Study And Analysis Of The Function Of Pi-PIPK-D8 In Phytophthora Infestans

Phytophthora infestans is a widely distributed and destructive pathogen of potato. The sexual reproduction is very important for the survival and evolution of the species, but the molecular mechanism of this process has not been elucidated. In Phytophthora, a new family of G protein coupled receptors (GPCR) is found, in which an N-terminal 7-TM domain is combined with a C-terminal phosphatidylinositol phosphate kinase (PIPK) domain. This family is named GPCR-PIPKs (abbreviated as GKs). In this study, the function of a member of GKs, P1-PIPK-D8 gene, was studied.The strain HQK8-3 of P. infestans was uesd as the research material. The genetic transformation system was constructed by a green flouresent protein expression vector. The expression of the gene Pi-PIPK-D8 at the different stages was determined by real time RT-PCR. The coding region of P1-PIPK-D8 cDNA was cloned and used to construct a fluorescent protein fusion expression vector. The subcellular localization of P1-PIPK-D8 was detected. The RNA interference vector pSTORA (A1935) of the gene Pi-PIPK-D8 was transformed into the strain HQK8-3 of P. infestans by the electro-transformation method. The phenotypes of the interferent strains were studied to elucidate the function of the gene.The results showed that the straightforward electroporation method was feasible for P. infestans and the transformation effciency of the electro-tansformation was approximatly 2 times higher than that of the protoplast transformation, and the straightforward electro-transformation was easier and more rapid than the protoplast transformation. The coding region of P1-PIPK-D8 cDNA of the strain HQK8-3 was 3045 bp long, coding a protein containing 1015 amino acids, which is the same as the sequence published on NCBI web. There were 9 different bases between the cDN A reported here with the cDNA published on NCBI web and 3 different amino acids between their coding proteins. The expression level of the gene Pi-PIPK-D8 was the highest in the cysts and decreased gradually in the germinating cysts, sporangia, zoospores, and growing mycelia of P. infestans. The Pi-PIPK-D8 protein may locate in the granular structure of mycelium. After Pi-PIPK-D8 gene silencing, the growth of hypha became thin and the number of sporangia and zoospores was decreased. The number of oospores was also decreased when the interferent strains were cultured with the the opposite mating type strain. The results indicated that the gene Pi-PIPK-D8 may be involved in the growth, sporangium production, and sexual reproduction of P. infestans.This study reveals that the Pi-PIPK-D8 of P. infestans may be involved in the growth and development of P. infestans, which is of great significance to discover the molecular mechanisms of some important biological phenomena and prevent and treat the diseases caused by this pathogen in the future.