Cloning And Functional Analysis Of RxLR Effectors From Phytophthora Capsici

Pepper Phytophthora Blight(Phytophthora capsici Leonian)is an important soilborn diseases,and it can cause devastating disease on hosts.The symptoms on pepper are mainly root rot,blight,stem blight,fruit rot.Currently,the treatment of Phytophthora capsici mainly consists of chemical control,agricultural control in combination with other measures.The effectiveness of prevention is relatively low,and prevention work is also relatively slow,in suitable climate years,Phytophthora capsici can still happen in a large area.With the further study of oomycetes,it was found that RxLR effectors played a major role in the interaction process between plant and pathogenic oomycetes,therefore,studying RxLR effectors and host internal resistance mechanism become the current hotspot.As one of the important plant pathogenic oomycetes,Phytophthora capsici is appropriate for further study,while there are also a lot of RxLR effect factors in Phytophthora capsici,resently the research of RxLR mostly focus on Phytophthora infestans and P.sojae,RxLR effectors also show variety function,so we think it is necessary and meaningful to do capsici RxLR effectors research.We use advanced molecular biology techniques to analyse the function of RxLR effectors and expect to find the meaningful RxLR effectors for further studying,in order to provide theoretical and practical basis for the control of Phytophthora capsici.In this study,we use high-virulent Phytophthora capsici strain SD33 as experimental material witch was collected and preserved in our lab.According to the crystal structure study of RxLR in our lab we selected and cloned three RxLR effectors from Phytophthora capsici strain SD33,then we do the research of them as following:1.Tne bioinformatic analysis and cloned of RxLR genes from Phytophthora capsici strain SD33.According crystallography study of Phytophthora capsici in our laboratory,we selected three RxLR genes and succeed cloned them from Pepper Phytophthora genome.The three RxLR genes were RxLR129044,RxLR133393,RxLR116645.Use variety of bioinformatics software to do prediction for them.2.Testing RxLR129044,RxLR133393,RxLR116645 genes transient expression in N.benthamiana.We use Western blotting to check the expression of RxLR129044,RxLR133393,RxLR116645.The result showing that RxLR133393,RxLR116645 can express,RxLR129044 need to remove RxLR-dEER for normal expression.3.Subcellular localization of RxLR129044,RxLR133393,RxLR116645 genes.To further investigate RxLR129044,RxLR133393,RxLR116645 function,we first analyzed their subcellular localization.The study found that there was no clear position of RxLR129044 while RxLR133393 localized to the cell membrane and the nucleus and RxLR116645 localized to the cytoplasm.4.Functional study of RxLR129044,RxLR133393,RxLR116645.We analyzed pathogenicity,immunity and affection against Phytophthora capsici infection of the three genes.The results showed that RxLR116645 can cause cell death in N.benthamiana and pepper while RxLR129044,RxLR133393 can not.5.The localization nuclear signal and nuclear export signal to the C-terminal of RxLR116645 could change its localization and function.The RxLR116645 was added a localization nuclear signal and nuclear export signal,and its function was studied again.We found that the localization nuclear signal make function lost,nuclear export signal can enhance cell necrosis.Through this study,we can speculate that RxLR116645 plays a certain role in interaction of Phytophthora capsici and host plant.Agrobacterium-mediated transient expression technology was used successfully in this study.The results of the study also define our next research directions.In the future,we can reveal its pathogenesis by yeast two-hybrid technology finding interacting protein of RxLR116645.