Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T. atroviride exudates. In contrast, P. simiae and P. putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δtmk3) gene of T. atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T. atroviride, bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δtmk3 mutant. Unexpectedly, exudates from wild-type T. atroviride and the Δtmk3 mutant rescued purine auxotrophic mutants of H. seropedicae, K. michiganensis and P. simiae. Metabolomic analysis on exudates from wild-type T. atroviride and the Δtmk3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma-metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

中文翻译：

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全基因组适应性分析揭示了细菌与深绿木霉外代谢物相互作用的分子机制。

木霉属 是无处不在的根际真菌，能够产生几类次生代谢物，这些代谢物可以改变植物相关微生物组的动态。然而，介导这些相互作用的细菌-真菌机制尚未得到充分表征。在这里，采用随机条形码转座子位点测序（RB-TnSeq）方法来鉴定对深绿木霉渗出物存在下的健康重要的细菌基因。我们选择了三种具有 RB-TnSeq 突变文库的可促进植物生长的根际细菌：固氮菌密歇根克雷伯菌 M5aI 和 Herbaspirillum seropedicae SmR1 以及假单胞菌 WCS417。作为非根际物种，恶臭假单胞菌 KT2440 也被包括在内。从 RB-TnSeq 数据来看，固氮细菌主要竞争铁，并且需要铁载体运输系统 TonB/ExbB 在 T. atroviride 渗出物存在的情况下实现最佳适应性。相比之下，猿假单胞菌和恶臭假单胞菌高度依赖与膜脂修饰相关的机制，而膜脂修饰是抵抗阳离子抗菌肽（CAMP）所需的。T. atroviride 的 Hog1-MAP 激酶 (Δtmk3) 基因的突变体显示许多具有潜在抗生素活性的非核糖体肽合成酶 (NRPS) 生物合成基因簇的表达模式发生改变。与野生型 T. atroviride 的渗出物相比，当 RB-TnSeq 文库暴露于 Δtmk3 突变体的渗出物时，含有与抗生素耐药性相关的基因损伤的细菌突变体没有表现出适应性缺陷。出乎意料的是，野生型T. atroviride和Δtmk3突变体的渗出物拯救了H. seropedicae、K. michiganensis和P. simiae的嘌呤营养缺陷型突变体。对野生型 T. atroviride 和 Δtmk3 突变体渗出物的代谢组学分析表明，两种菌株均分泌嘌呤和复杂的代谢物；产生其中一些代谢物需要功能性 Tmk3。这项研究强调了木霉代谢物和土壤细菌之间复杂的相互作用，揭示了有益和拮抗的作用，并强调了这种关系的复杂性和多方面性。