Introduction

Ectomycorrhizal (ECM) symbiosis is one of the most predominant forms of plant–microbe interactions in boreal and temperate forest ecosystems (Read et al., 2004; Qu et al., 2010; McGuire et al., 2013) in which it plays a key role in plant health through mineral nutrient cycling. During symbiosis establishment, ECM fungi form a sheath around the lateral root tips and colonise the apoplastic space between epidermal and cortical root cells (Kottke & Oberwinkler, 1987; Abras et al., 1988), forming the Hartig net, a nutritional exchange site between plant and fungus. Formation of this symbiotic interface requires modification of plant and fungal cell walls (Brundrett, 2004; Martin et al., 2017) and has interested researchers for > 30 yr (Paris et al., 1993; Balestrini et al., 1996; Balestrini & Bonfante, 2014). In situ immunolabelling-based characterisation of the cell-wall polymer epitopes (pectin, cellulose, hemicellulose) in mature ECM in comparison with nonmycorrhizal roots, has underpinned our understanding of how the plant cell wall is altered during the establishment of hyphae in the apoplastic space. The suggested key mechanisms for Hartig net formation are (1) mechanical force from the hyphal tip (Garcia et al., 2015), (2) auxin secretion from root tips to promote cell-wall loosening (Gea et al., 1994) and (3) secretion of fungal cell-wall-degrading enzymes (CWDEs) (Bonfante & Genre, 2010; Veneault-Fourrey et al., 2014). The two former hypotheses await further proof through functional studies, whereas the latter has largely benefitted from the availability of fungal genomes and the possibility of using RNAi techniques in the ectomycorrhizal model fungus L. bicolor to identify and functionally characterise potential fungal plant cell-wall degrading (PCWDE) enzymes (Zhang et al., 2018, 2022). Whole-genome sequences of several ECM species have revealed that ECM fungi have a reduced set of PCWDEs compared with their saprotrophic ancestors (Martin et al., 2008; Kohler et al., 2015; Miyauchi et al., 2020). ECM fungi are likely to employ these PCWDEs to modify the plant cell-wall matrix for apoplastic accommodation to establish bi-directional nutrient transport. A significant increase of PCWDE expression during ECM interactions further supports this hypothesis (Sebastiana et al., 2014; Veneault-Fourrey et al., 2014; Kohler et al., 2015).

