Introduction

The brown planthopper (Nilaparvata lugens, BPH), a monophagous sap-sucking arthropod herbivore, is considered the most destructive pest of rice (Oryza sativa) throughout Asia. Understanding and utilizing BPH resistance genes is considered to be the most economical and efficient strategy for BPH management. Several BPH resistance genes have been cloned and studied (Du et al., 2009; Liu et al., 2015; Zhao et al., 2016; Hu et al., 2017). Bph14 and Bph9 encode a nucleotide-binding and leucine-rich repeat (NLR)-containing protein (Du et al., 2009; Zhao et al., 2016; Hu et al., 2017). Bph3 is a cluster of three genes encoding lectin receptor kinases (OsLecRK1-OsLecRK3) (Liu et al., 2015). These genes are supposed to induce immune responses and mediate downstream signalling events, but the mechanism remains unclear. Bph6 is a new type of resistance gene encoding a previously uncharacterized protein. Current research shows that Bph6 in rice mediates resistance to BPH through multiple signalling pathways. Bph6 alters the action of hormones (cZ-type CKs, JA and SA) and positively regulates phytoalexin production. Bph6 also functions in the physical defence against BPH attack by reinforcing the accumulation of cellulose and hemicellulose in cell walls and maintaining the cell wall thickness. BPH6 localizes to the exocyst complex and enhanced exocytosis. It interacts with OsEXO70E1 instead of other exocyst subunits (OsSEC3, OsSEC5, OsSEC6, OsSEC8, OsSEC10, OsSEC15 and OsEXO84). Knocking down OsExo70E1 expression would decrease the resistance of Bph6-NIL plants to BPH (Guo et al., 2018).

Exocytosis mediated by exocysts is an important trafficking event involved in plant cell processes (Zarsky et al., 2013). Disease resistance in the plant is determined by the secretion of defence proteins and antimicrobial metabolites at infection sites (Collins et al., 2003; An et al., 2006; Du et al., 2018). The exocyst complex is composed of eight subunits (SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70 and EXO84) (Hala et al., 2008; Fendrych et al., 2010). In contrast to one or a few copies of other subunits, EXO70 forms a family of paralogues in plants: 13 in moss (Physcomitrella patens), 23 in Arabidopsis and 47 in rice (O. sativa) (Chong et al., 2009; Zhang et al., 2010; Cvrckova et al., 2012). The multiplication of EXO70 proteins in plants may partly respond to the extensive demands for unusual cargo sorting and secretory traffic, reflecting specific biological contexts and functions (Li et al., 2010; Zhang et al., 2010; Martin-Urdiroz et al., 2016). EXO70 family genes were found to be involved in plant organ development. For instance, OsEXO70A1 is required for normal vascular bundle differentiation and primary nutrient assimilation (Tu et al., 2015). The SR1 gene encoding the EXO70L2 protein is critical to cell division and tracheary element development in rice (Xing et al., 2021). EXO70 isoforms also function in plant immunity. For instance, OsEXO70B1 acts as an essential regulator in rice immunity by interacting with the receptor-like kinase OsCERK1, an essential component for chitin reception (Hou et al., 2020). OsEXO70F3/F2 specifically forms a complex with the Magnaporthe oryzae effector AVR-Pii and is necessary for NLR protein Pii-dependent resistance (Fujisaki et al., 2015). Although EXO70 members contribute in regulating many processes important for plant development and immunity, the influence restricting herbivore attacks is still ignored .

