Functional Analysis Of Polygalacturonases From Phytophthora Capsici And Effect Of N-Glycosylation On The Pathogenesis

Pepper blight is a worldwide disease, which badly impacts on agriculture of China andother countries and caused enormous loss on economy. The disease is caused byPhytophthora capsici. P. capsici is a soilborn oomycete and can cause stem rot, root rot andwilt of pepper. The host range of the pathogen is wide including the pepper, pumpkin,cucumber, tomato, and so on. Plant pathogens produce a diverse range of cell walldegradation enzymes during host-pathogen interactions. Polygalacturonase (EC3.2.1.15) isone of important pectic enzymes that can degrade the pectin and middle lamella of the plantcell walls to destroy the mechanism of host defense, which in favour of the infectionstructures of pathogenic fungi, various of pathogenic enzymes or pathogenic toxins infect thehost cells to induce pathogenesis. The gene family of P. capsici can produce a lot ofpolygalacturonases.N-linked glycans are attached to the side chain of Asn residues in Asn-X-Ser/Thr, whereX can not be Pro. The primary structures of polygalacturonases have various number ofpotential N-glycosylation sites. The N-glycosylation states of various polygalacturonases mayplay important roles in pathogenesis, as there is growing evidence that glycosylation can havedramatic effects on the structure and function of proteins, as well as on protein-proteininteractions. In the present work the N-glycosylation of fungi is little studied. N-linkedglycosylation does not occur at every potential site and the role played by glycosylation indifferent proteins is highly variable and depends on the individual protein, so there is veryimportant to analyze single N-glycosylation site function for the stability and function ofenzyme. As a gene family, polygalacturonases of P. capsici exist in multiple forms. In thestudy, various N-linked glycosylation structures are found in the polygalacturonase genesthrough the N-linked glycosylation site prediction by the software of NetNGlyc1.0Server.The number and location of N-linked glycosylation site, the evolution of N-glycosylation inthe gene family may directly or indirectly affect the activity or stability of enzymes, andinfluence the pathogenesis of polygalacturonases during P. capsici infecting host. The mainresearch as follow:As the target gene, Pcipg5was selected to analyze the relationship of the function andpathogenesis. Pcipg5was detected at transcription level using RT-PCR, Northern Blot andReal-time qRT-PCR during P. capsici infecting host. Pcipg5was expressed by Pichiapastoris and purified by Ni-affinity chromatography. Pcipg5was detected the destruction of the pepper leaves and the degradation of pepper cell-wall in the purified protein inoculatingpepper leaves. The results suggested that Pcipg5was a virulence factor during P. capsiciinfecting host.By inoculating the pepper leaves with zoospore suspensions, we analyzed thetranscription level of nine polygalacturonase genes of P. capsici through Real-time qRT-PCR.These polygalacturonase genes and N-glycosylation mutations were analyzed by transcientexpression. The results showed that Pcipg5, Pcipg2and Pcipg21were expressed in theprophase of plant infection; Pcipg16, Pcipg18, Pcipg13, Pcipg15and Pcipg20were expressedin the metaphase of plant infection; Pcipg11were expressed in the anaphase of plant infectionby Real-time qRT-PCR. The genes and N-glycosylation mutations over-expressed plantsshowed severe necrosis after virus infection.The phenotypes around the inoculated sitesinfection PVX/Pcipg15-N68, PVX/Pcipg2-NQ were more serious than those infected withPVX/Pcipg15and PVX/Pcipg2by PVX transcient expression. The results showed thatN-glycosylation sites may play important role in PG degrading pectin during P. capsiciinfecting hosts.N252was upregulation in PCIPG5by mutating N-glycosylation sites mutation, PVXtransient expression, eukaryotic expression, pH stability activity analysis, thermostabilityactivity analysis and so on. Single N34，N76，N137played a direct role in PCIPG2, but allthree N-glycosylation sites play downregulation role. The N-glycosylation site (N186, N322)was upregulation in PCIPG21, N374was downregulation in PCIPG21, and all these tenN-glycosylation sites play upregulation role. The studies displayed that variousN-glycosylation site had different functions in protein activity and pathogenesis.The work analyzed the functions of Pcipg5gene and the influence of N-glycosylationsites in P. capsici polygalacturonase genes. We can analyze the relationships ofN-glycosylation sites and pathogenesis by PVX agroinfection. Through eukaryotic expressionand activity analysis, we can analyze the relationship of the folding, secretion, activity andpathogenesis of expressed proteins and single N-glycosylation site.