The extracellular route that fungal hyphae take during Hartig net formation is rich in pectic polysaccharides; in particular, homogalacturonan (HG) is predominant in the middle lamella between adjacent root cells (Daher & Braybrook, 2015). Pectic polysaccharides make up to 35% of the total cell-wall dry weight and play versatile roles in plant physiological processes including cell growth and differentiation. Their dynamic alterations can modify cell-wall chemistry and rheology (Bidhendi & Geitmann, 2016). HG modifying enzymes (HGMEs) play a central role in cell-to-cell adhesion and cell separation (Sénéchal et al., 2014; Daher & Braybrook, 2015) and include multigenic families such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), pectate lyases (PLs) and polygalacturonases (PGs) (Sénéchal et al., 2014). The ECM basidiomycete Laccaria bicolor has a restricted set of HGMEs comprising only four PMEs and six PGs (Martin et al., 2008), of which the ECM-induced LbGH28A has recently been functionally characterised for its activity on pectin and polygalacturonic acid and is suggested to contribute to Hartig net formation (Zhang et al., 2022). Conversely, its host plant Populus tremula has a large set of HGMEs comprising 73 PMEs, 12 PAEs, 28 PLs and 38 PGs (Sundell et al., 2015). Despite the reduced HGME set, L. bicolor HGMEs, that is LbPMEs and LbPGs, are transcriptionally upregulated at various time points during an interaction with Populus (Veneault-Fourrey et al., 2014; Kohler et al., 2015; and the current study) suggesting their involvement in ECM development. PMEs seem to act as first modifiers of HG modification (Pelloux et al., 2007; Manmohit Kalia, 2015; Sénéchal et al., 2015) and catalyse the de-esterification of the C6-linked methyl-ester groups of HG chains. Although the exact modes of action of PMEs are still debated, PME-mediated HG de-esterification patterns can be random or blockwise, with enzyme isoforms and cell wall pH being determining factors (Catoire et al., 1998; Denès et al., 2000; Kim et al., 2005). Therefore, depending on physiological conditions, PME activity regulates both cell-wall loosening and cell-wall stiffening (Micheli, 2001; Sénéchal et al., 2014). During cell-wall loosening, PME de-esterification activity makes HG more accessible to depolymerising enzymes such as PLs and PGs (Micheli, 2001; Pelloux et al., 2007; Sénéchal et al., 2014; Manmohit Kalia, 2015). As it has recently been shown that a fungal PG (LbGH28A) is involved in Hartig net formation (Zhang et al., 2022), we are here hypothesising that preceding steps of HG modification also receive a fungal contribution. Several fungal pathogens employ PMEs to overcome pectin-rich cell-wall barriers (Lionetti et al., 2012; Sella et al., 2016; Fan et al., 2017). ECM fungi encoding PMEs may apply similar strategies to modify cell walls to form the Hartig net. We hypothesise that L. bicolor PMEs facilitate cell-wall loosening during ECM development. Therefore, we investigated the functional role of L. bicolor PMEs in ECM symbiosis. Using L. bicolor and P. tremula × P. tremuloides interactions in an in vitro culture system, we leveraged whole-genome transcriptomics, microscopy coupled with immunolabelling and transgenic approaches targeting LbPMEs to explore the methylesterification state of plant cell walls in ECM and the potential role of L. bicolor PMEs during ECM formation.

中文翻译：

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Laccaria bicolor 果胶甲基酯酶参与美洲山杨×美洲山杨外生菌根的发育

介绍

外生菌根 (ECM) 共生是北方和温带森林生态系统中植物与微生物相互作用的最主要形式之一（Read等人，  2004 年；Qu等人，  2010 年；McGuire等人，2013 年），它在其中发挥着重要作用通过矿物质养分循环在植物健康中发挥关键作用。在共生建立过程中，ECM 真菌在侧根尖周围形成一个鞘，并在表皮和皮质根细胞之间的质外体空间定植（Kottke & Oberwinkler，  1987 年；Abras等人，  1988 年）)，形成 Hartig 网，这是植物和真菌之间的营养交换场所。这种共生界面的形成需要对植物和真菌细胞壁进行修饰（Brundrett，  2004 年；Martin等人，  2017 年），研究人员对其感兴趣已有 30 年以上（Paris等人，  1993 年；Balestrini 等人，  1996 年；Balestrini &邦凡特，  2014 年）。就地与非菌根根相比，成熟 ECM 中细胞壁聚合物表位（果胶、纤维素、半纤维素）的基于免疫标记的表征巩固了我们对植物细胞壁在质外体空间中菌丝建立过程中如何改变的理解。Hartig 网形成的建议关键机制是 (1) 来自菌丝尖端的机械力 (Garcia et al .,  2015 )，(2) 从根尖分泌生长素以促进细胞壁松动 (Gea et al .,  1994 ) 和(3) 真菌细胞壁降解酶 (CWDE) 的分泌 (Bonfante & Genre,  2010 ; Veneault-Fourrey et al ., 2014). 前两个假设有待通过功能研究进一步证明，而后者在很大程度上受益于真菌基因组的可用性以及在外生菌根模型真菌L. bicolor中使用 RNAi 技术的可能性，以识别和功能表征潜在的真菌植物细胞壁降解(PCWDE) 酶（Zhang等人，  2018 年，2022 年）。几种 ECM 物种的全基因组序列表明，与其腐生祖先相比，ECM 真菌的 PCWDE 数量减少（Martin等人，  2008 年；Kohler等人，  2015 年；Miyauchi等人，  2020 年）). ECM 真菌很可能使用这些 PCWDEs 来修饰植物细胞壁基质以进行质外体调节以建立双向养分运输。ECM 相互作用期间 PCWDE 表达的显着增加进一步支持了这一假设（Sebastiana等人，  2014 年；Veneault-Fourrey等人，2014 年；Kohler等人，  2015 年）。

