Screeningand Functional Studies Of CFEM EffectorInteraction Proteins In Colletotrichum Fructicola Camellia Oleifera

The common in several fungal extracellular membrane(CFEM)domain is a fungus-specific domain that typically encodes 60 amino acids containing 8 cysteine(aa)protein domains that are characterized by distinguishing it from known cysteine-rich domains.Relevant studies have shown that CFEM protein plays an important role in the formation and development of attached cells,maintaining cell wall integrity and tolerance,maintaining the stability of free iron in cells,signaling between cells,forming biofilms,inducing plant cell necrosis and pathogenicity.Our research group screened and predicted 7 CFEM effectors of Colletotrichum fructicola,and found that 3 CFEM effectors could simultaneously inhibit the necrosis of Benthamhama leaves caused by BAX and INF1,and promote the infection of Phytophthora capsici,and it was preliminarily judged that these three effector proteins could affect the immune response of plants.On this basis,the known effector proteins were screened by yeast two-hybrid technology,which provided a basic basis for studying the role of these host proteins in the pathogenesis of anthrax bacteria,and further elucidating the mechanism of inhibition of the immune molecules of Camellia oleifera by anthrax bacteria,laying a theoretical foundation for the proposed targeted prevention and control scheme of Camellia oleifera anthracnose and the optimal breeding of Camellia oleifera varieties.The main findings are as follows:1.Bait carrier self-activation and toxicity detection:In this study,BD-CFEM6/CFEM19/CFEM21 three yeast two-hybrid bait proteins were constructed on the basis of the existing laboratory,and self-activation and toxicity detection were carried out,and the results showed that BD-CFEM6/CFEM19 could grow normally on the two-deficient medium,but could not grow on the third-deficient medium,indicating that there was no autoactivation reaction between the two.However,BD-CFEF21 can grow normally on the three-deficiency medium,indicating that it can spontaneously activate the transcription of downstream reporter genes,and it needs to be added with inhibitors,which are inhibited when the working solution concentration of 3-AT inhibitor is 5 mM on the three-deficiency medium.Moreover,on SD/-Trp medium,the number of yeast transferred by the three was roughly the same as that of BD empty carrier,indicating that the bait carrier was not toxic to yeast,and the two results showed that the effectors CFEM6,CFEM 19 and CFEM21 could be subjected to yeast two-hybrid screening tests.2.Screening and verification of interactive proteins:Using yeast two-hybrid technology,the interaction proteins of CFEM6,CFEM19 and CFEM20 were screened,and a total of 65 candidate proteins were obtained by BLAST analysis.Fourteen target proteins related to plant immunity were selected from 65 positive clones for yeast two-hybrid one-to-one verification of the interaction relationship between CFEM6,CFEM19,CFEM21 and candidate proteins.The yeast one-to-one verification results showed that after the BD-CFEM6/CFEM19/CFEM21 were co-converted into yeast cells with 14 proteins,7 of them could have blue yeast colony growth on the defective medium(containing X-α-Gal),and continued to use bimolecular fluorescence complementary technology(BIFC)to continue the next step of verification for the candidate proteins,and it was found that only three proteins emitted yellow fluorescence signals.P1 in CFEM19 and N4 and N5 in CFEM21,respectively,indicating that these three proteins can interact with CFEM effectors and interact on the cell membrane.3.Functional analysis of interacting proteins:Using bioinformatics analysis software,after NCBI carried out Blast comparative identification,it was found that its functions were annotated,including chitinase related to plant disease resistance,defense response and growth and development,and NAC domain proteins that participate in plant immunity and play an important role in regulating plant abiotic stress responses.The subcellular localization analysis of three interacting proteins showed that they were all located on the cell membrane,and the BIFC results showed that CFEM and P1,N4 and N5 interacted with each other in the cell membrane,and it was speculated that when effectors invaded plant cells,the interaction proteins played an anti-stress effect on the cell membrane.The experimental results of the effect of interaction protein on plant resistance showed that the disease spots in the P1,N4 and N,regions were smaller than those in the Pgr106 empty carrier Agrobacterium area,indicating that these three interaction proteins could enhance the disease resistance of Bac.benthama and inhibit the infection of Bacoccus anthracis when anthracnose invaded plants.The results of fluorescence PCR showed that the expression of the three interacting proteins increased significantly with the increase of infection time,and the relative expression of the interaction proteins was upregulated in the later stage,which was consistent with the results of the interaction proteins affecting the pathogenicity of pathogenic bacteria,and it was speculated that plants produced a large amount of chitinase to resist the invasion of pathogenic bacteria.The NAC domain protein was analyzed by real-time PCR and pathogenicity of pathogenic bacteria,and it was found that the tobacco leaf disease spot was smaller than that of Pgr 106 empty carrier disease spot at 48 h,and the relative expression was upregulated after 48 h,indicating that the interaction protein positively regulated plant immunity and promoted plant disease resistance.In summary,this study found that chitinase and NAC domain proteins in Camellia oleifera can improve plant disease resistance and positively regulate plant immune responses.