Functional Analysis And Utilization Of A Quorum-Quenching Gene From Ralstonia Solanacearum

Bacterial wilt caused by Ralstonia solanacearum, is one of the most important and widespread bacterial diseases of plants. R. solanacearum is a devastating plant pathogen with an unusually wide host range of > 200 specieies in 50 families. Some of its economically important hosts are potato, tomato, eggplant, capsicum, papaya, peanut, tobacco, banana, mulberry, eucalyptus, and olive.AHLs(N-acyl homoserine lactones), quorum-sensing signals regulate virulence gene expression in a range of plant and animal (including human) bacterial pathogens. However, AHL-inactivation approach can attenuate the plant pathogenicity of pathogens.1. Cloning of a quorum-quenching gene from R. solanacearum and bioactivity assay of its fusion protein expressed in E. coliThe R. solanacearum probable aculeacin A acylase (AAC) gene was cloned by PCR amplification and constructed into bacterial expressing vector. AAC fusion protein was obtained in E. coli system. It was observed that recombinant Escherichia coli producing AAC proteins had AHL-degrading activity and could attenuate the plant pathogenicity of pathogen.2. Mutant construction of R. solanacearum aac gene and estimation its pathogenicityTo determine the function of aac gene in bacterial wilt disease development, a suicide vector of aac gene was constructed and introduced into wild-type R. solanacearum by electroporation. After homologous recombination, the aac mutants were generated and identified. The result of soil inoculation showed that the aac mutants were much less virulent on tomato than the wild-type, which indicated that the aac gene is a very important factor in the pathogenesis of R. solanacearum.3. Breeding transgenic tobacco with aac gene against bacterial wiltTo further analysis of the function of aac gene, a plant high-efficient expression plasmid of aac gene, pBI121-Ω4A-aac was successfully constructed. And the plasmid was then transformed into Agrobacterium tumefaciens and then introduced into leaf disk of tobacoo and tomato. Under Kanamycin selection pressure, 35 and 15 resistant regenerated plants of NC89 and'zhongshu-5'were obtained, respectively. The results of PCR, RT-PCR, Northern, and ELISA identification indicated that the aac gene was successfully integrated into the genomes of tobacco and tomato plants and transcribed correctly. The result of plant inoculation showed that ransgenic tobacco and tomato expressing AAC exhibit significantly enhanced resistance to R. solanacearum comparing to non-transgenic ones. The transgenic plants could delay the wilt symptom development and make disease index reduced. All of the results indicated that the resistance of plants could be enhanced by introducing aac gene into the genomic.4. Interactions analysis between the Gsp proteins of Type II secretory system in R. solanacearumTo identification the interactions between the Gsp proteins of Type II Secretory System in R. solanacearum controlled by the quorum-sensing, the gspC,E,M,L genes were cloned into different bacteria expression plasmids, and the fusion proteins were obtained. After refolding, the refolded proteins with biological activity were gained. The results of Pull-down assay show that GspE-GspL and GspL-GspM can be interact, respectively.