TQA毒素在柑橘绿霉菌与寄主互作过程中的作用

Penicillium digitatum (Pers.:Fr) Sacc, causing green mold decay, is the most devastating pathogen of postharvest citrus fruits. It contributes up to 90% of total losses after harvesting. Crucial roles of many proteins in P. digitatum pathogenesis have been revealed, like transport protein Pdmfs1, transcription factor PacC and some cell wall degrading enzymes. Mycotoxin is one of the main factors during infection of plant pathogenic fungi. For many mycotixins,biosynthetic pathways and the their roles in host-pathogen interaction have been characterized in recent years with the help of developed techniques. However, our knowledge about the role of mycotoxin in P. digitatum pathogenesis is scarce. Tryptoquialanine(TQA) is a major component of P. digitatum produced mycotoxins. Its biosynthetic gene cluster has been identified in P. digitatum Pd01 during the whole genome sequencing project. We have found the up-regulation of all the TQA biosynthetic genes while Pd01 growing on the citrus peel. Moreover, we found that TQA would damage the tissues while exposure to citrus peel. These facts therefore suggest a potential role of TQA in P. digitatum pathogenesis. In order to reveal the mechanism in detail, deletion mutant of PdtqaA, the key gene in TQA biosynthesis, will be constructed in this work. And also, the mutant and the wild strain induced gene expression profiles of citrus host will be investigated, with the purpose to identify the targeted genes of TQA. We believe that this work would increase the knowledge about the biological roles of mycotoxin in pathogen-host interaction, and would also promote the development of drug design and biocontrol.

柑橘绿霉菌是柑橘采后病害最主要的病原菌，其造成的损失占柑橘采后腐烂损失的90%以上。对该菌致病机理的研究已鉴定出部分致病因子，但真菌毒素在该菌致病过程中的作用尚无明确报道。Tryptoquialanine(TQA)是柑橘绿霉菌产生主要的真菌毒素之一。申请人已明确柑橘绿霉菌中TQA生物合成的遗传基础，同时发现其合成受柑橘表皮营养成分的诱导，并已初步证实TQA能伤害柑橘表皮组织，但其作用机制尚不明确。本项目的研究拟通过构建TQA合成关键基因的缺失突变体，了解TQA毒素对柑橘绿霉菌致病性及菌株形态、产孢等方面的影响；并通过比较分析突变体和野生型菌株作用下寄主细胞的基因表达差异，了解TQA毒素对寄主细胞产生伤害的作用机制。本项目的开展将有利于加深对真菌毒素生物学功能的认识，促进病原-寄主互作机制的了解，并为植物病原真菌新型防治手段的开发提供理论基础。

真菌次级代谢产物在药物研发以及病原菌与宿主的互作过程中发挥重要作用。在本项目中，我们对柑橘绿霉菌中的真菌毒素TQA(tryptoquialanine)的合成调控和功能进行了研究。我们首先构建了预测的TQA合成途径中主要组成基因tqaA和tqaI的突变体，明确在二者敲除的情况下，TQA均不能正常合成。我们对突变体的生长发育及致病性等表型进行了检测，表明TQA毒素不影响柑橘绿霉菌的正常生长发育，这与对真菌次级代谢产物的现有认识一致。同时，TQA的缺失也没有引起柑橘绿霉菌致病性和毒力的显著变化。但我们明确了TQA的合成受酸性pH值和柑橘果皮营养成分的影响，且其合成与柑橘绿霉菌侵染宿主的过程密切相关。为进一步了解TQA合成的调控机制，我们构建了全局调控因子LaeA的敲除和过表达突变体，并通过转录组学的方法对突变体的基因表达进行了系统分析。结果鉴定了包括TQA合成途径在内的3个受LaeA调控的次级代谢产物合成基因簇，同时确认了LaeA突变引起柑橘绿霉菌产孢的下降(14%)，并增强了该菌对碱性pH和细胞壁抑制剂的敏感性。此外，我们在研究过程中优化了真菌次级代谢途径预测的方法，结合比较基因组学和基因差异表达的数据，有效提高了次级代谢途径边界定位的准确性。.综上，本项目验证了TQA毒素的合成途径，明确了酸性pH和柑橘果皮等环境因子对其合成的诱导作用，以及LaeA对其合成具有调控作用，但TQA毒素不影响柑橘绿霉菌的正常生长和发育，不参与致病和毒力机制。此外，本项目的工作同时表明LaeA是柑橘绿霉菌发育过程的重要调控因子，对其产孢以及环境压力响应机制有重要影响。本项目的完成，促进了我们对TQA毒素和LaeA功能的认识，鉴定出的基因簇为柑橘绿霉菌中其它次级代谢产物功能的研究提供了基础，而对真菌次级代谢基因簇预测方法的优化也为相关研究提供了新的策略。