Cloning Of Phytophthora Capsici RxLR Effector And Functional Study Of RxLR 23 Homologues

Phytophthora capsici,causing great damage to vegetable production all over the world every year,is a widespread soil-borne soil disease.The earliest record about pepper blight was Leonian(1892),who discovered this plant disease in Mexico and identified the pathogen as P.apsici.Presently,chemical pesticides is mainly used to control P.apsici.At the same time,it also brings a series of problems such as numerous pesticide residues and plant pesticide resistance improvement.On the other hand,due to the strong variation of P.apsici,it's getting harder to develop resistant varieties.On the whole,the prevention and control of hot pepper disease is a huge problem in China and even in the world.It needs further research.P.apsici is an important pathogen in plant pathogenic oomycetes,and RxLR effector proteins are presumed to contributed to virulence.During infection,a lot of RxLR effectors are secreted to destroy host immune response.Therefore,studying the function and structure of the RxLR effector of P.apsici is of great importance to understand the pathogenic mechanism and control the development of the disease.This study was based on the highly pathogenic P.apsici strain SD33,which was collected,isolated and preserved in the laboratory.Associated with the former researches,we conducted the following studies to P.apsici RxLR effectors.1.According to the JGI Phytophthora genome database,the RxLR genes of P.apsici were screened,cloned and analyzed by bioinformatics.Aligned by NCBI,supported with gene function to confirm the RxLR effectors.2.The DNA library of P.apsici SD33(preserved in our laboratory)was constructed,and the effectors were cloned for bioinformatics prediction and analysis.The P.apsici strains from Fujian and Jiangxi province were aligned with the RxLR23 screened from SD33.RxLR3-1,RxLR6-2,and RxLR8-2 were selected as homologous genes for this study.3.The effectors RxLR84,RxLR23,RxLR3-1,RxLR6-2,and RxLR8-2 were fused to the pBIN-GFP2 expression vector.These genes were transferred to Agrobacterium tumefaciens and infected different hosts,such as pepper and Nicotiana benthamiana to observe phenotype.Western blot and bimolecular fluorescent confocal were used to detected the expression level.RxLR84?RxLR23 were located at both nuclear and cytoplasm,while RxLR3-1?RxLR6-2and RxLR8-2 just located at the cytoplasm..4.The effect factors RxLR23,RxLR3-1,and RxLR6-2 were inoculated with N.benthamiana,and the P.capsici zoospores were inoculated 24 h later.The results were observed after 3days.RxLR23 and RxLR6-2 can promote the infection of P.capsici zoospores but inhibited by RxLR3-1.5.To further investigate the function of RxLR23,NES and NLS were fused to the c-terminal of RxLR23,the detection methods were as same as the former.The results suggested that RxLR23 NLS could trigger cell death,but RxLR23 NES rarely could.The infection analysis of P.capsici zoospores revealed that this phenomenon was inhibited by RxLR23 NLS,but promoted by RxLR23 NES.Through the above studies,we found that the function of RxLR effectors are vary from one to another.Some of the effectors play a role in inhibiting cell death meditated by INF1 or Bax.The homologous genes are polymorphic to some amino acids and function differently,nuclear export signal will alert the function of RxLR23,such as inhibit the of infection triggered by P.capsici SD33.After a series of in-depth discussions,we have found that RxLR23 is a toxic effector that can cause plant cell death(PCD).Protein interaction is a common pathogenic mechanism of effectors,so,only by screening out the corresponding interaction proteins and research the intrinsic relationship can we explain the pathogenic mechanism of RxLR effector more complete.