Screening Of Target Proteins In Grape That Interact With Effector LtCre1 In Lasiodiplodia Theobromae

Grape（Vitis vinifera L.）is an economic fruit with extremely high nutritional value,but it is easy to cause diseases because of infected by pathgens.Plant diseases seriously affect the quality and yield of grapes.Botryosphaeria dieback is one of the important dieases caused by botryosphaericeae pathogens,moreover Lasiodiplodia theobromae is one of the major pathogens causing this disease happened.When pathogens infect host plants,they release a set of virulence determinants are regarded as “effectors” that act as inhibition of plant defense responses and promoted pathogen colonization in plants.In order to reveal the pathogenic mechanism of L.theobromae on the host and lay the foundation for disease prevention and cultivate of grape disease-resistant varieties.But now there are few reports on the research of L.theobromae effector.In this study,we research the LtCre1 effector that plays a pathogenic role during the infection of L.theobromae screening proteins interacting with grape and also analysis of the unique effector LT₃97 of L.theobromae transcription level under adversity treatment.The main findings are as follows:（1）Using yeast two-hybrid technology to pre-screen effector LtCre1 from L.theobromae interact with grape cDNA library.It found that more than 210 candidate proteins interact with LtCre1 full-length effector and LtCre1-N terminal effector.After that,extraction and transformation of candidate proteins yeast plasmid DNA.Sequencing the monoclonal bacterial liquid and using NCBI（http://www.ncbi.nlm.nih.gov）and The Grape Genomes database（http://www.genoscope.cns.fr/blat-server/cgi-bin / vitis / webBlat）analysis of candiate genes name and functional annotation.Eighty candidate genes were selected and verified by yeast self-activation and candidate proteins interact with effector.It was obsvered that a total of 10 candidate proteins interact with LtCre1 full-length effector and LtCre1-N terminal effector.（2）Full-length sequence clones of candidate genes were constructed on the pGAD-T7 vector and yeast two-hybrid of technology was used to demonstrated that a total of 3 candidate proteins interacted with LtCre1 full-length effector and LtCre1-N terminal effector.The three candidate proteins were named as VvCIP2,VvCIP4 and VvCIP5.（3）It demonstrate that the candidate protein VvCIP2 and VvCIP4 interact with effector LtCre1 in tobacco expression system by using bimolecular fluorescence complementary technology.（4）To construct prokaryotic expression vectors of effector LtCre1 and candidate protein,recombinant plasmids were transformed into E.coli BL21 and target proteins were induced and expression with IPTG.This experimental results showed that target proteins were expressed in the form of soluble protein.In study,laid the foundation for the next step to use GST-pull down technology to verify the interaction mechanism between candidate proteins and effector LtCre1 in vitro.（5）The transient expression of Nicotiana benthamiana indicated that the effector LT₃97 could inhibit the hypersensitive response（HR）induced by Burkholderia glumae.Therefore,this effector inhibits the plant defense response.Under adversity treatment,the results of qPCR showed that the transcription level of LT₃97 gene was increased in nutritional stress,oxidative stress,high temperature stress and UV treatment.