The Roles Of Two Receptor-like Cytoplasmic Kinase Genes OsRLK And OsNRRB During The Interactions Between Rice And The Pathogen Of Bacterial Leaf Streak And Drought

Rice is not only an important food crop in the world,but also a model plant of monocotyledonous investigation,its importance is self-evident.With the increase of population,cultivated land area per capita is less and less,food problem becomes more and more serious,at the same time,biotic and abiotic stress on food crops is becoming more and more serious.Biotic stress mainly refers to various diseases,rice bacterial leaf streak(BLS)is a major biotic constraint to rice production.Investigation of differentially expressed protein genes by analysis of the proteomes,one of them including the receptor-like cytoplasmic kinase genes OsRLK that are expressed differentially at transcription levels,was chosen in this study to further investigate its roles during rice-Xoc interactions.OsRLK encodes a putative receptor-like cytoplasmic kinase,and belongs to LRK10L-2 gene subfamily.The expression profile of OsRLK under adversity revealed that the gene could be induced by many stresses such as high salt,H202,ABA,SA and IAA.By analyzing cis-elements in the promoter region(1kb)upstream of OsRLK gene,we found that many pathogens responsive elements and putative stress response-related cis-elements were located in the promotor,implying that OsRLK may involve in responding to abiotic stresses.Sequence alignment showed OsRLK has high sequence homology with resistance gene Lr10.The OsRLK protein is localized in cytoplasm that confirmed by expression of OsRLK-GFP fusion protein in rice root cells.Most defense-related proteins have the same subcellular localization,implying OsRLK participate rice and Xoc interaction.The inoculation experiment shows that overexpression of OsRLK resulted in enhanced tolerance to Xoc.In order to study the genetic mechanisms of resistance to Xoc,we use quantitative RT-PCR analysising the expression of marker genes.The results showed that the expression of PR5and PAL1 were significantly induced;antioxidant-related genes Fe-SOD and SodCc2 also was induced;OsRLK also induce the expression of the transcription factor genes WRKY77and WRKY13.OsRLK-OE plants accumulated less H202,DAB staining also proved this point;Consistently,MDA content was reduced,compared with controls.In addition to,the activity of CAT,SOD and POD in wild-type and transgenic plants were determined.The results showed that over expression OsRLK significantly increased antioxidant enzyme activity,on the other hand,the activity of CAT,SOD and POD of OsRLK-RNAi transgenic plants was significantly lower than the wild type.In summary,the differentially expressed protein genes OsRLK play important roles during rice and Xoc interaction,they are involved in the regulation of rice and bacterial leaf streak resistance,Inducible expression of defense-related genes and regulating antioxidant system may one of the main mechanisms to improve the resistance of rice to Xoc.These would do us a favor for further understanding the complex defense network of rice under Xoc infection.Abiotic stress such as:drought,salt,low temperature and high temperature stress are adverse factors affecting seriously growth,development,and productivity of rice.Hence,it is of great theoretical and realistic significance to clone rice resistant genes and to make clear the resistant mechanism of such genes.And drought is a major stress that is commonly encountered by plants during their development Thus,it has an important significance to explore the mechanism of drought stress,to separate and clone drought resistant genes,and to improve the drought tolerance of rice.In recent years,it has become a hotspot in the modern rice breeding research.Based on the analysis of gene chips and bioinformatics,a receptor-like cytoplasmic kinase gene OsNRRB was isolated from rice,which contains a cytoplasmic protein kinase domain and a U-box domain,and is a member of RLCK-IXb gene subfamily.Quantitative RT-PCR(qRT-PCR)data showed that OsNRRB express in all tissues and organs of rice and the expression of OsNRRB gene exhibited significant tissue specificity in rice.The expression of OsNRRB was repressed by salt,drought,ABA,and H202 stresses,Lots of abiotic stresses responsive elements such as ABA,drought and cold responsive elements present in the 1.5kbp promoter region upstream of OsNRRB,implying that OsNRRB may involve in responding to abiotic stresses.There are also a lot of carbon metabolism-related elements,light responsive elements and tissue-specific regulatory elements,OsNRRB may also play other functions in rice.In order to make further research on the thorough function of OsNRRB,we constructed their overexpression,RNAi,subcellular localization and tissue localization expression vectors,and obtained transgenic plants by agrobacterium mediated genetic transformation methods.Select overexpressing and RNAi transgenic plants and expression profiling analysis using qRT-PCR,all of the examined stress-responsive genes,including ABA biosynthesis-related genes OsNCED4,regulatory genes OsbZIP23 and DREB2A and two functional genes OsLea3 andP5CS.P5CS,DREB2A,OsLea3 and OsbZIP23 were found to be upregulated in OsNRRB-overexpressing rice compared to corresponding wild type rice,On the contrary,the plants which inhibited the expression of OsNRRB gene changed little.The expression of OsNCED4 was significantly reduced.The research on drought resistance in the T1 generation of transgenic plants indicated that over expressing OsNRRB improves resistance of the transgenic plants to drought,and suppressing its expression enhanced susceptibility of the transgenic plants to drought,indicating that OsNRRB positively regulates resistance of rice to drought.Overall,OsNRRB positively regulated rice drought tolerances by downregulating the expression level of ABA biosynthesis-related genes OsNCED4,and upregulated stress-responsive genes,including regulatory genes OsbZIP23 and DREB2A and functional genes P5CS and OsLea3.