Tn5转座子插入α-酮戊二酸脱氢酶基因sucA对香蕉细菌性软腐病菌毒力的影响

Tn5 transposon insertion of α-ketoglutarate dehydrogenase gene sucA affects the virulence of the pathogen causing bacterial soft rot of banana

摘要:

【背景】玉米迪克氏菌(Dickeya zeae)可引起作物细菌性软腐病，严重威胁香蕉、水稻等重要作物的生产安全。本研究在香蕉细菌性软腐病菌D. zeae MS1的Tn5突变体库中筛选到4个毒力显著减弱的突变体。【目的】进一步鉴定Tn5转座子插入突变体的具体基因位点，并明确该基因对细菌毒力的影响。【方法】通过热不对称交错PCR (thermal asymmetric interlaced polymerase chain reaction, TAIL-PCR)鉴定转座子插入位点；利用同源重组方法构建插入基因的敲除突变体及其互补菌株，并比较它们与野生型在细菌生长、相关代谢产物活性、植物细胞壁降解酶(plant cell wall-degrading enzymes, PCWDEs)分泌、运动性及毒力等方面的差异。【结果】TAIL-PCR鉴定出4个突变体的转座子插入位点均是位于α-酮戊二酸脱氢酶(α-ketoglutarate dehydrogenase, α-KGDH)基因sucA内，该基因编码三羧酸(tricarboxylic acid, TCA)循环过程的关键酶组分。基因敲除突变体ΔsucA在细菌生长曲线上受到一定程度的影响，但是其α-KGDH活性完全丧失。基因突变可显著减少PCWDEs的分泌并降低pelB和pelC等基因的转录表达，而不影响细菌的涌动、游动能力。对比野生型，ΔsucA在烟叶上产生的过敏性反应明显减弱，对香蕉幼苗的致病性也显著降低。回补菌株则可将上述细菌表型均恢复至与野生型接近的水平。【结论】本研究利用Tn5转座子插入突变鉴定到可影响细菌毒力的TCA关键酶基因sucA，该基因的突变可减弱重要毒力因子PCWDEs的分泌，但不影响细菌运动性。

Abstract:

[Background] Dickeya zeae is a phytopathogen causing bacterial soft rot and has seriously threatened the production of a wide variety of crops including banana and rice worldwide. In this study, four Tn5 transposon mutants of D. zeae MS1 isolated from diseased banana plants were obtained and found to exhibit attenuated virulence in banana plants. [Objective] To determine the gene loci inserted with the transposon in the four mutants and examine the effect of this gene on bacterial virulence. [Methods] Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was applied to identify the transposon-inserting genes; further, homologous recombination was used for the construction of in-frame deletion mutants and the complementation strains of target genes, which were compared with the wild-type strain D. zeae MS1 in terms of growth, metabolite activity, secretion of plant cell wall-degrading enzymes (PCWDEs), motility, and virulence. [Results] TAIL-PCR showed that all the four mutants were produced by the insertion in the same gene, sucA, which encoded an α-ketoglutarate dehydrogenase (α-KGDH) in the tricarboxylic acid (TCA) cycle. An in-frame deletion mutant ΔsucA showcased partially affected growth, and its α-KGDH activity was completely deprived. ΔsucA showed significantly reduced secretion of PCWDEs and was repressed at the transcription levels of genes encoding PCWDEs, such as pelB and pelC. However, mutation of sucA did not affect the swarming or swimming motility. In comparison with the wild-type, ΔsucA induced weakened hypersensitive responses (HR) in tobacco leaves and demonstrated significantly attenuated pathogenicity in banana seedlings. The complementation of this gene restored all these phenotypes to the levels as the wild-type. [Conclusion] Generation of Tn5 transposon mutants revealed that sucA was involved in virulence of D. zeae, which encoded a key enzyme in the TCA cycle. Mutation of this gene could weaken the secretion of key virulence factors PCWDEs but did not affect bacterial motility.