PDF(Pubmed)
Abstract：
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 the 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.
摘要：
灰霉病菌的小RNA（sRNA）可以进入植物细胞并劫持宿主Argonaute蛋白1（AGO1）以沉默宿主免疫基因。然而,这些真菌sRNAs分泌并进入宿主细胞的机制尚不清楚。这里,我们证明了灰霉病菌利用细胞外囊泡（EV）分泌Bc-sRNAs，然后通过网格蛋白介导的内吞作用（CME）被植物细胞内化。灰霉病菌四跨膜蛋白，无冲头1(BcPLS1),作为EV生物标志物，在真菌致病性中起着至关重要的作用。我们观察到许多拟南芥网格蛋白包被的囊泡（CCV）周围的灰化芽孢杆菌感染部位以及灰化芽孢杆菌EV标记BcPLS1和拟南芥CLATHRIN轻链1的共定位，这是CCV的核心成分之一。同时,感染后，在纯化的CCV中检测到BcPLS1和灰霉病菌分泌的sRNA。拟南芥基因敲除突变体和CME途径关键组分的诱导型显性阴性突变体表现出对灰霉病菌感染的抗性增加。此外，在那些CME突变体中,装载到拟南芥AGO1中的Bc-sRNA和宿主靶基因抑制减弱。一起,我们的结果表明真菌通过电动汽车分泌sRNAs，然后主要通过CME进入宿主植物细胞。
