Screening,Identification And Functional Analysis Of Effector Interaction Proteins In Colletotrichum Fructicola

In recent years,with Camellia oleifera industry fast development,Camellia diseases is also increasingly serious.Colletotrichum fructicola,a one of the most harmful diseases of Camellia oleifera,has seriously affected the healthy development of Camellia oleifera industry.Therefore,it is urgent to understand the interaction mechanism between oil tea anthrax and oil tea,and "suit the remedy to the case" to accurately control oil tea anthrax.The effector protein is a small molecule protein secreted into the host cell or outside the host cell by changing the structure or function of the host cell,thereby promoting the infection or colonization of pathogenic bacteria.In recent years,a large number of effector proteins have been identified and cloned to explore the interaction mechanism between pathogens and plant hosts.Through previous research,our research group found that C.fruticola was the dominant strain of oil tea anthracnose;Through the analysis and prediction of 7890 differentially expressed protein sequences in the transcriptome data of Camellia oleifera,51 effector proteins meeting the requirements were obtained,and six of them were verified to have the function of causing programmed cell death of plant cells,among which the effectors CfEP4 and CfEP 15 were the most prominent.In this study,the cDNA library of camellia oleifera infected by C.fruticola was constructed,and the functional sites of effector proteins CfEP4 and CfEP15 in host cells were determined by GFP fusion expression vector method.The yeast two-hybrid system bait vectors pGBKT7-CfEP4 and pGBKT7-CfEP15 were constructed for yeast selfactivation detection.The proteins interacting with the effector of C.fruticola in camellia oleifera were screened by yeast two-hybrid technique,and the conserved domain and subcellular localization of the proteins were predicted and analyzed.Finally,the expression levels of the proteins interacting with C.fruticola in different stages of infection were analyzed.The purpose is to lay a theoretical foundation for further clarifying the internal mechanism of C.fruticola infection of Camellia oleifera,the precise control of Anthracnose of Camellia oleifera and the breeding of high-yield and disease resistant Camellia oleifera varieties.The main resμlts of this experiment are as follows:1、In this experiment,the cDNA library of oil tea leaves infected by C.fruticola was constructed by gateway technology for the first time.The capacity of the primary library reached 1.44×107cfu/mL,much higher than the capacity of the standard library;The gene recombination rate of 24 clones randomly selected was 100%,and the inserted fragments were equal to or greater than 1KB,indicating that the long fragment genes accounted for a large proportion in the library,with high fragment effectiveness and high integrity of genetic information.It is a cDNA library that can fμlly reflect the genetic information of Camellia oleifera in the infected state.Secondly,based on the primary cDNA library,we connected different vectors and transformed them into a secondary nuclear membrane system,to build a yeast cDNA library for subsequent mining of the interaction proteins in response to Camellia oleifera.2、The action sites of effectors CfEP 4 and CfEP 15 in plant host cells were defined.Subcellular localization experiment showed that cfep4 was expressed on cell membrane and nucleus after entering plant host cells;CfEP 15 only acts on the nucleus.The bait carrier proteins BD-CfEP 4 and BD-CfEP15 were constructed respectively.The yeast selfactivation test showed that there was no self-activation of CfEP 15;CfEP 4 had a slight selfactivation reaction.When the 3-AT(3-Amino-1,2,4-triazole)self-activation inhibitor in the medium was at 5,the self-activation reaction of CfEP4 was inhibited.3、Using yeast two hybrid technology,ten proteins interacting with effector CfEP4 in camellia oleifera,and identified as Endochitinase,Mannitol dehydrogenase,MTD,Metallothionein(MTs),Sulfite Oxidase,β-Cyanoalanine synthase(β-CAS),1-minocyclopropane-1-carboxyl oxidase(ACO),Myo-inositol oxygenase,which is involved in plant cell wall synthesis(MIOX),THAumatin-like protein 1,ap-1 complex subunit(AP-1);Six proteins interacting with effector CfEP 15,of which two were unknown proteins with unclear functions,and the remaining four were uncharacterized DDB proteins,Mannitol dehydrogenase,MTD),L-asparaginase and 1-aminocy clopropane-1-carboxyl oxidase(ACO).4、The expression levels of 16 interacting proteins in Camellia oleifera leaves infected by C.fruticola(0h、24h、48h、72h)were analyzed.It was found that the expression levels of sulfite oxidase interacting with cfep4 and isoasparagine peptidase interacting with cfep1 5 were significantly lower than those in normal leaves;The expression of other interacting proteins after infection was significantly higher than that of normal leaves,but the up regulation time was different,indicating that they played a role in different time periods of infection.