On facing pathogens, plant cell walls form an important line of defence. Pathogens secrete an array of enzymes to collapse the cell wall polysaccharides to penetrate and colonize host tissues. To counter these attacks, plants have evolved many compounds to inhibit these enzymes and maintain and strengthen the structural integrity of the cell wall (Juge, 2006; Lagaert et al., 2009). Rice upregulates cellulose, hemicellulose and lignin synthesis to strengthen cell walls to resist BPH (Guo et al., 2018; He et al., 2020; Shi et al., 2021). One of the main components of the cell wall is lignin, which improves its rigidity and hydrophobicity. Lignin forms a protective barrier hindering mechanical penetration by pathogenic microorganisms or degradation by microbial enzymes (Walter, 1992). Lignin concentration is closely related to disease resistance (Delgado et al., 2002; Bonello et al., 2003; Peltier et al., 2009). Lignin biosynthesis starts with the deamination of phenylalanine and involves successive hydroxylation and methylation reactions (Wout et al., 2003; Barros et al., 2015). S-adenosylmethionine synthetase (SAMS)-catalysed S-adenosylmethionine (SAM) acts as a methyl donor involved in numerous transmethylation reactions including the synthesis of lignin monomers (Takusagawa et al., 1996; Kota et al., 2004; Roje, 2006). It has been claimed that SAMS is preferentially accumulated in lignified cells in the tomato xylem and dermal tissue (Sanchez-Aguayo et al., 2004). A mutation in the Arabidopsis SAMS3 results in high free methionine and decreased lignin content (Shen et al., 2002). These observations indicate that SAMS contributes to lignification in the secondary cell wall biosynthesis.

The finding that BPH6 associated with OsEXO70E1 paves the way for exploring the role of EXO70 in insect resistance. So, we further screened BPH6-binding proteins from EXO70 paralogues. This study identified another EXO70 member, OsEXO70H3, which interacts with BPH6 and has a positive role in Bph6-mediated BPH resistance. Further, we found S-adenosylmethionine synthase-like (SAMSL) that interacts with OsEXO70H3 from the apoplastic proteome. OsEXO70H3 recruits SAMSL and promotes the delivery of SAMSL into the cell exterior. OsExo70H3-RNAi transgenic plants and samsl mutants display significantly weakened resistance to BPH attack and decrease the lignin content in plant leaf sheaths. Our results suggest that OsEXO70H3 enhances the trafficking of SAMSL to regulate lignin deposition, thus participating in Bph6-mediated resistance.

中文翻译：

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OsEXO70H3 调控 SAMSL 排泄和细胞壁木质素沉积的水稻抗飞虱的必要性

介绍

褐飞虱 ( Nilaparvata lugens , BPH) 是一种单食性吸汁节肢动物食草动物，被认为是整个亚洲对水稻 ( Oryza sativa ) 最具破坏性的害虫。了解和利用 BPH 抗性基因被认为是 BPH 管理最经济有效的策略。已经克隆和研究了几个 BPH 抗性基因（Du et al ., 2009 ; Liu et al ., 2015 ; Zhao et al ., 2016 ; Hu et al ., 2017）。Bph14和Bph9编码一种核苷酸结合和富含亮氨酸重复 (NLR) 的蛋白质 (Du等人，2009 年；赵等，2016；胡等人，2017）。Bph3是一组编码凝集素受体激酶 (OsLecRK1-OsLecRK3) 的三个基因 (Liu et al ., 2015 )。这些基因被认为可以诱导免疫反应并介导下游信号事件，但机制仍不清楚。Bph6是一种新型抗性基因，编码一种以前未鉴定的蛋白质。目前的研究表明，水稻中的Bph6通过多种信号通路介导对 BPH 的抗性。Bph6改变激素（cZ 型 CKs、JA 和 SA）的作用并正向调节植物抗毒素的产生。Bph6还通过增强细胞壁中纤维素和半纤维素的积累并保持细胞壁厚度，在物理防御 BPH 攻击中​​发挥作用。BPH6 定位于胞外囊复合体并增强胞吐作用。它与 OsEXO70E1 相互作用，而不是与其他外囊亚基（OsSEC3、OsSEC5、OsSEC6、OsSEC8、OsSEC10、OsSEC15 和 OsEXO84）相互作用。敲低OsExo70E1的表达会降低Bph6 -NIL 植物对 BPH 的抗性 (Guo et al ., 2018 )。

