Introduction

Beneficial root-endophytic fungi are major players within the consortia of plant-associated microorganisms collectively referred to as ‘plant microbiota’ (Glynou et al., 2016, 2018; Trivedi et al., 2020; Mahdi et al., 2022). While the composition of plant microbiota varies between different host species and depends on environmental factors (Tkacz et al., 2015; Strullu-Derrien et al., 2018), a balanced microbiota contributes to plant performance by improving host nutrient uptake and increasing resistance to biotic and abiotic stress (Raaijmakers et al., 2009; Hermosa et al., 2012; Finkel et al., 2017; Mahdi et al., 2022). Beneficial properties have been observed in plant interactions with ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi, as well as fungal endophytes (Zuccaro et al., 2014). These symbiotic interactions have evolved over millions of years, giving rise to fine-tuned relationships not only between fungi and their host plants but also among the various members of the microbiota (Mesny et al., 2023). The health of plants is directly influenced by intermicrobial relationships. This is illustrated by microorganisms that manifest high pathogenic potential in mono-associations but are effectively restrained in a microbial community context (Sarkar et al., 2019; Mesny et al., 2021; Mahdi et al., 2022). Host priming, along with direct intermicrobial competition and cooperation, collectively contribute to this phenomenon.

Root endophytes of the order Sebacinales, notably Serendipita indica (Si) and Serendipita vermifera (Sv), provide protection to various plant species against the aggressive phytopathogenic fungus Bipolaris sorokiniana (Bs) (Kumar et al., 2002; Sarkar et al., 2019; Y. Li et al., 2023). This well-adapted cereal pathogen poses a significant threat, causing diseases such as common root rot and spot blotch, which lead to substantial yield losses, particularly in warmer agricultural regions (Kumar et al., 2002). Recent research has shown that Sebacinales not only protect plants directly but also cooperate with other microbiota members. Notably, Sv has been shown to act synergistically with beneficial bacteria in the plant microbiota to enhance the protection of barley and Arabidopsis roots against Bs (Mahdi et al., 2022).

Through a split-root system, we previously demonstrated that Sv reduces Bs biomass in barley roots through a combination of systemic and local effects. These effects did not depend on extensive host transcriptional reprogramming but correlated with the downregulation of the phytopathogen effector repertoire in planta. Direct fungal confrontation experiments in soil revealed an induction of hydrolytic enzymes and effectors in Sv in the presence of Bs. Such an antagonistic response was not activated during the tripartite interaction in barley roots. This indicates that Sv effectors reduce the virulence potential of Bs in the rhizosphere before host colonization, enabling the endophyte to act as a host-protective barrier against the phytopathogenic intruder (Sarkar et al., 2019). A similar capacity for effector-induced host microbiota manipulation has been reported in pathogenic fungi. The soilborne fungus Verticillium dahliae, for instance, secretes the antimicrobial effector VdAve1, which suppresses antagonistic bacteria and thereby facilitates the infection of tomato plants (Snelders et al., 2020, 2023). While effectors were originally described as small-secreted proteins (SSPs), which suppress plant immunity and manipulate host metabolism in order to promote microbial colonization and reproduction (De Wit et al., 2009), these recent findings call for an expansion of the traditional effector concept toward supporting a role of effector secretion in shaping the niche (Veneault-Fourrey & Martin, 2011; Hemetsberger et al., 2012; Win et al., 2012; Lo Presti et al., 2015; Snelders et al., 2022).

Sv expresses distinct sets of effectors during bipartite confrontation with Bs in soil and tripartite interactions with barley. As of now, it remains unclear whether this differential expression of effectors is linked to diverging functions in host colonization and niche defense. The genomes of Sv and its close relative Serendipita indica (Si) encompass large repertoires of genes encoding for proteins involved in carbohydrate binding, plant cell wall degradation, and protein hydrolysis, as well as numerous SSPs with effector-like properties (Zuccaro et al., 2011, 2014). While for some of these proteins, roles in the evasion and suppression of plant immunity have been reported, the function of most of these proteins remains elusive (Jacobs et al., 2011; Lahrmann et al., 2013). In this study, we conducted a time-resolved transcriptomic analysis of Si and Sv, examining their transcriptional responses when exposed to monocot and dicot host plants, the phytopathogen Bs, or a synthetic community of beneficial root-associated bacteria. We investigated the expression profiles of putative effector genes with a focus on SSPs and carbohydrate-active enzymes (CAZymes). Our aim was to identify Sebacinales effectors induced specifically in response to host plants and/or microbes in order to discern their roles in host colonization and niche protection. We identified a GH18-CBM5 chitinase that was specifically induced in response to Bs in both Si and Sv and characterized the enzyme as a novel antimicrobial effector of Sebacinales involved in plant protection through fungal antagonism in the rhizosphere.

