Studies On The Response Of Heat Shock Proteins And Heterotrimeric G Proteins In Rice (Oryza Sativa L.) Under Blast Stress

Rice blast is one of the most destructive disease of rice (Oryza sativa L.). It is caused by the fungal pathogen (Magnaporthe oryzae), and has become one of the major constraints to the stable crop production. Understanding how rice plants sense and conduct the pathogen attacking signal is fundmental for the development of new blast-resistant rice cultivars to overcome rice blast. The previous researc work indicated that Heat Shock Proteins (Hsps) and Heterotrimeric G protein (G protein) were the key proteins in the signal conducting process. However, to date, the role of HSPs and heterotrimeric G proteins in rice in response to rice blast is not fully determined.In this paper, rice (Oryza sativa cv Nipponbare) and fungal pathogen GUY11 were employed as materials, we found that rice Oryza sativa cv Nipponbare displayed resistant phenotype after inoculated with fungal pathogen GUY11. To investigate the underlying blast resistance mechanisms, bioinformatics tools were used. The Two-dimension etrophoresis (2-DE) and MALDI-TOF-TOF-MS were used to screen the differentially expressed proteins, while the microarray chips and qRT-PCR were aplied to identify the differential expressed genes in the rice leaves in response to blast stress. Afer that, the interacted proteins of Hsps were analyzed by 1D Native-PAGE,2D SDS-PAGE and Pull-down methods. The following results were reached in this paper.1. The cloned blast gene sequences were retrieved from NCB1 database, and searched against genomes of Nipponbare and CO39 to identify physical locations and SNPs by BLAST and BWA. Compared with GUY11 sensitive rice CO39, Pish, Pi-d3, Pi-56, Pi-21and Pi-37 resistance genes and associated SNPs were found only in rice Nipponbare.2. By 2-DE and MALDI-TOF-MS/MS methods, the differentially expressed proteins in rice in response to 2d,3d and 4d rice blast stresses were analyzed. Total 22 differential proteins were identified, of which 17 proteins were constitutively up-regulated and 5 proteins were constitutively down-regulated in rice under 2d,3d and 4d rice blast stresses. These proteins could be divided into 3 classes based on Gene Ontology (GO) terms, including resistance-related proteins, photosynthesis-related proteins and protein-synthetic proteins. Meanwhile,6 HSPs, including Hsp90, Hsp70, Cpn60, Hsp17.9A, Hsp17.4 and Hsp16.9A, were found to be up-regulated in rice under blast stress. The result illustrated that rice Hsps might play significant roles in rice leaves in response to rice blast.3. The microarray chip data (GSE9450) of rice in response to 48h GUY 11 stress was downloaded and analyzed by using GE02R online tool. The result showed that 4 differential genes were matched to 4 of the above 22 differential proteins. These genes showed higher mRNA abundance in blast resistant rice cultivar than in the susceptible ones. Two of them were Hspl7.9A and Cpn60 respectively, which indicated that the expression abundance of Hsps might be related to the different ability of rice cultivars to resist rice blast.4. The mRNA expression level of G protein subunits was detected by qRT-PCR. The results showed that the expression of G protein subunits was inhibited by rice blast fungi. However, some exception was founnd in the expresison change of G protein subunits in rice in response to different stage of rice blast stress. It turned out that rice plants might regulated the expression of G protein subunits to transduct the blast-stress signal under the inhibition of rice blast fungi.5. Three different protein complexes in the leaves of rice in response to 2d,3d and 4d blast stress were found by using Native-PAGE method, in which there were 4 energy metabolism related proteins and 2 stress related proteins. These interaction was confirmed by Gene Ontology. Besides, His Pull-down was used to screen interacting proteins of Hspl7.9A in rice in response to rice blast stress. The results showed that the up-regulation of Hsp17.9A transduct blast stress regulated signal to its downtrean interacting proteins, then regulated amino acid metabolism, carbon metabolism and immunity process in rice.In summary, the results in this paper were consistent with the finding in our previous research work, and provided a clue for the study on the function of Hsp17.9A in rice..