9 locus would promote breeding of resistance rice cultivars.ResultsIn this study, we aim to identify resistant germplasm containing novel R genes at or tightly linked to the Pi2/9 locus using a molecular marker, designated as Pi2/9-RH (Pi2/9 resistant haplotype), developed from the 5' portion of the Pi2 sequence which was conserved only in the rice lines containing functional Pi2/9 alleles. DNA analysis using Pi2/9-RH identified 24 positive lines in 55 shortlisted landraces which showed resistance to 4 rice blast isolates. Analysis of partial sequences of the full-length cDNAs of Pi2/9 homologues resulted in the clustering of these 24 lines into 5 haplotypes each containing different Pi2/9 homologues which were designated as Pi2/9-A5, -A15, -A42, -A53, and -A54. Interestingly, Pi2/9-A5 and Pi2/9-A54 are identical to Piz-t and Pi2, respectively. To validate the association of other three novel Pi2/9 homologues with the blast resistance, monogenic lines at BC3F3 generation were generated by marker assisted backcrossing (MABC). Resistance assessment of the derived monogenic lines in both the greenhouse and the field hotspot indicated that they all controlled broad-spectrum resistance against rice blast. Moreover, genetic analysis revealed that the blast resistance of these three monogenic lines was co-segregated with Pi2/9-RH, suggesting that the Pi2/9 locus or tightly linked loci could be responsible for the resistance.ConclusionThe newly developed marker Pi2/9-RH could be used as a potentially diagnostic marker for the quick identification of resistant donors containing functional Pi2/9 alleles or unknown linked R genes. The three new monogenic lines containing the Pi2/9 introgression segment could be used as valuable materials for disease assessment and resistance donors in breeding program.

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Identification of miRNAs Contributing to the Broad-Spectrum and Durable Blast Resistance in the Yunnan Local Rice Germplasm

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