荧光假单胞菌HN58抑制甘蔗鞭黑粉菌有性配合的作用机制

The sugarcane whip smut which caused by plant pathogen Ustilago scitaminea is a severe disease, seriously affecting the sustainable development of sugarcane plants and sugar manufacturing industry in China. Pseudomonas fluorescens strain HN58 has been screened for the biocontrol proteins characterized by their high inhibitory activities on sexual mating and formation of the pathogenic dikaryotic hyphae of Ustilago scitaminea, which presents an effective way for smut control. To explore the mechanism behind the biocontrol of Ustilago scitaminea by strain HN58, we used an integrated approach involving genetics and molecular biology techniques. Random Tn5 transposon insertion mutants of P. fluorescens that displayed alterations in mating inhibitory abilities were isolated and the genes disrupted were identified via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). The full sequences of some target genes encoding related active proteins are obtained and annotated by bioinformatic analysis based on the genomic sequences of P. fluorescens, and the amino acid sequences are analyzed for protein functional prediction, domain analysis and homolog alignment. Gene knockout, functional complementation and prokaryotic overexpression are further employed to determine the roles of both the functional genes and regulatory genes in the inhibitory activity against the Ustilago scitaminea's sexual mating. Gene deletion or point mutations were also employed to identify the functional domains and sites on the functional proteins. After comprehensive analyses of the changes of the mRNAs, protein profiles and inhibitory activities of the functional gene expression in the context of the regulatory gene mutations, their regulation on the functional genes are revealed. Through this research, the biocontrol mechanism of strain HN58 against Ustilago scitaminea's sexual mating will be clear. Furthermore, blocking the infection process in contact period will provide a new model for the sugarcane smut biocontrol and also benefit the protection of the population ecology and microbial diversity.

甘蔗鞭黑穗病是甘蔗最严重的真菌性病害。荧光假单胞菌HN58产生的活性蛋白，能有效地抑制甘蔗鞭黑粉菌的有性配合，阻止具有侵染致病能力的双核菌丝体形成，但其抑制机理尚不清楚。为此，本项目采用转座子插入突变技术，获得HN58菌株功能缺失突变体库，运用基因克隆、遗传互补、基因产物表达及其抑制病原菌有性配合活性分析来确定该细菌的功能基因与其调控因子。同时，通过分析HN58菌株效应蛋白在基因片段敲除或点突变背景下对抑制有性配合功能的影响，来明确功能域及功能位点。在调控基因突变的背景下，通过综合分析HN58菌株功能基因的mRNAs表达特征、蛋白谱以及抑制有性配合的活性变化，来揭示调控基因对功能基因的调控方式。通过上述研究，明确HN58菌株抑制甘蔗鞭黑粉菌有性配合的作用机制。本研究以阻断病原菌致病过程的侵入环节为切入点，进行甘蔗鞭黑穗病的生防研究，将可能为该病的生物防治开辟一个新的方向。

甘蔗鞭黑穗病是甘蔗最严重的真菌性病害。荧光假单胞 HN58 菌株产生的胞外活性物质，对甘蔗鞭黑穗病菌表现出较好的生防潜力。本项目主要是验证其生防机理是抑制了菌丝本身的形态生长还是抑制了调控其有性配合的生物信息素的产生或传导。黑粉菌有性配合包括信息素及其受体的相互识别（由a位点控制）和双核菌丝体的形成与维持（由a位点和b位点控制）及其致病性（由b位点控制）。生物活性测定和电镜扫描结果表明：生防菌HN58胞外活性物质对病原菌的单倍体菌系形态生长没有影响，但对单倍体菌系接合管的形成和双核菌丝体的形成有明显影响。其中，接合管不能形成表明信息素的产生即a位点受到影响；双核菌丝体不能形成则表明b位点受到影响。实时荧光定量PCR的结果进一步表明：a位点的pra1基因（信息素受体基因）和b位点bE（控制双核菌丝形成及其致病性的基因）基因受HN58胞外活性物质的影响而相对表达量明显下降；因此，其双核菌丝体不能形成或不稳定。从而证明了该生防菌HN58对甘蔗鞭黑穗病菌的生防基础是抑制了该病原菌的有性配合，其作用靶标不是病原菌形态本身，而是病原菌产生的信号物质。这对生物防治具有重要意义。这也为进一步研究该生防菌的相关功能基因及其开发应用奠定了基础。同时，采用Tn5 转座子插入突变技术，对生防菌 HN58 菌株进行了突变，获得了4个对甘蔗鞭黑穗病菌抑制作用降低或丧失的功能突变体，并通过 hiTAIL-PCR 获得其突变基因序列和功能注释。