The Transcriptome Analysis Of Rice Treated With Protein Elicitor MoHrip2 And Preliminary Identification Of The Interacting Proteins In Rice

MoHrip2 is a kind of protein elcitor isolated from fermentation broth of Magnaporthe oryzae.The protein is composed of 152 amino acids and its coding gene reading frame contains 459 bases(GenBank serial number: JQ815555.1).Studies have shown that MoHrip2 can cause hypersensitivity reactions in tobacco leaves and promote the deposition of phenolic compounds and the production of superoxide compounds in tobacco cells.In order to reveal the immune pathway of rice in response to MoHrip2,we perform RNA-seq and interaction experimets techniques.The results are as follows:(1)Eukaryotic Expression and Purification of MoHrip2The eukaryotic expression vector PICZ?A-MoHrip2 was successfully constructed and MoHrip2 protein is correctly expressed proved by SDS-PAGE and Western-blot,the activity detection result shows the protein's activity is good.(2)Transcriptome sequencing and analysisThe transcriptome was sequenced by spraying 30?M MoHrip2 solution on rice which has 3-4 leaves,and samples were taken at 0h,24 h,and 48 h.Genes with different expression is screened at the time of 24 h and 48 h.GO and KEGG analysis show that DEGs mainly involve in biological metabolism,secondary product metabolism process,and enriched in the pathway of isoflavonoid biosysthesis.(3)Transcriptome data validationQuantitative validation showed that genes associated with isoflavone biosynthesis such as IFR,IFS were significantly up-regulated at 48 h.Meanwhile,salicylic acid biosynthesis related genes PAL and salicylate transduction related genes PR-1a,PR-10,and PR5 were all up-regulated significantly.We compared the content of salicylic acid between the control group and treated group,the result show that SA content in the treated group is upper than the control group,indicating that MoHrip2 can induce rice salicylic acid gene expression and salicylic acid synthesis.It is speculated that MoHrip2 may be induced first by inducing the expression of salicylic acid-related genes in rice.After synthesizing salicylate hormone in rice,it induces the expression of disease-resistant transcription factors and specific genes,and the key enzyme in the pathway of salicylic acid synthesis,PAL,may participates in the isoflavone biosynthesis pathway.(4)Validation of interaction between MoHrip2 and rice OsOlp in vivoBioinformatics analysis revealed that OsOlp shares high homology with Mo2BP1,and OsOlp has a transmembrane domain,indicating that there may exist an interaction between OsOlp and MoHrip2.The results of yeast two-hybrid experiment showed that only the yeasts on DDO plates containing AD-OsOlp and BD-MoHrip2 grew,indicating that there exists the interaction between MoHrip2 and OsOlp.The resuts make the foundation for the further research of rice immune mechanism in response to MoHrip2.