Lin L, Xu X, Zheng Y, Zhang C
Department of Bioengineering, School of Medicine, Southeast University, Nanjing, China.
Division of Electronic Microscopy, School of Medicine, Southeast University, Nanjing, China.
Lett Appl Microbiol. 2018 Sep;67(3):270-277. doi: 10.1111/lam.13019. Epub 2018 Jul 17.
The biosynthesis of phytotoxin thaxtomin A (TXT) constitutes the major pathogenicity determinant in Streptomyces scabies, the most widely studied phytopathogen causing scab disease in potato and other root crops. It is recognized that S. scabies regulates its pathogenicity via γ-butyrolactone (GBL)-dependent quorum sensing (QS) signalling. AttM, from Agrobacterium tumefaciens C58 strain, has recently been proposed to have GBL-assimilative capacity. Here, we presented the introduction of A. tumefaciens-derived attM gene into S. scabies using the Escherichia coli-Streptomyces shuttle vector pIJ8600 via intergeneric conjugation, followed by the investigation of secondary metabolism (mycelium growth, TXT production and pathogenicity) in S. scabies attM exconjugants (S.s/attM) in comparison with their wild-type parent strain (S.s/WT). Among the resultant S.s/attM exconjugants, attM was found to be integrated into S. scabies chromosome as analysed by Southern blotting. Moreover, S.s/attM failed to evoke the disease symptoms in planta and displayed altered morphological differentiation in contrast to S.s/WT. The abolishment of TXT production in S.s/attM substantiated the loss of pathogenicity and also implied that attM, when constitutively expressed in S. scabies, could paralyse its GBL signalling pathway. Altogether, lactonase-coding gene attM would be useful in a quorum quenching strategy for plant protection via suppressing TXT production and pathogenicity of S. scabies.
This study provides an efficient means to introduce the lactonase gene attM from Agrobacterium tumefaciens into Streptomyces scabies for evaluating the role of γ-butyrolactone (GBL) in thaxtomin A production and pathogenicity, etc. Our results showed that pathogenicity was abrogated in attM-expressing S. scabies exconjugants. Although there are gene knockout approaches to inactivating GBL signalling and thus pathogenicity in S. scabies, they are not only time consuming due to refractory host but also possibly incomplete in view of gene redundancy. Our work is the first report for a kind of lactonase affecting pathogenicity and/or virulence of scab-causing Streptomyces species.
植物毒素thaxtomin A（TXT）的生物合成是疮痂链霉菌致病的主要决定因素，疮痂链霉菌是研究最广泛的引起马铃薯和其他块根作物疮痂病的植物病原体。人们认识到，疮痂链霉菌通过γ-丁内酯（GBL）依赖性群体感应（QS）信号传导来调节其致病性。最近有人提出，根癌农杆菌C58菌株的AttM具有GBL同化能力。在这里，我们通过属间接合，利用大肠杆菌-链霉菌穿梭载体pIJ8600将根癌农杆菌来源的attM基因导入疮痂链霉菌，随后研究了疮痂链霉菌attM接合后体（S.s/attM）与野生型亲本菌株（S.s/WT）相比的次生代谢（菌丝生长、TXT产生和致病性）。在所得的S.s/attM接合后体中，通过Southern印迹分析发现attM已整合到疮痂链霉菌染色体中。此外，与S.s/WT相比，S.s/attM在植物中未能引发疾病症状，并且表现出形态分化改变。S.s/attM中TXT产生的消除证实了致病性的丧失，这也意味着attM在疮痂链霉菌中组成型表达时，可能会使其GBL信号通路瘫痪。总之，编码内酯酶的基因attM在通过抑制疮痂链霉菌的TXT产生和致病性来保护植物的群体猝灭策略中可能会发挥作用。
本研究提供了一种有效的方法，将根癌农杆菌的内酯酶基因attM导入疮痂链霉菌，以评估γ-丁内酯（GBL）在thaxtomin A产生和致病性等方面的作用。我们的结果表明，表达attM的疮痂链霉菌接合后体的致病性被消除。虽然有基因敲除方法来使疮痂链霉菌中的GBL信号失活从而消除致病性，但由于宿主难治，这些方法不仅耗时，而且鉴于基因冗余可能并不完全。我们的工作是关于一种内酯酶影响引起疮痂的链霉菌物种致病性和/或毒力的首次报道。
