Functional Analysis Of Osmesl In Leaf Senescence And Disease Resistance In Rice

Premature leaf senescence at the late heading stage of indica hybrid rice varieties seriously restricts rice yield,and understanding the molecular mechanism of leaf senescence not only provides a theoretical basis for dissecting the problem of premature leaf senescence,but also provides candidate genes for creating premature senescence resistant varieties.Previous studies in our laboratory found that OsMESL(methylesteraselike)gene(i.e.,A12 gene)was involved in the rice leaf senescence process.In this study,a new gene OsMESL that negatively regulates leaf senescence after heading in rice was identified by molecular biology,physiology,and genetics using the T-DNA insertion mutant osmesl as the entry point on the basis of previous studies,and it was found during the study that the gene mutation showed significant resistance to bacterial diseases bacterial blight and fungal diseases sheath blight and blast.The main results in this study are as follow:1.We purchased the homozygous T-DNA insertion mutant osmesl of the OsMESL gene from the Kyung Hee University of Korean.After heading,the mutants showed a green-holding phenotype,with significantly higher chlorophyll content than ZH11 in wild-type(WT)control,significantly lower expression levels of positive senescence regulation related genes Osh36,Osl55 and OsWRKY42 than WT,and significantly higher expression levels of negative senescence regulation Marker gene OsDOS than WT.Chlorophyll degradation related genes OsNOL,OsNYC1,OsNYC4 and OsPAO were significantly lower than the control group.osmesl showed delayed senescence induced by Me JA,and also showed delayed senescence during dark treatment.Complementary plants can recover the delayed senescence phenotypes of osmesl,and RNAi plants delayed leaf senescence,and over-expression plants accelerated leaf senescence.2.Rice protoplast subcellular localization experiment found that OsMESL was localized in cell membrane and chloroplast,and an interacting protein OsBI-1 was screened by membrane system of yeast two-hybrid.Co-localization studies found that OsMESL and OsBI-1 could co-localize locally in the cell membrane.Further Bi FC and bimolecular luciferase activity recovery experiments confirmed that OsMESL could interact with OsBI-1 in vitro and in vivo.The expression level of OsBI-1 in osmesl was significantly increased.Compared with WT,the expression level of OsBI-1 was higher when osmesl was infected by pathogenic bacteria.Trypan blue staining results showed that the cell mortality in osmesl leaves was lower than WT under normal conditions,and the cell death induced by pathogenic bacteria was affected in osmesl.Transmission electron microscope results showed that autophagosome was less in osmesl.Hormone determination showed that IAA content in osmesl was significantly higher than WT after heading,but there was no significant difference in ABA content.Meanwhile,the expression levels of IAA synthesis related genes,OsYUCCA1,OsYUCCA2,OsYUCCA4 and OsYUCCA6,were increased,while the expression levels of genes related to IAA transport,OsSAUR39 and OsSAUR45,were decreased.The above results indicated that the senescence delay of osmesl leaves was involved in the cell death inhibitor OsBI-1 pathway and the IAA pathway.3.Inoculated with Xoo(Xanthomonas oryzae Pv.oryzae),R.solani(Rhizoctonia Solani)and M.oryzae(Magnaporthe Oryzae)respectively,osmesl showed significant resistance to these three diseases.The pathogen inoculation experiment was carried out on the complementary lines at booting stage,and the phenotypes was recovered.Meanwhile,both RNAi plants and OsMESL-CRISPR-Cas9 plants showed resistance to these pathogens.4.The membrane yeast two-hybrid system was used to screen a thioredoxin OsTrxm localized in the chloroplast.The interaction in yeast,luciferase activity recovery in rice protoplast,Bi FC and tobacco immunoprecipitation experiments all proved that the two can interact in vitro and in vivo.NBT staining showed that ROS content in the leaves of osmesl and RNAi plants was higher than WT after pathogen infecting.Quantitative H2O2 determination was measured on the leaves of WT,osmesl and RNAi plants before and after inoculation with pathogens,and the results showed that H2O2 content in osmesl and RNAi plants were significantly higher than WT.In osmesl,the expression level of OsWORKY45-1,a gene associated with negative regulation of disease resistance,was significantly lower than WT,while the expression level of OsPR1 b,a gene associated with positive regulation of disease resistance,was significantly higher than WT.In addition,the content of JA in osmesl,an important hormone in pathogen resistance pathway,was significantly higher than WT.OsTrxm overexpression plants showed no resistance to pathogens during booting stage,but OsTrxm gene knockout plants showed significant resistance to pathogens.These results suggest that OsMESL regulates ROS levels through the thioredoxin pathway,thus regulating resistance to pathogens.