| The tomato bacterial speak disease is the main disease which can affect the tomato's quantity and quality. Pseudomonas syringae pv. tomato(Pst)is the pathogen of bacterial speck disease in tomato and Arabidopsis. The interaction between Pst and tomato has been established as a typical model system of studying plant-pathogen interaction. There are two types of avirulence genes in Pst: AvrPto and AvrPtoB. Their encoded proteins can separately interact with the resistance protein Pto of tomato which is Ser-Thr kinase protein specificly. It matches with Flor's hypothesis"gene for gene". Lescpth5 is a member of Pto gene family, and is Pto orthology in resistance tomato lines, which can encode active kinase protein. The AvrPto and AvrPtoB elicit immunity-associated programmed cell death (PCD) when shows in tomato plants carring Pto. However, when it is in the lacks of Pto plants, they functions to suppress immunity in plant and promote bacterial growth.This ressearch clarified that transcription of Lescpth5 can identify by RT-PCR. It used the yeast two-hybrid system to analyse Lescpth5's interaction with AvrPto/AvrPtoB. It used AvrPto/AvrPtoB as bait to screen genes which can interact with AvrPto/AvrPtoB in Zhongshusihao cDNA library.The objective of the research was finding the targets of AvrPto/AvrPtoB as virlence effectors, and analysed the interaction between AvrPto/AvrPtoB and Lescpth5.PCR was used to amplify reverse-transcibed mRNA from tomato Zhongshusihao in order to identify transcribed Lescpth5. The amplification product was sequenced and we did bioinformatic analysis to this gene from both nucleotide level and amino acid level. This provided fundament to analysis of the interaction between Lescpth5 and AvrPto/AvrPtoB.AvrPto,AvrPtoB and Lescpth5 sequences were amplified with primers that designed according to the published AvrPto,AvrPtoB and Lescpth5 sequences and incorporated appropriate restriction enzyme sites. The amplifications were cloned into both bait vector(pGBKT7) and prey vector(pGADT7) separately. By the yeast two-hybrid system, cotransformed yeast strain AH109 with pGBKT7-AvrPto/ pGBKT7-AvrPtoB and pGADT7/Lescpth5. We found Lescpth5 can neither interact with AvrPto nor AvrPtoB .Therefore, it suggests that Lescpth5 is not the target of AvrPto/AvrPtoB, However, the function of Lescpth5 is not clear.We successfully established Zhongshusihao cDNA library in order to screen genes which AvrPto or AvrPtoB could interact with. Finally we selected 19 positive clones which could interact with AvrPto and classified them into 4 types. We also found 11 potive clones which can interact with AvrPtoB and classified them into 2 different types. However, we could not find any Pto gene family members. The proteins which interacted with AvrPto/AvrPtoB were mostly effectors participating in photosynthesis. These data indicate that Pto homologs is not the target of AvrPto/AvrPtoB.This paper was aim to find the targets of AvrPto and AvrPtoB in susceptible tomato cultivar zhongshusihao.It was proved that Lescpth5 can neither interact with AvrPto nor AvrPtoB. We also screened some positive clones which can interact with AvrPto or AvrPtoB and most of them were proteins which participate in host photosynthesis. We supposed that AvrPto and AvrPtoB ,as virlence factors ,interacted with proteins which participate in photosynthesis, can influence Calvin cycle or synthesis of chlorophyll and other proteins in chloroplast. Consequently, it led to the leaf cells death. These results can explain a little bit of why zhongshusihao is susceptible to Pst. It helped us to build up the fundamental theory of controlling the tomato bacterial speak disease. |
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