Cloning And Functional Verification Of Rice Blast Resistance Gene Pb-jnw1 And Functional Analysis Of OsNPSN11 Protein

Rice blast,a severe and destructive disease in rice production,causes significant yield losses ranging from 10-30%annually.The breeding of new rice varieties with disease resistance remains the most economical,effective,and sustainable approach to combat this disease.Rice blast can manifest in all growth stages of rice,but panicle blast has a more pronounced impact on yield and quality.While over 30 rice blast resistance genes have been identified,only a few,such as Pb1,Pigm,Pi64,and Pi25 and their alleles Pid3 and Pid3-A4,confer panicle blast resistance.Therefore,it is crucial to search for more broad-spectrum panicle resistance genes and analyze their resistance mechanisms.This study cloned a gene Pb-jnw1 conferring both leaf blast and panicle blast resistance from a local japonica variety from Taihu region,Jiangnanwan,based on preliminary laboratory study,and preliminarily analyzed its functions.Furthermore,the molecular mechanism of an SNARE protein OsNPSN11 involved in the process of rice blast resistance was also preliminarily deciphered.The main research results are as follows:1.Cloning and functional verification of the panicle blast resistance gene Pb-jnw1from Jiangnanwan.（1）A residual heterozygous line（NIL18）containing the panicle blast resistance gene Pb-jnw1 was used to produce BC₂F₂ to BC₂F₄ populations through 1-3 generations of selfing.1150 individuals from the BC₂F₂ population,1456 individuals from the BC₂F₃population and 5769 individuals from the BC₂F₄ population were used to narrow down the Pb-jnw1 locus to a genomic region between the markers JS57 and JS4-5.Homozygous lines produced by self-crossing were inoculated with spore suspension at the booting stage to identify panicle blast resistance.The Pb-jnw1 locus was found in a 16 kb region that contained four predicted genes.After gene sequence alignment and domain analysis between Jiangnanwan and Suyunuo,two genes were identified as the most probable candidate genes.LOC＿Os11g45600 and LOC＿Os11g45620 encoded a LRR protein and a CC-NBS protein,respectively.（2）GWAS was performed based on the panicle blast phenotype of 232 materials with700K SNPs,including Jiangnanwan and Suyunuo.A 1.47 Mb panicle blast resistance repeat locus was then identified between SNP＿26984329 and SNP＿28459253 at the end of chromosome 11（PBRL10 and PBRL18）.Through LD heatmap,polymorphic SNP grouping,haplotype,expression,and functional domain analysis,LOC＿Os11g45600 was finally identified as a candidate gene.（3）Two candidate genes were cloned.After sequencing and comparison,it was found that LOC＿Os11g45600 was part of the coding region of LOC＿Os11g45620,which was named Pb-jnw1.Protein encoded by Pb-jnw1 contains a complete CC-NBS-LRR domain.The allele of Pb-jnw1^Su has a single base substitution,which results in premature termination and deletion of 526 amino acids（58.92 KDa）,leading to the destruction of the integrity of the NB-ARC domain.Complementation or overexpression of Pb-jnw1 could effectively reduce the percentage of diseased grains and lesions number,but it did not affect the average lesion length compared to the wild type（WT）.The expression of Pb-jnw1 was induced by the M.oryzae Hoku1,and endosperm blue staining directed by the GUS reporter gene was observed,indicating that Pb-jnw1 was highly expressed in grains at the booting stage.The results of evolutionary analysis showed that Pb-jnw1 has a very short genetic distance from the alleles in Oryza rufipogon.（4）Statistical analysis of agronomic characters of transgenic materials showed that knockout,complementation,and overexpression of Pb-jnw1 did not affect agronomic traits such as plant height,tillering,grain-setting rate,and 1000-grain weight.A molecular marker JS4-5 linked to Pb-jnw1 was developed and used to introduce the Pb-jnw1 gene into popular varieties in Jiangsu province,such as Ningdao 1,Nanjing 9108,Nanjing 44,Wuyunjing 7,and Zhendao 88,through marker-assisted selection（MAS）.These materials have been tested in both field disease nurseries and greenhouses with inoculation to confirm that Pb-jnw1 confers high resistance to both leaf and panicle blast,providing a useful resource for MAS breeding of rice blast-resistant varieties.2.The mechanism of OsNPSN11 involved in rice blast resistance has been elucidated.（1）The protein encoded by OsNPSN11 contains a Qb-SNARE domain and a C-terminal transmembrane domain.Based on the publicly available microarray database,OsNPSN11 has a high expression level during the full growth period of rice.Expression analysis of OsNPSN11 after inoculation with M.oryzae Hoku1 showed that OsNPSN11was significantly induced in the rice blast-resistant material Heikezijing,reaching a peak at48 hpi,while the expression level in the susceptible material Suyunuo decreased until 24hpi.（2）Identification of rice blast resistance of overexpressing transgenic lines（S1-2 and S11-4）and interfering suppressor-expressing transgenic lines（A11-49 and A11-72）.The results showed that the average number of lesions in S11-2 and S11-4 were 11.4±2.3 and16.5±3.1,respectively,which were significantly lower than that of the WT,while the average number of lesions in A11-49 and A11-72 was 60.3±7.5 and 58.9±6.8,respectively,which were significantly higher than those in the overexpression materials and WT.Compared with WT,there was no significant difference in the lesion length between the overexpressing transgenic lines and the interfering suppressor-expressing transgenic lines.（3）The subcellular localization showed that OsNPSN11 was localized on the cytoplasmic membrane.Comparing the proteomics and microarray of S11 with WT,the differential expressed proteins were mainly involved in nitrogen metabolism,primary metabolism,chlorophyll synthesis,organic matter transport,ion migration and catalytic activity.Up-regulated genes were mainly involved in plant abiotic stress,hydrogen peroxide decomposition,hormone response,detoxification,antioxidant,plant-pathogen interaction and the MAPK signaling pathway,while down-regulated genes were mainly involved in nitrogen metabolism,autophagy and diterpenoid biosynthesis.Further research found that the content of H₂O₂ near the infection point of interfering suppressor expressing transgenic lines was lower than that of WT.Determination of the total H₂O₂ content in plant tissues showed that the H₂O₂ content in WT and A11-49 increased significantly at 12hpi,but decreased significantly in the two overexpressing transgenic lines.It was shown that OsNPSN11 promoted the accumulation of ROS near the infection site.（4）In order to study the relationship between OsNPSN11 and ROS bursts,the related genes involved in the regulation of ROS bursts were analyzed.Through a LUC transient expression system,it was verified that OsNPSN11 interacted with receptor-like cytoplasmic kinases Os RLCK118,Os RLCK185,and chitin elicitor-binding protein Os CEBi P to participate in ROS regulation.q PCR analysis showed that inhibiting the expression of OsNPSN11 promoted the expression of Os RLCK118,Os RLCK185,Osrboh B,Os CEBi P,and Os CERK1,while overexpression of OsNPSN11 had no significant effect on these genes.In conclusion,when rice was infected with M.oryzae,OsNPSN11 promoted the accumulation of H₂O₂ near the infection point to inhibit the further invasion of M.oryzae.Meanwhile,it activated peroxidase family genes and pathogenesis-related genes,while suppressing the expression of ROS burst-related genes.