Sequence Analysis And Functional Verification Of RXLR Effector PITG₁6427.2 In Potato Late Blight

Potato is the fourth largest food crop in the world which plays an important role in food security.Potato late blight caused by Phytophthora infestans is an oomycete disease which is regarded as the largest crop disease in the world with threatened effect on the production of potatoes.During the infection process,a large number of RXLR effectors are secreted to inhibit and destroy the immune response of host plants,achieving successfully colonization and infection life cycles.Therefore,it is of great significance to identify the important RXLR effectors and explore the pathogenic mechanism for effective control of potato late blight.In this study,we have screened and identified an important effector PITG₁6427.2^HLJof Phytophthora infestans HLJ strain from construced library that contained 251 RXLR effectors.We obtained the following research results:?1?Using the method of transient expression,an effector PITG₁6427.2^HLJ was verified to inhibit INF1-induced HR in N.benthamiana.At the same time,this gene was also able to inhibit the HR response induced by Bax,PsojNIP,SFI2,Avh238 and Avh241,indicating that PITG₁6427.2^HLJ can broadly inhibit PTI and ETI of plant.?2?Taking PITG₁6427.2 in Phytophthora infestans strain T30-4 as a reference,PITG₁6427.2 was cloned in 17 different oomycete pathogen strains?Phytophthora infestans,Phytophthora sojae,Phytophthora capsici and Phytophthora parasitica?and they were performed in bioinformatics analysis.The results showed that there were 11 specific amino acid sequences in PITG₁6427.2 between different oomycetes,and the amino acid sequence identity was over 93%,indicating that PITG₁6427.2 is highly conserved and it may be important in pathogenic oomycetes factor.Inhibition of cell death assays showed that they all inhibited HR induced by INF1 and Avh241.And natural variation did not affect pathogenic differentiation.?3?Using the method of real-time PCR,it was proved that the effector PITG₁6427.2 can significantly up-regulate in the expression of the Phytophthora infestans HLJ strain infecting the potato variety Désirée in nutrition stage which is similar to most of the currently identified omycete pathogens,suggesting that PITG₁6427.2 may play an important role in the infection process.?4?Excessive expression of PITG₁6427.2^HLJ in N.benthamiana and susceptible potato Désirée can significantly improve the host susceptibility to Phytophthora infestans HLJ strain.This indicates that PITG₁6427.2^HLJ is susceptible factor.?5?DAB staining method was used to detect the accumulation of hydrogen peroxide in N.benthamiana after infected by Phytophthora infestans.The results showed that the accumulation of hydrogen peroxide in the leaves expressing PITG₁6427.2^HLJ was significantly lower than empty control.This suggests that PITG₁6427.2^HLJ may inhibit immunity of plant and enhance the susceptibility by inhibiting active oxygen bursts in the host.?6?Plant hormones play an important role in plant disease resistance and stress resistance.The expression of key genes in the synthetic pathways of SA and ET was detected by real-time PCR to preliminarily analyze the mechanism which PITG₁6427.2^HLJ could promote plant disease after infected by Phytophthora infects.After 48 hours of infected by Phytophthora infestans,SA signal marker gene PR1b was significantly down-regulated and ET signal marker gene EFR1 were significantly up-regulated.This suggests that PITG₁6427.2^HLJ may play a pathogenic role by inhibiting the SA signal pathway and enhancing the ET signal pathway during infection of plant with Phytophthora infestans.?7?It was observed by laser confocal microscopy that PITG₁6427.2^HLJ was localized in the membrane and nucleus of the plant.In summary,our study have analyzed and identified a key effector PITG₁6427.2 of Phytophthora infestans HLJ strain.We have explored the function of PITG₁6427.2 and initially analyzed pathogenicity in the process of infecting plants which provides clues for a further analysis of the role of effector molecules in the process of Phytophthora infestans infecting potatoes.