The endocytic pathway for absorption of exogenous RNAs in <i>Verticillium dahliae</i>

RNAi technologies have been exploited to control viruses, pests, oomycetes, and fungal phytopathogens that cause disasters in host plants, including many agronomically significant crops. Double-stranded RNA (dsRNA) or small interfering RNA (siRNA) has been applied as a trigger for trans-kingdom RNAi between hosts and fungi. However, it is unclear what process mediates RNA uptake by fungi. In this study, by using live-cell imaging, we determined that exogenously synthesized RNA or small RNA (sRNA) was indiscriminately absorbed into <i>Verticillium dahliae</i>, a notorious pathogenic fungus. Moreover, the application of endocytic inhibitors or deletion of endocytic-related genes reduced RNA uptake efficiency, showing that RNA absorption by fungal cells occurs mainly through endocytosis. In addition, we found that the endocytosed fluorescence-labeled RNAs were partly colocalized with endosome marker genes. Overall, our research concluded that exogenous RNA could be assimilated by <i>V. dahliae</i> through the endocytic pathway. Unraveling this cytological mechanism underlying trans-kingdom RNAi holds significant importance, especially considering the fact that RNAi-based strategies targeting pathogenic fungi are increasingly prevalent in the realm of crop protection.