Phylogenetic And Necrosis-inducing Functional Analysis Of NPP1-like And PcF/SCR-like Genes In Phytophthora Infestans

Late blight caused by Phytophthora infestans was the most serious disease inpotato production. By secreting an arsenal of effector molecules into the apoplast andcytoplasm of host cells, pathogen may suppress or accelerate the death of plantcells.With the development of bioinformatics, which can provides opportunities for thesystem evolution and functional analysis of P. infestans genome. In this study, weconducted a phylogenetic and necrosis-inducing functional analysis of NPP1-like andPcF/SCR-like genes to provide theoretical basis for high mutability and pathogenesis ofP. infestans. The main results in this study were as follows:1. Most genes in NPP1-like and PcF/SCR-like family existed as cluster.Phylogenetic trees were generated by neighbor-joining, as implemented in Mega5.0.The result showed that131NPP1sequences from different sources can be clustered into6categories. Most NPP1genes from oomycetes and fungi were classified into differentclades, while some genes are intertwined in two groups. This result implied that theevolution of oomycetes and fungi was independent, but they may have a convergentevolution trend. Forty-one genes in oomycetes PcF-like and SCR74family can beclassified into2categories. Genes from P. infestans SCR74family and onePcF/SCR-like gene were clustered into one group; the remaining PcF-like genes fromdifferent sources of oomycetes were clustered into another clade. Although genesequence from SCR74family and PcF-like family are very similar, they belong todifferent evolutionary types.2. The genes from NPP1-like and PcF/SCR-like family showed different ability forinducing plant necrosis. The full-length cDNA of genes were obtained by RT-PCR andfunctional analysis was conducted through heterologous expression in Nicotianabenthamiana by agro-infiltration method. Six genes choosed from NPP1-like familyshowed two different symptoms. Gene PiNLP17, PiNLP29and PiNLP36showedserious collapse and necrosis symptoms, but gene PiNLP24, PiNLP39and PiNLP35only induced chlorosis in N. benthamiana. Genes in PcF/SCR-like family also showeddifferent symptoms after agro-infiltration depending on the number of SS-bridge. It was found that PcF/SCR.6, PcF/SCR.10and PcF/SCR.1, each containing3conservedSS-bridges, were biologically active, while the other gene PcF/SCR.8has no conservedSS-bridge, showed the weakest necrosis activity and only induced etiolation of N.benthamiana leaves.3. Site-directed mutagenesis of PiNLP17was carried out by over-lap PCR.Recombinant plasmids of mutants were constructed and expressed in N. benthamiana todetermine the roles of mutated sites. The result showed that3amino acid mutants ofD9A, E22A and D20A only caused slightly yellow symptoms in N. benthamiana leaves,which suggested that D9, E22and D20amino site may play an important role innecrosis-inducing function of PiNLP17. While mutant of H17A site amino acid did notaffect the function of PiNLP17, and led to the same serious collapse symptom as wildtype after7days post inoculation (dpi). It proved that the active site plays a veryimportant role in the necrosis-inducing function of NPP1-like protein.4. In PcF/SCR-like family, the necrosis-inducing function was related with thenumber of disulfide bonds. To clarify the function of disulfide bonds, site directedmutagenesis was used to destroy each disulfide bridges of PcF/SCR.1by replaced fourcysteines with alanine at amino acid sites at99,100,104and110. The resultingplasmids were then heterologously expressed in N. benthamiana. The agro-infiltrationassay results showed that a cysteine point mutation at aa110, or double mutations at aa99and aa110had no effects on the necrosis-inducing function of PcF/SCR.1. However,three cysteine mutations (C99/100/110) or four (C99/100/104/110) of PcF/SCR.1onlycaused slightly yellow symptoms in leaves in contrast to that of PcF/SCR.1.5. Genes from NPP1-like family and PcF/SCR-like family have differentexpression pattern in P. infestans infection potato. The relative expression level of genePiNLP17, PiNLP29, PiNLP35and PcF/SCR.1was up-regulated stably at vegetative andearly infection stage, and the expression peak appeared at a certain stage of lateinfection (IF48and IF60), especially gene PiNLP17. The expression of PiNLP36andPcF/SCR.8in vegetative and the infection stage was up-regulated, but no particularexpression peak appeared. PiNLP24, the up-regulated expression of small amplitude,and the expression peak appeared at zoospore stage. The different expression patternindicated that genes in NPP1-like and PcF/SCR-like family may have different functionin plant-pathogens interaction. The expression level of genes was also related to thenecrosis-inducing ability. The expression quantity of genes (PiNLP24、PiNLP35andPcF/SCR.8) without necrosis ability was lower than that functional genes.