Cloning And Quantitative RT-PCR Analysis Of Pathogenicity Genes And MAPK Pathway Genes Of Oidium Heveae

Powdery mildew in rubber tree, caused by obligate parasitic fungus Oidium heveae B A. Steinmann, greatly reduced rubber production while serious harm. However,the study of Oidium heveae focused on observation to pathogen biology and cell biology, very little research have been done on O. heveae about the molecule, especially gene function on regulate development of infecting structure. For exploring the molecular regulated mechanism and gene functions in infection of Oidium heveae, we conducted transcriptome sequencing of rubber powdery mildew interactions during infection, find out the genes of pathogenicity-related and MAPK pathway. Induced Oidium heveae B.A.Steinmann HO-73strain infecting structure development by substratum and analyzed genes expression of pathogenicity-related and MAPK pathway by qRT-PCR, preliminary analyzed of the regulation of pathogenicity-related and MAPK pathway genes for O.heveae infecting structures.The main results as follows:1Germination and appressorium formation of12d post inoculation fresh conidium was better than the30d,and leaf age7to10d rubber leaves suitable conidium in detail. Total RNA was extracted from Oidium heveae at four infection stages,0,1,3and30dpi. Based on the genome of Oidium heveae, we used GO to predict the functions of O. heveae genes related to pathogenicity, and have characterized11pathogenicity-related genes. These11genes were cloned and analyzed by qRT-PCR. The results indicated that, at1dpi, appressoria formation, Oh-Imp-. Oh-hypothetical protein-4were up-regulated. They may be relevant with the growth of infect organ at the early infection stage. At3dpi, the stage of haustoria develop, metabolism-related Oh-PC2、 Oh-G protein beta subunit/AC1. Oh-EKA were up-regulated, growth gene related Oh-AAA peroxin, Oh-RNP and Oh-Imp were up-regulated, the hypothetical protein-1,2,3were up-regulated. They may be be relevant with haustoria development. At30dpi, the Oh-PC2, Oh-RNP, Oh-hypothetical protein-1,2,3were up-regulated. They may be relevant with conidiospores production.2Identification of MAPK cascade pathway from wide genome, and analysis with using blast, simple modular architecture research tool and phylogenetic in Oidium heveae will be useful for further research on the functions of MAPK cascade pathway in plant pathogenic fungus. Based on the conserved amino acid sequences of MAPK,30genes were identified from Oidium heveae database which homologous to three MAPK cascade signaling pathway genes from Blumeria graminis by Blast. One MAP K genes, one MAPKK genes and three MAPKKK genes in O.heveae genome were found.Summarized the process of infecting structure development of0-24hpi under parafilm surface as4morphological developmental stage, such as no germinated (Ohpi), germination (4hpi), appressorium formation (6,8hpi) and secondary appressorium (16,24hpi). We analysis the expression of the MAPK pathway under parafilm surface. When the germ tube developed at4hpi, all the genes were up-regulated.20892、18727、8311and4961were high fold expression at this time, indicated the Fus3/Kss1MAPK pathway had important function. When appressorium formation at8hpi, all the genes were up-regulated.1505.4241、4865、5368、18727、20892were high fold expression at this time, indicated the MEK1、Bkk1、Bck1and Wis4had important function. When the haustoria developed at16hpi,4961was down-regulated, other genes were up-regulated.1505、4241、9052、20892were high fold expression at this time, indicated the Hogl MAPK pathway had important function. When hyphae developed at24hpi, all the genes were up-regulated.4241、4961、8311、18727、9052and20892were high fold expression at this time, indicated the Hogl MAPK pathway had important function. Exogenous U0126treatment suggested that O.heveae could formed Appressorium in the non-induced agar surface. Added at a concentration of10μmol/L-30μmol/L concentration gradient U0126to1.0%agar culture medium. U0126with increased concentration of the inhibitor, spore germination rate decreased significantly, and there was a significant difference compared with control with no addition of inhibitor(P<0.05). At24hpi time,10μmol/L final germination rate of57%,20μmol/L final germination rate was57.33%,30μmol/L final germination rate was33.33%, lower than the control, the final germination rate of control is83.33%.