VBNC状态番茄溃疡病菌的抗逆机制研究

Bacteria in the viable but non-culturable (VBNC) state fail to grow on routine culture media on which they would normally grow and develop into colonies, but are alive and capable of renewed metabolic activity. The bacterial cells in VBNC state can resuscitate, return to an actively metabolizing and culturable form, and retain virulence in some adaptable conditions. However, the molecular mechanism is still unsolved after more than thirty years of VBNC state research. Clavibacter michiganensis subsp. michignensis (Cmm) is one of the most important quarantine phytopathogenic bacteria in China. As the causal agent of bacterial canker disease of tomato, its occurrence has caused substantial economic losses for seed production and fresh/processing tomato production worldwide. In previous studies, we proved that the culturable Cmm cells can be induced into VBNC state by copper and low pH condition. The VBNC-Cmm cells resuscitated and regained their culturability and pathogenicity when they were grown in tomato seedling homogenate media or inoculated on tomato seedlings. In this project, we plan to reveal the mechanism of VBNC-Cmm cells, based on the transcriptome RNA-Seq analysis, bacterial morphological characteristics observation, the cell wall peptidoglycan architecture and structure analysis, as well as the resistance and pathogenicity tests of VBNC-Cmm cells, and disclose the key genes involved in VBNC state and confirm their functions by gene knockout and complementation technology. It is the first study on VBNC state for Gram-positive phytopathogenic bacteria, as well as the first VBNC study on phytopathogenic bacteria in China. The expected results of this project will contribute to bacterial biology and resistance mechanism, and provide novel viewpoints for detection of plant pathogenic bacteria, invasion of quarantine pests and management of crop diseases.

“有活力但不可培养”(VBNC)状态是指细菌失去了在常规培养基上生长并形成菌落的能力，但仍然具有活性和复苏能力。VBNC状态的病原细菌复苏后能够重新形成菌落、恢复代谢活性和致病性。然而，VBNC概念提出距今已经30多年，其机制仍未得到全面的阐述。本项目在前期已经证实番茄溃疡病菌在铜离子和酸性条件诱导下进入VBNC状态，并确证可复苏的基础上，计划以番茄溃疡病菌这一重要的检疫性革兰氏阳性植物病原细菌为材料，通过转录组测序、VBNC菌体的形态变化研究、细胞壁组成分析、抗逆性和致病性测定，阐明参与VBNC状态的关键基因，并进行敲除和互补验证，全面解析番茄溃疡病菌VBNC 状态的机制。预期结果将填补国内外植物病原细菌VBNC状态机制研究的空白，拓展对革兰氏阳性植物病原细菌存活状态的认识，解析VBNC状态番茄溃疡病菌的抗逆机制及其潜在风险，为控制检疫性有害生物入侵和植物细菌病害综合防治提供新思路。

有活力但不可培养（VBNC）状态是指细菌失去了在常规培养基上生长并形成菌落的能力，但仍具有活性和复苏能力。文献报道多种逆境条件能使菌体进入VBNC状态，但其机制仍不明确。番茄溃疡病菌（Cm）是一种革兰氏阳性菌，实验室前期研究已经明确在铜离子诱导下能使其进入VBNC状态。.本研究通过转录组测序、结合qPCR分析差异表达基因，筛选出与VBNC状态相关的33个基因，分为9类：与抗铜相关、严谨反应、抵抗逆境、细胞壁结构、延伸因子与σ因子、转录因子、代谢与蛋白翻译、细胞分裂、致病力。.与指数期菌体相比，VBNC菌体表现出明显对超声波、高温、抗性素等抗性显著增加。在扫描电镜与透射电镜下，VBNC状态菌体表现为长宽比变小、表面粗糙，并随诱导时间增长变化更明显。此外，VBNC状态菌体肽聚糖交联程度增强；在对与肽聚糖合成相关基因青霉素结合蛋白研究中发现，在VBNC形成过程中，这些基因表达量上调，基因缺失突变体生长分裂受到影响，更不耐受盐胁迫、溶菌酶处理、超声胁迫，且突变体ΔpbpC更快丧失可培养性，不能维持VBNC状态。对突变体肽聚糖的研究结果表明其肽聚糖层变薄，且交联程度降低。.对于严谨反应，我们分析了信号分子ppGpp合成相关基因，并获得敲除与回补菌株，通过测定突变体的表型，发现ppGpp能够影响菌株生长、抗逆、胞外多糖分泌、致病力。通过检测VBNC状态ppGpp的含量，发现随VBNC诱导时间的延长，ppGpp含量先提高后下降，并且突变体提早丧失可培养性，而活菌量与总菌量无明显差异。.本项目揭示了VBNC状态Cm的形成及抗逆机制，丰富了对于G+植物病原细菌的VBNC机制和植物病原细菌的生理状态的理解，为植物病害初侵染来源和细菌病害综合防治研究提供了新思路。