中文翻译：

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转录组学揭示了一种生态位防御机制：两种有益的根内生真菌部署抗菌 GH18-CBM5 几丁质酶来保护其宿主

 介绍

有益的根内生真菌是统称为“植物微生物群”的植物相关微生物联盟中的主要参与者（Glynou等 人，2016 年，2018 年;Trivedi等 人，2020 年;Mahdi et al.， 2022）。虽然植物微生物群的组成因不同宿主物种而异，并取决于环境因素（Tkacz等 人，2015 年;Strullu-Derrien et al.， 2018），平衡的微生物群通过改善宿主养分吸收和增加对生物和非生物胁迫的抵抗力来促进植物性能（Raaijmakers et al.， 2009;Hermosa等 人，2012 年;Finkel等 人，2017 年;Mahdi et al.， 2022）。在植物与外生菌根 （ECM） 和丛枝菌根 （AM） 真菌以及真菌内生菌的相互作用中观察到有益特性（Zuccaro et al.， 2014）。这些共生相互作用已经进化了数百万年，不仅在真菌与其寄主植物之间，而且在微生物群的各个成员之间也产生了微调的关系（Mesny等 人，2023 年）。植物的健康直接受到微生物间关系的影响。这在单关联中表现出高致病性潜力但在微生物群落环境中被有效抑制的微生物可以说明（Sarkar等 人，2019 年;Mesny等 人，2021 年;Mahdi et al.， 2022）。 宿主引发以及直接的微生物间竞争和合作共同导致了这种现象。

Sebacinales 目的根内生菌，特别是 Serendipita indica （Si） 和 Serendipita vermifera （Sv），为各种植物物种提供保护，使其免受侵略性植物病原真菌 Bipolaris sorokiniana （Bs） 的侵害（Kumar 等 人，2002 年;Sarkar等 人，2019 年;Y. Li et al.， 2023）。这种适应性强的谷物病原体构成了重大威胁，会导致普通根腐病和斑点等疾病，从而导致大量的产量损失，特别是在温暖的农业地区（Kumar 等 人，2002 年）。最近的研究表明，Sebacinales 不仅直接保护植物，而且还与其他微生物群成员合作。值得注意的是，Sv 已被证明与植物微生物群中的有益细菌协同作用，以增强大麦和拟南芥根对 B 的保护（Mahdi等 人，2022 年）.

通过分裂根系，我们之前证明 Sv 通过系统效应和局部效应的结合降低了大麦根中的 Bs 生物量。这些效应不依赖于广泛的宿主转录重编程，而是与植物中植物病原体效应子库的下调相关。土壤中的直接真菌对抗实验显示，在 Bs 存在下，Sv 中水解酶和效应子的诱导。在大麦根的三方相互作用期间，这种拮抗反应没有被激活。这表明 Sv 效应子在宿主定植之前降低了 Bs 在根际的毒力潜力，使内生菌能够作为抵御植物病原入侵者的宿主保护屏障（Sarkar等 人，2019 年）。在病原真菌中已报道了类似的效应子诱导的宿主微生物群操纵能力。例如，土壤真菌大丽轮枝菌分泌抗菌效应器 VdAve1，它抑制拮抗细菌，从而促进番茄植物的感染（Snelders等 人，2020 年，2023 年）。虽然效应器最初被描述为小分泌蛋白（SSPs），它们抑制植物免疫并操纵宿主代谢以促进微生物定植和繁殖（De Wit等 人，2009），但这些最近的发现呼吁扩展传统的效应器概念，以支持效应器分泌在塑造生态位中的作用（Veneault-Fourrey & Martin， 2011 年;Hemetsberger 等 人。，2012 年;Win et al.， 2012;Lo Presti et al.， 2015;Snelders等 人，2022 年）。

Sv 在土壤中与 Bs 的二分对抗以及与大麦的三方相互作用中表达不同的效应子集。截至目前，尚不清楚效应子的这种差异表达是否与宿主定植和生态位防御的不同功能有关。Sv 及其近亲 Serendipita indica （Si） 的基因组包含大量编码参与碳水化合物结合、植物细胞壁降解和蛋白质水解的蛋白质的基因库，以及许多具有效应子样特性的 SSP（Zuccaro et al.， 2011， 2014).虽然对于其中一些蛋白质，已经报道了在逃避和抑制植物免疫中的作用，但这些蛋白质中的大多数的功能仍然难以捉摸（Jacobs等 人，2011 年;Lahrmann et al.， 2013）。在这项研究中，我们对 Si 和 Sv 进行了时间分辨转录组学分析，检查它们在暴露于单子叶植物和双子叶植物寄主植物、植物病原体 Bs 或有益根相关细菌的合成群落时的转录反应。我们研究了推定效应基因的表达谱，重点是 SSP 和碳水化合物活性酶 （CAZymes）。我们的目标是鉴定专门响应寄主植物和/或微生物诱导的 Sebacinales 效应子，以区分它们在宿主定植和生态位保护中的作用。 我们鉴定了一种 GH18-CBM5 几丁质酶，该酶在 Si 和 Sv 中均响应 Bs 而被特异性诱导，并将该酶表征为皮脂霉属的新型抗菌效应物，通过根际的真菌拮抗作用参与植物保护。