Sequence Analysis And Functional Verification Of RXLR Effector PITG₂1645.2

Potato（Solanum tuberosum）,an annual herb,belongs to Solanaceae.Phytophthora infestans [Phytophthora infestans（Mont.）de Bary] is a kind of filamentous oomycete of plant,which belongs to semi-biotrophic type.It can cause potato late blight,the most devastating disease of potato.In the long process of evolution,the mutation rates of the P.infestans isolates is fast,and the diversity of isolates is great.By studying the polymorphism analysis and the functional verification of the sequence of PITG₂1645.2 homologous genes from different oomycetes,we hope to provide clues in exploring the role of RXLR effectors in P.infestans’ s pathogenicity.Transient expressing the elicitor of P.Infestans--INF1,can trigger the programmed cell death in Nicotiana benthamiana.PITG₂1645.2,an effector of P.infestans isolates Heilongjiang（HLJ）,could inhibit the programmed cell death induced by INF1 in N.benthamiana,with the polymorphism in the amino acid sequence between isolates Heilongjiang（HLJ）and Pattern isolates T30-4.To identify that it is one of the important effectors,the experimental was basised on the Pattern isolates T30-4 to design primers.The homologous genes of PITG₂1645.2 were cloned from 10 isolates of P.infestans and three other oomycetes species including Phytophthora capsici,Phytophthora sojae and Phytophthora parasitica.The sequences of DNA were translated into amino acid sequences by using ExPASy-Translate tool（http://web.expasy.org/translate/）.And the comparative of amino acid sequences were analysised by Clustal W（http://www.genome.jp/tools/clustalw/）.The analysis of sequence showed that,they shared 93% to 100% identity with each other for the amino acid sequence.After co-infiltration with INF1,only PITG₂1645.2MP903,which contains the Serine differentiated to Asparagine of those homologues at the position of 31,couldn’t suppressed the PCD triggered by INF1.And the site directed mutagenesis of the 31 th amino acid of PITG₂1645.2MP903 from Serine to Asparagine could restored the inhibition function.These implied that the polymorphic amino acid at position 31 is the key site to suppress the INF1-triggered PCD.Meanwhile,we identified that PITG₂1645.2HLJ but not PITG₂1645.2MP903 could also suppress the PCD triggered by BAX,or other two secreted peptides PsojNIP and Avh238 of P.sojae,in N.Benthamiana,further proving that PITG₂1645.2 can inhibit plant’s defense system from multiple pathway.Similarly,the site directed mutagenesis of the 31 th amino acid of PITG₂1645.2MP903 from Serine to Asparagine could also restored the inhibition function,which further verifies that the polymorphic amino acid at position 31 is the key site to inhibitory function.Moreover,transient expression of PITG₂1645.2HLJ could also enhance the susceptibility to P.infestans in N.Benthamiana by half-leaf method.This is indicated that PITG₂1645.2plays important roles during the infection process for P.infestans.In summary,by using the method of homologous comparison to analyse the polymorphism of homologous sequence of PITG₂1645.2,analysing the key sites,exploring the role of PITG₂1645.2 in inhibiting plant’s PTI and ETI,we hope to provide the foundation for further research in exploring the function of RXLR effector.