柑橘溃疡病菌一个单功能域反应调控蛋白的功能研究

Functional analysis of a single-domain response regulator in Xanthomonas citri subsp.citri

由柑橘黄单胞柑橘亚种(Xanthomonas citri subsp.citri,Xcci)引起的柑橘溃疡病是我国柑橘生产上的一种重要病害。我们在前期研究中发现,Xcci广西菌株N8中有一个与306菌株的XAC3126同源的基因,可能参与调控病原菌的致病性。预测该基因编码一个单功能反应调控蛋白。为明确该基因的生物学功能,本研究对该基因进行缺失突变和功能互补,获得了相应的缺失突变体和互补菌株。致病性测定发现,该基因缺失导致Xcci在柑橘上的致病性显著下降,并且其胞外多糖产量、运动性,以及生物膜形成能力均显著降低,而互补菌株的这些表型恢复至野生型水平。进一步通过RT-qPCR分析该基因缺失对Xcci运动性相关基因及胞外多糖相关基因转录的影响,发现相比野生型菌株,缺失突变体在丰富培养基NB中培养时,这些基因的表达均下降,表明该基因的产物是通过调控多种致病因子的表达来影响病原菌在柑橘上的致病性。据此,我们将该基因命名为embR(Extracellular polysaccharide,motility and biofilm formation related regulator)。

Citrus bacterial canker caused by Xanthomonas citri subsp.citri(Xcci)is an important bacterial disea-se of citrus plants.Our previous work revealed that the homologous of XAC3126(Accession no.AAM37971.1)of Xcci strain 306,which was predicted to be a single-domain response regulator,might be involved in the bacterial virulence in Xcci Guangxi wild-type strain N8.To investigate the biological functions of this gene(named embR),a deletion mutant was constructed with strain N8 as the starting strain.Simultaneously,a complemented strain was constructed using a recombinant plasmid harboring this innate gene.Phenotypic analysis revealed that the embR deletion mutantΔembR showed obviously reduced virulence on the host plant Citrus reticulata Blanco'Orah'compared with the wild-type strain,while the complemented strain exhibited similar virulence with that of the wild-type strain.Additionally,theΔembR mutant displayed an obvious reduction in extracellular polysaccharide(EPS)production,cell motility and cell aggregation.Reverse transcription quantitative real-time PCR(RT-qPCR)revealed that the transcript level of a set of genes involved in EPS production and cell motility in theΔembR mutant was decreased compared with that in the wild-type strain.These combined data indicate that the embR gene is required for multiple cellular processes including virulence in Xcci and modulates the expression of a series of virulence factor-related genes.