He Baoye, Wang Huan, Liu Guosheng, Chen Angela, Calvo Alejandra, Cai Qiang, Jin Hailing
Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, CA, USA.
State Key Laboratory of Hybrid Rice, College of Life Science, Wuhan University, Wuhan, China.
bioRxiv. 2023 Jun 29:2023.06.15.545159. doi: 10.1101/2023.06.15.545159.
Small RNAs (sRNAs) of the fungal pathogen Botrytis cinerea can enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate that B. cinerea utilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). The B. cinerea tetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerous Arabidopsis clathrin-coated vesicles (CCVs) around B. cinerea infection sites and the colocalization of B. cinerea EV marker BcPLS1 and Arabidopsis CLATHRIN LIGHT CHAIN 1, one of the core components of CCV. Meanwhile, BcPLS1 and the B. cinerea-secreted sRNAs are detected in purified CCVs after infection. Arabidopsis knockout mutants and inducible dominant-negative mutants of key components of CME pathway exhibit increased resistance to B. cinerea infection. Furthermore, Bc-sRNA loading into Arabidopsis AGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME.
灰霉病菌的小RNA（sRNA）能够进入植物细胞并劫持宿主AGO1蛋白以沉默宿主免疫基因。然而，这些真菌sRNA的分泌及进入宿主细胞的机制仍不清楚。在此，我们证明灰霉病菌利用细胞外囊泡（EV）来分泌Bc-sRNA，随后这些Bc-sRNA通过网格蛋白介导的内吞作用（CME）被植物细胞内化。灰霉病菌的四跨膜蛋白Punchless 1（BcPLS1）作为EV的生物标志物，在真菌致病性中发挥着重要作用。我们观察到在灰霉病菌感染位点周围有大量拟南芥网格蛋白包被囊泡（CCV），并且灰霉病菌EV标志物BcPLS1与CCV的核心成分之一拟南芥网格蛋白轻链1共定位。同时，在感染后纯化的CCV中检测到BcPLS1和灰霉病菌分泌的sRNA。拟南芥CME途径关键成分的敲除突变体和诱导型显性负突变体对灰霉病菌感染表现出增强的抗性。此外，在那些CME突变体中，Bc-sRNA加载到拟南芥AGO1中以及宿主靶基因抑制作用均减弱。总之，我们的结果表明真菌通过EV分泌sRNA，然后这些sRNA主要通过CME进入宿主植物细胞。
