The Subcellular Localization Of Cell Cycle Regulator Protein Pcsdal Gene And Functional Analysis Of RxLR Effector Genes From Phytophthora Capsici

Phytophthora capsici is a kind of destructive filamentous plant pathogen,its distribution is widespread,which can cause pepper blight disease in the world-wide,has important effects on the global agriculture and natural ecosystems(Fisher et al.,2012).Nowadays,it mainly use chemical agents to prevent Phytophthora capsici,which brought huge threat and harm to pepper production,health and environment safety of human life,whereas excessive heavy metals,pesticide residues and other problems have become increasingly prominent.Therefore,the use of molecular biology techniques to explore important pathogenic factor of phytophthora capsici and its pathogenic mechanism has increasingly become a hot research.Improvement of the host plant disease resistance is one of the most effective measures to control Phytophthora capsici.Phytophthora capsici infection process of host pathogen can secrete large amounts of Rx LR effector to promote disease resistance and destroy host plants.So,the study of Rx LR effector gene function and protein structure,to understand the pathogenic mechanism of phytophthora capsici and effective control of Phytophthora capsici is important.In this study,the highly pathogenic strain of Phytophthora capsici SD33 saved in our lab was used as experimental material,and the work we did for the Rx LR effector gene was as follows:1.we screened nucleotide and amino acid sequence about Rx LR effector molecule gene by using software from the biology of Phytophthora capsici JGI genome database and had an alignment by NCBI BLASTn and BLASTp to determine the correctness of selected genes.2.To extract gDNA from the highly pathogenic P.capsici SD33,then we cloned and analyzed the Rx LR effector gene,we named it as RxLR effector.3.Using bioinformatiics software to analyze the gene sequence.4.We found that the Pcsda1-GFP fusion protein located in the nucleus in capsici SD33,then we speculate that Pcsda1 affect normal growth and development of sporangia and mycelium.5.we examined the actin cytoskeleton in mycelia and sporangia of the Pcsda1-silenced,overexpression and control lines using phalloidin–rhodamine staining,and we found the actin plaques and actin filaments displayed irregularities in distribution and were brighter and more abundant in both the mycelia in silenced lines and overexpression lines,became elongated,clavate and intensely stained.Therefore,Pcsda1 appears to regulate the abundance,distribution and morphology of actin plaques and actin filaments,especially in mycelia.6.We study the Rx LR effector of Pepper Phytophthora by using p BIN-GFP2 expression vector for expressing four candidate effectors in N.benthamiana,and wefound that 129113 can induce cell death in benthamiana,129113,101860 can inhibit hypersensitive response of Bax,INF1,CRN4 induced.7.To further investigate the function of Rx LR,we got the subcellular localization of four Rx LR effectors,and we found GFP in the nucleus and cytoplasm.We merge the C-terminal of 129113 gene and nuclear export signal(NES),a nuclear localization signal(NLS1 and NLS2).Then we found NLS2,NES has changed the subcellular localization of 129113,but,NLS1 has not changed the subcellular localization of 129113.We change the location of 129113,and affect its function in the host plant cells.Then we speculate the function of 129113 may occur in the cytoplasm in a plant cell.Through this study,we can speculate four RxLR effector molecule in pepper Phytophthora infection process,enhance the sensitivity of plants or improve plant disease resistance,and lay the foundation for the study of Phytophthora capsici infection mechanisms.