真菌菌丝在 Hartig 网形成过程中所采取的细胞外途径富含果胶多糖；特别是，同型半乳糖醛酸 (HG) 在相邻根细胞之间的中间薄层中占主导地位 (Daher & Braybrook,  2015 )。果胶多糖占细胞壁总干重的 35%，在细胞生长和分化等植物生理过程中发挥多种作用。它们的动态变化可以改变细胞壁化学和流变学（Bidhendi & Geitmann，  2016 年）。HG 修饰酶 (HGME) 在细胞间粘附和细胞分离中起着核心作用（Sénéchal等人，  2014 年；Daher 和 Braybrook，  2015 年) 并包括多基因家族，例如果胶甲酯酶 (PME)、果胶乙酰酯酶 (PAE)、果胶酸裂解酶 (PL) 和多聚半乳糖醛酸酶 (PG)（Sénéchal等人，  2014 年）。ECM 担子菌Laccaria bicolor具有一组受限制的 HGME，仅包含四个 PME 和六个 PG（Martin等人，  2008），其中 ECM 诱导的LbGH28A最近因其对果胶和聚半乳糖醛酸的活性而在功能上得到表征，并被建议有助于 Hartig 网的形成 (Zhang et al .,  2022 )。相反，其寄主植物欧洲山杨具有大量 HGME，包括 73PME、12 个 PAE、28 个 PL 和 38 个 PG（Sundell等人，  2015 年）。尽管 HGME 集减少，但L. bicolor HGME，即 LbPME 和 LbPG，在与Populus相互作用期间的不同时间点转录上调（Veneault-Fourrey等人，  2014 年；Kohler等人，  2015 年；以及当前的研究) 表明他们参与了 ECM 开发。PME 似乎是 HG 修饰的第一修饰剂（Pelloux等人，  2007 年；Manmohit Kalia，  2015 年；Sénéchal等人，  2015 年）) 并催化 HG 链的 C6 连接的甲基酯基团的去酯化。尽管 PME 的确切作用模式仍有争议，但 PME 介导的 HG 去酯化模式可以是随机的或块状的，酶同种型和细胞壁 pH 值是决定因素（Catoire等人，  1998 年；Denès等人，2000 年） ；Kim等人，  2005 年）。因此，根据生理条件，PME 活动调节细胞壁松动和细胞壁硬化（Micheli，  2001 年；Sénéchal等人，  2014 年）). 在细胞壁松动过程中，PME 去酯化活性使 HG 更容易接触解聚酶，例如 PL 和 PG（Micheli，  2001 年；Pelloux等人，  2007 年；Sénéchal等人，  2014 年；Manmohit Kalia，  2015 年）。由于最近表明真菌 PG (LbGH28A) 参与了 Hartig 网的形成（Zhang等人，  2022 年），我们在此假设 HG 修饰的前面步骤也有真菌的贡献。几种真菌病原体使用 PME 来克服富含果胶的细胞壁屏障（Lionetti等人，  2012 年；Sella等人， 2016 年；范等人，  2017）。编码 PME 的 ECM 真菌可以应用类似的策略来修改细胞壁以形成 Hartig 网。我们假设L. bicolor PME 在 ECM 发育过程中促进细胞壁松动。因此，我们研究了 L. bicolor PME 在 ECM 共生中的功能作用。利用L. bicolor和P. tremula × P. tremuloides在体外培养系统中的相互作用，我们利用全基因组转录组学、显微镜结合免疫标记和针对 LbPME 的转基因方法来探索 ECM 中植物细胞壁的甲酯化状态及其潜力的角色ECM 形成过程中的L. 双色PME。