The endocytic pathway for absorption of exogenous RNAs in Verticillium dahliae
image: (A) Fluorescence microscopy observation of V. dahliae hyphae after coincubation with synthesized RNA or sRNA. Green fluorescence represents fluorescein-labeled RNAs, and red fluorescence represents Cy3-labeled sRNAs. UTP was used as a negative control. (B) Model of endocytic pathway for exogenous RNAs absorption in V. dahliae. Exogenous RNAs
Credit: Yun Jin, State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences
Small RNAs (sRNAs) not only play essential roles within their own cells but also act as signaling molecules, inducing trans-kingdom gene silencing between plants, fungi, and other organisms. Building on this naturally occurring trans-kingdom communication, RNA interference (RNAi) technologies have become powerful tools for combating plant viruses, pests, and fungal pathogens. When it comes to the pathway for these trans-kingdom RNAs taken up by recipient cells, previous studies have shown that exogenous RNAs can be internalized by animal cells via surface binding or other uptake mechanisms, and in plants, both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) have been reported. However, for filamentous fungi, the mechanisms by which fungal cells uptake exogenous RNAs remain largely unknown.
To address this question, Prof. Hui-Shan Guo (Institute of Microbiology, Chinese Academy of Sciences) and her team made great efforts to visually depict the process of exogenous RNA uptake in fungal cells. Her team has previously shown that sRNAs from plants or rhizospheric beneficial fungi can enter V. dahliae hyphae and downregulate the expression of virulence genes, thus reducing their pathogenicity. In this study, the researchers employed live-cell imaging technology combined with molecular biology experiments and revealed how those exogenous RNAs enter fungal cells.
Single- or double-stranded RNAs of various origins and lengths could enter the hyphae of V. dahliae as their observation under a confocal microscope. However, treatment with LatB, the endocytosis inhibitor, significantly reduced RNA uptake, indicating that endocytosis is involved in this process.
Endocytosis involves actin dynamics and the formation of endosomes. Small GTPases Rab5 and Rab7 are known to associate with early and late endosomes, respectively. In V. dahliae, the homologs of these two endosomal markers are VdVPS and VdYPTC5, which partially colocalize with the entering exogenous RNAs in fungal cells. Additionally, exogenous RNAs could also colocalize with the fungal actin proteins VdCapA and VdEND3. Further exploration revealed that these two genes are crucial components of V. dahliae endocytosis and their knockout mutants exhibited significantly reduced RNA uptake efficiency.
Endocytosis can be categorized into clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). By treating fungal cells with specific inhibitors of CME or CIE, the researchers found that chlorpromazine hydrochloride (CPZ), the CME inhibitor, significantly impaired the uptake of exogenous RNA in both hyphae and protoplasts, which highlights CME as the primary pathway for RNA uptake in V. dahliae.
This study elucidated how exogenous RNAs enter fungi and identified the core components involved in fungal endocytic pathway. The successful entry of exogenous RNAs into recipient cells is a critical step for RNAi-based crop protection technologies. In the future, more efficient RNAi-based disease resistance strategies may be developed by enhancing endocytosis in recipient cells to promote fungal uptake of small RNAs. This study provides new theoretical insights for the application and development of RNAi technologies, holding significant promise for environmentally sustainable crop protection and human disease therapies against pathogenic fungi.
How to cite this article:
Liu C, Cui C, Zhou G, Gao F, Zhao J, Guo H, et al. The endocytic pathway for absorption of exogenous RNAs in Verticillium dahliae. mLife. 2025;4:45–54.