Function Analysis Of The PlMPK14 Gene In Phytophthora Litchii

Many species of Phytophthora genus are very serious plant pathogenic bacteria which can harm the series of ormamental plants and crops.Phytophthora sojae infection which causes stem and rot rot is one of destructive disease of soybean production,while Phytophthora litchii(past for Peronohthora litehii)causes lots of litchi fruit drop,fruit rot,so Phytophthora litchii has become one of the most serious litchi diseases.Although P.sojae and P.litchii with other Phytophthora genus have similar mycelial morphology to fungus,and they are far from fungi in terms of evolutionary relationship.It has a unique set of characteristics of genome and regulation of development and pathogenic mechanisms.However,genome structure of Phytophthora is diploid and complex,its molecular genetic manipulation is more difficult.Studies on its development and molecular pathogenic mechanism lags behind,especially Phytophthora litchii,its molecular genetic system has not been established,yet its functional genes has not been identified.In the regulation of growth and pathogenic process of ftongus,mitogenic protein kinase(MAPK)pathway is a very conservative and important Pathway.More and more research and evidence shows that MPK signal pathway plays an important role in the process of zoospores' development and release,sexual reproduction and pathogenicity.In our prophase research,14 protein kinases were identified in P.sojae genome.And it was found that PsSAK1(PsMPKS)was involved in the regulation of zoospores development and pathogenic process.The silented mutations shows zoospores repose fast and lower percentage of cystcspore germination,germ tube becomes longer and pathogenicity reducing;protein kinase PsMPK1 participated in regulation morphogenesis and pathogenic process ofP.sojae;silencing PsMPK10 and PsMPK7 affected cystcspore germination,and affected the patihogenicity by regulating the ability of scavenging of ROS;besides,PsMPK7 also participated in the regulation of the strain stress tolerance.In this study,we identified PsMPK1 4 through the new genome analysis of P.sojae genome,and through the fungus relationship between one to one correspondence of orthologous genes,we identified P1MPK14 of PR litchii.And compared MPK14 biological functions in two kinds of Phytophthora by gene silencing method.Since there is no report about successful genetic technology in P.litchii such as gene knockout to study genes' biological function.This study would be the first success in stable expression GFP(green fluorescent protein)in P.litchii,which proved that exogenous genes stable expression and RNAi gene silencing both can be successfully conducted in P.litchii through CaC12-PEG mediated genetic transformation system,and our study could be the preliminary exploration and locading on gene function of P.litchii.Through CaCl2-PEG mediated genetic transformation,elective medium containing G418 and real-time quantitative PCR,we obtained 3 strains of PlMPK14-silented transformants which efficiency is around 50%as well as 3 strains of PsMPKI4-silented rtransformants.Then we verified the purpose genes are already successfully inserted into the genome of each silented transformant by extracted DNA and PCR amplification.And through the analysis of biological character of the mutanlts,we found that PlMPK14 involved in regulation of zoospores release and pathogenic process in P.litchii.PlMPK14-silented transfornats hade less zoospore production than the wild type and CK under the appropriate cold stimulation,the percentage of incomplete zoospore release is also higher,the abnormal sporangium released incompletely and some zoospores couldenft swim;besides that,PlMPK14-silernted transformats shew that ability to infect the host litchi leaf was decreased;while PsMPK14-silented transformats also existed the same phenotype.The results of the above illusstrate that the initial protein kinase MPK14 has conserved biological functions in this two kinds of Phytophthora,and MPK14 is involved in the regulation of zoospores release and pathogenic process.