Screening Of Proteins Interacting With Xcc8004Type Ⅲ Effectors From Cabbage By Yeast Two-Hybrid System

Chinese cabbage(Brassica campestris) is a cultivated vegetable in China. The amount of cultivated acreage is the largest, and cabbage is the widely distributed between the vegetable cultivars. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of Chinese cabbage, and it often leads to a cabbage yield decreased, and lower quality. This study preliminary screened the target proteins which have interactions with pathogenic type Ⅲ effectors of plant pathogen Xcc8004by yeast two-hybrid system. In this research, we have constructed bait vectors with seven type Ⅲ effector genes (XC1210, XC2081, XC2082, XC2995, XC3802, XC3177and XC4273) which are deleted lead to reducing the plant pathogen Xcc8004virulence. The bait vectors include pGBKT7-1210, pGBKT7-2081, pGBKT7-2082, pGBKT7-2995, pGBKT7-3177, pGBKT7-3802and pGBKT7-4273. The yeast strains Y187containing the bait plasmid were used to the detection of their self-activation and toxicity testing. We found that the expression of bait vectors for type Ⅲ effectors did not have self-activation and toxicity to the yeast strains Y187. We have constructed a cDNA library by the wild-type Xcc8004infecting the cabbage leaves that is used for yeast two-hybrid system. The transformation efficiency of the cDNA library is6.2×106transformants/3μg pGADT7-Rec. The titer is2.1×109pfu/mL. The recombination rate of cDNA library is96%.Cotransformed yeast strains Y187containing the bait plasmids and cDNA library yeast AH109. We found that the type Ⅲ effectors including XC2082and XC2995have positive yeast clones; otherwise type Ⅲ effectors including XC1210, XC2081, XC3177, XC3802and XC4273did not have positive yeast clones. We screened the clones as the following steps.(1) Transfered the clones onto SD/-Trp+X-a-gal plates after screening the cDNA library to do phenotypic test.(2) Excluding the repetitive clones by yeast clone-PCR.(3) Identifying the existing of insertion sequence by the double digestion with restriction enzyme.(4) Having got the insertion sequence of positive library plasmids.(5) Transforming the bait plasmid and the corresponding library plasmids into yeast AH109to test the interaction. And we tested the beta-galactosidase activity to determine the existence of the interaction strength. Analysising the sequences by bioinformatics analysis, we found that XC2082have interactions with11kinds of target proteins including F-box protein, aspartyl protease, ribonuclease, glyceraldehyde-3-phosphate dehydrogenase, guanine nucleotide-binding protein subunit beta; XC2995have interactions with6kinds of target proteins including ribonuclease, binding protein, glyceraldehyde3-phosphate dehydrogenase. This research provides important information to pathogenic mechanism of plant pathogenic Xcc8004infecting the cabbage by analysising the known biological functions of target proteins. And it also provides a theoretical basis for the prevention and treatment of black rot disease and the breeding new varieties of disease-resistant Chinese cabbage.