Function Of Loricrin-like Protein Gene PiLLP And Analysis Of Cytochrome P450 In Oomycetes

Phytophthora infestans（Mont.）de Bary is the causal agent of potato late blight,which is a devastating disease worldwide.Sexual reproduction of Phytophthora plays an important role in disease cycle and pathogen genetic variation.Based on data of differential expressions of genes in sexual reproduction obtained by Prakob,we investigated the function of PiLLP,a significantly up-regulated gene in sexual reproduction using gene silencing and overexpression technique.Moreover,on the basis of chemical structure of hormone and catalytic reaction mechanism of CYP450,we analyzed the sequence and expression levels of CYP450 genes that may involve in hormone synthesis in nine oomycete species including P.infestans.Loricrin-like protein is a glycine rich protein.The sequence and phylogenetic analysis of PiLLP showed that LLP is highly conserved in oomycetes and close to Class V GRPs（Glycine Rich Proteins）in plants.PiLLP-silenced transformants mated with A2 mating-type isolate produced aborted oospores.The result of subcellular localization showed that PiLLP was mainly distributed in oogonial walls,indicating that LLP is a structural component in the ooginial wall.Compared with the wild type,PiLLP-silenced transformants had slower colony expansion rates,produced less sporangia with lower germination and zoospore-release rates,had severely diminished virulence levels and catalase activities.PiLLP-silenced transformants were more sensitive to hydrogen peroxide;virulence could be partially restored with DPI treatments.The results showed that PiLLP play important roles in asexual,sexual reproduction and plant infection;PiLLP affected the pathogenicity of isolates through affecting the catalase activity.We aslo analyzed CYP450 genes amounts,classes and secondary structures in oomycetes through bioinformatics approaches and found that CYP450 genes in oomycetes were classified into 4 families CYP5014,CYP5015,CYP5016,CYP5017,28 subfamilies,and CYP5015 is common.More CYP450 genes were found in the facultative parasitic oomycetes than in the obligate parasitic ones.P.infestans includes 20 CYP450 genes,which were classified into 3 families and 14 subfamilies.Analysis of CYP450 in P.infestans showed that CYP450 genes in the same superfamily had similar SRS sequences,indicating that CYP450 genes in the same superfamily involve in catalyzing the same kind substrate.We detected the expression levels of CYP450 genes in P.infestans in the presence of phytol,found that three candidate CYP450 genes（PITG₁6499、PITG₀6554 and PITG₁3865）were significantly up regulated in sexual reproduction,suggesting that these CYP450 genes may participating in hormone formation of P.infestans.Our study unveiled that PiLLP play an important role in sexual reproduction of P.infestans and plant infection.Sequence analysis and expression level of CYP450 genes in oomycetes provides useful inputs to help explore hormone synthesis genes.Results of this study provide a foundation for future investigation on the mechanism of sexual reproduction in oomycetes.