ABSTRACT

Autophagy is critical for plant defense against necrotrophic pathogens, which causes serious yield loss on crops. However, the post-translational regulatory mechanisms of autophagy pathway in plant resistance against necrotrophs remain poorly understood. In this study, we report that phosphorylation modification on ATG18a, a key regulator of autophagosome formation in Arabidopsis thaliana, constitutes a post-translation regulation of autophagy, which attenuates plant resistance against necrotrophic pathogens. We found that phosphorylation of ATG18a suppresses autophagosome formation and its subsequent delivery into the vacuole, which results in reduced autophagy activity and compromised plant resistance against Botrytis cinerea. In contrast, overexpression of ATG18a dephosphorylation-mimic form increases the accumulation of autophagosomes and complements the plant resistance of atg18a mutant against B. cinerea. Moreover, BAK1, a key regulator in plant resistance, was identified to physically interact with and phosphorylate ATG18a. Mutation of BAK1 blocks ATG18a phosphorylation at four of the five detected phosphorylation sites after B. cinerea infection and strongly activates autophagy, leading to enhanced resistance against B. cinerea. Collectively, the identification of functional phosphorylation sites on ATG18a and the corresponding kinase BAK1 unveiled how plant regulates autophagy during resistance against necrotrophic pathogens.

Abbreviations: 35s: the cauliflower mosaic virus 35s promoter; A. thaliana: Arabidopsis thaliana; A. brassicicola: Alternaria brassicicola; ABA: abscisic acid; ATG: autophagy-related; ATG18a: autophagy-related protein 18a in A. thaliana; ATG8a: autophagy-related protein 8a in A. thaliana; ATG8–PE: ATG8 conjugated with PE; B. cinerea: Botrytis cinerea; BAK1: Brassinosteroid insensitive 1-associated receptor kinase1 in A. thaliana; BiFC: biomolecular fluorescence complementation; BIK1: Botrytis-insensitive kinase 1 in A. thaliana; BKK1: BAK1-like 1 in A. thaliana; BR: brassinosteroid; Co-IP: coimmunoprecipitation; dai: days after inoculation; DAMPs: damage-associated molecular patterns; E. coli: Escherochia coli; ER: endoplasmic reticulum; ETI: effector-triggered immunity; GFP: green fluorescent protein; HA: hemagglutinin; IP: immunoprecipitation; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LCI: luciferase complementation imaging; MPK3: mitogen-activated protein kinase 3 in A. thaliana; MPK4: mitogen-activated protein kinase 4 in A. thaliana; MPK6: mitogen-activated protein kinase 6 in A. thaliana; N. benthamiana: Nicotiana benthamiana; NES: nuclear export sequence; PAMP: pathogen-associated molecular pattern; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PRR: pattern recognition receptor; PtdIns(3,5)P_2: phosphatidylinositol (3,5)-biphosphate; PtdIns3P: phosphatidylinositol 3-biphosphate; PTI: PAMP-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; SnRK2.6: SNF1-related protein kinase 2.6 in A. thaliana; TORC1: the rapamycin-sensitive Tor complex1; TRAF: tumor necrosis factor receptor-associated factor; WT: wild type plant; Yc: C-terminal fragment of YFP; YFP: yellow fluorescent protein; Yn: N-terminal fragment of YFP

摘要

自噬对于植物防御坏死性病原体至关重要，这会导致作物严重的产量损失。然而，自噬途径在植物抗坏死营养物质中的翻译后调控机制仍然知之甚少。在这项研究中，我们报告了 ATG18a 的磷酸化修饰，ATG18a 是拟南芥自噬体形成的关键调节因子，构成了自噬的翻译后调节，从而减弱了植物对坏死性病原体的抗性。我们发现 ATG18a 的磷酸化抑制了自噬体的形成及其随后向液泡的传递，这导致自噬活性降低并损害了植物对灰霉病菌的抗性. 相反，ATG18a 去磷酸化模拟形式的过表达增加了自噬体的积累，并补充了atg18a突变体对灰霉病菌的植物抗性。此外，植物抗性的关键调节因子 BAK1 被鉴定为与 ATG18a 物理相互作用并使其磷酸化。BAK1的突变阻断B. cinerea感染后五个检测到的磷酸化位点中的四个处的 ATG18a 磷酸化，并强烈激活自噬，导致对B. cinerea的抗性增强. 总的来说，ATG18a 和相应的激酶 BAK1 上功能性磷酸化位点的鉴定揭示了植物在抵抗坏死性病原体的过程中如何调节自噬。

缩写： 35s：花椰菜花叶病毒35s启动子；A. thaliana：拟南芥；A. 芸苔属：链格孢属芸苔属；ABA：脱落酸；ATG：自噬相关；ATG18a：拟南芥中的自噬相关蛋白 18a ；ATG8a：拟南芥中的自噬相关蛋白 8a ；ATG8–PE：ATG8 与 PE 缀合；B. cinerea：灰葡萄孢；BAK1：拟南芥中油菜素类固醇不敏感的 1 相关受体激酶 1 ；BiFC：生物分子荧光互补；BIK1：拟南芥中对葡萄孢菌不敏感的激酶 1 ；BKK1：拟南芥中的 BAK1 样 1; BR：油菜素内酯；Co-IP：免疫共沉淀；dai：接种后天数；DAMPs：损伤相关的分子模式；大肠杆菌：大肠杆菌；ER：内质网；ETI：效应触发免疫；GFP：绿色荧光蛋白；HA：血凝素；IP：免疫沉淀；LC-MS/MS：液相色谱-串联质谱；LCI：荧光素酶互补成像；MPK3：拟南芥中的丝裂原活化蛋白激酶 3 ；MPK4：拟南芥中的丝裂原活化蛋白激酶 4 ；MPK6：拟南芥中的丝裂原活化蛋白激酶 6 ；N.benthamiana：本氏烟草; NES：核输出序列；PAMP：病原体相关分子模式；PCR：聚合酶链式反应；PE：磷脂酰乙醇胺；PRR：模式识别受体；PtdIns(3,5)P _2：磷脂酰肌醇 (3,5)-二磷酸；PtdIns3P：磷脂酰肌醇 3-二磷酸；PTI：PAMP 触发的免疫；qRT-PCR：定量逆转录PCR；SnRK2.6：拟南芥中的 SNF1 相关蛋白激酶 2.6 ；TORC1：雷帕霉素敏感的 Tor 复合物1；TRAF：肿瘤坏死因子受体相关因子；WT：野生型植物；Yc：YFP的C端片段；YFP：黄色荧光蛋白；Yn：YFP的N端片段