Prediction,Identification And Application Trial Of Phytophthora Capsici Effectors With Host Nucleus Localization

Crop blight caused by pathogenic Phytophthora spp.is one class of devastating diseases in agricultural production,which seriously threatens the global ecological,food security and causes huge losses to the global economy.Due to the great genetic difference between Phytophthora spp.and plant pathogenic fungi,there is no efficient and effective agrochemical control method for crop Phytophthora blight management.With the development of transgenic breeding technology in recent years,breeding transgenic resistant varieties can provide an environmentally friendly and effective strategy for the management of crop Phytophthora blight.Nucleomodulins are one class of very important pathogenicity-related proteins in plant bacterial pathogens.They can enter the nuclei of host cells and regulate the expression of plant defense-related genes.At present,little is known about these proteins in oomycetes.In this dissertation,the following research was carried out in P.capsici:(1)In order to explore the presence of this group of effector proteins in P.capsici,the proteins with DNA binding domains and signal peptides(SPs)were predicted by bioinformatics tools.The results showed that 48 effector proteins contain SPs and DNA binding domains.(2)In order to verify whether the predicted effector proteins play a role in plant infection of P.capsici,the transcription levels of these 48 genes in different growth and infection stages of P.capsici were analyzed by quantitative fluorescence RT-PCR.The results showed that most of the genes(70.83%)were up-regulated in the infection stages,indicating that these effector proteins responded to P.capsici infection.(3)In order to further clarify the roles of these effector proteins in the infection process,two genes encoding the effector proteins PcSEP20 and PcSEP43 were cloned and further studied:(a)the SPs of PcSEP20 and PcSEP43 were found functional by yeast sucrase secretion experiments;(b)using laser scanning confocal microscope,it was found that PcSEP20 and PcSEP43 were localized in the host nuclei;(c)Agrobacterium-mediated transient expression analysis showed that the expression of PcSEP20 could promote the infection of P.capsici on Nicotiana benthamiana while the expression of PcSEP43 inhibited the infection of P.capsici.(4)The transgenic plants of PcSEP43 were made by Agrobacterium-mediated transformation and inoculated.It was found that the PcSep43-transgenic N.benthamiana showed very strong resistance against the infection of P.capsici,which confirmed that PcSEP43 could induce plant immune responses and therefore indicated that PcSep43 is a good candidate gene for breeding cultivars resistant against Phytophthora blight.In this study,potential nucleomodulins were predicted from P.capsici.Their gene expression levels were analyzed during the development and infection stages of P.capsici.The roles of PcSEP20 and PcSEP43 in the infection were elucidated.The results not only provided preliminary experimental data for understanding how Phytophthora pathogens modulate the gene expression of host plants,but also provided important gene source for breeding resistant crop varieties.