由外囊介导的胞吐作用是植物细胞过程中涉及的重要运输事件（Zarsky等人，2013 年）。植物的抗病性由感染部位防御蛋白和抗菌代谢物的分泌决定（Collins等人，2003；An等人，2006；Du等人，2018）。外囊复合体由八个亚基（SEC3、SEC5、SEC6、SEC8、SEC10、SEC15、EXO70 和 EXO84）组成（Hala等人，2008；Fendrych等人，2010）。与其他亚基的一个或几个拷贝相比，EXO70 在植物中形成了一个旁系同源物家族：苔藓中的 13 个 ( Physcomitrella patens )、拟南芥中的 23 个和水稻 ( O. sativa ) 中的47 个(Chong et al ., 2009 ; Zhang等人，2010 年；Cvrckova等人，2012 年）。植物中 EXO70 蛋白的增殖可能部分响应了对异常货物分类和分泌运输的广泛需求，反映了特定的生物学背景和功能（Li et al ., 2010 ; Zhang et al ., 2010 ; Martin-Urdiroz等人，2016 年）。EXO70 家族基因被发现参与植物器官发育。例如，OsEXO70A1 是正常维管束分化和初级营养吸收所必需的 (Tu et al ., 2015 )。编码 EXO70L2 蛋白的SR1基因对水稻的细胞分裂和气管元件发育至关重要（Xing等，2021）。EXO70 同工型也在植物免疫中起作用。例如，OsEXO70B1 通过与受体样激酶 OsCERK1（几丁质接收的重要成分）相互作用，在水稻免疫中发挥重要调节作用（Hou et al ., 2020）。OsEXO70F3/F2 与稻瘟病菌效应物 AVR-Pii特异性形成复合物，是 NLR 蛋白 Pii 依赖性抗性所必需的 (Fujisaki et al ., 2015 )。虽然 EXO70 成员在调节许多对植物发育和免疫很重要的过程中做出了贡献，但限制食草动物攻击的影响仍然被忽视。

在面对病原体时，植物细胞壁形成了一道重要的防线。病原体分泌一系列酶来破坏细胞壁多糖，从而穿透和定植宿主组织。为了对抗这些攻击，植物已经进化出许多化合物来抑制这些酶并维持和加强细胞壁的结构完整性（Juge，2006；Lagaert等人，2009）。水稻上调纤维素、半纤维素和木质素的合成以增强细胞壁以抵抗 BPH (Guo et al ., 2018 ; He et al ., 2020 ; Shi et al ., 2021）。细胞壁的主要成分之一是木质素，可提高其刚性和疏水性。木质素形成保护屏障，阻碍病原微生物的机械渗透或微生物酶的降解（Walter，1992）。木质素浓度与抗病性密切相关（Delgado等人，2002；Bonello等人，2003；Peltier等人，2009）。木质素生物合成从苯丙氨酸的脱氨开始，涉及连续的羟基化和甲基化反应（Wout等人，2003 年；Barros等人，2015 年）。S-腺苷甲硫氨酸合成酶 (SAMS) 催化的S-腺苷甲硫氨酸 (SAM) 作为甲基供体参与许多转甲基反应，包括木质素单体的合成（Takusagawa等人，1996 年；Kota等人，2004 年；Roje，2006 年） . 据称，SAMS 优先积聚在番茄木质部和真皮组织中的木质化细胞中（Sanchez-Aguayo等人，2004 年）。拟南芥SAMS3 中的突变导致高游离蛋氨酸和降低的木质素含量（Shen等人，2002）。这些观察表明SAMS有助于次生细胞壁生物合成中的木质化。

BPH6与OsEXO70E1相关的发现为探索EXO70在昆虫抗性中的作用铺平了道路。因此，我们进一步筛选了来自 EXO70 旁系同源物的 BPH6 结合蛋白。该研究确定了另一个 EXO70 成员 OsEXO70H3，它与 BPH6 相互作用并在Bph6介导的 BPH 抗性中起积极作用。此外，我们发现了与来自质外体蛋白质组的 OsEXO70H3 相互作用的S-腺苷甲硫氨酸合酶样 (SAMSL)。OsEXO70H3 募集 SAMSL 并促进 SAMSL 递送到细胞外部。OsExo70H3 -RNAi 转基因植物和samsl突变体对BPH攻击的抵抗力显着减弱，植物叶鞘中的木质素含量降低。我们的结果表明，OsEXO70H3 增强 SAMSL 的运输以调节木质素沉积，从而参与Bph6介导的抗性。