Histone modifications and chromatin-binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation-Sequencing (fCUT&Tag-Seq) protocol specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae, Neurospora crassa, Fusarium graminearum, and Sporisorium scitamineum, showing good signal-to-noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity.

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 Verticillium dahliae, 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 V. dahliae 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.

Small RNAs (sRNAs, including small interfering RNAs [siRNAs] and micro RNAs [miRNAs]) are key mediators of RNA silencing (or RNA interference), which play important roles in plant development and response to biotic and abiotic stimulation. Verticillium wilt is a plant vascular disease caused by the soil-borne fungal pathogens, such as Verticillium dahliae. We previously reported that V. dahliae infection increased two plant endogenous miRNAs that were exported to fungal cell to silence virulence genes. To investigate plant sRNAs in genome-wide response to V. dahliae infection, in this study, we constructed two sRNA libraries from Arabidopsis roots with and without V. dahliae infection, respectively. In total, 31 conserved miRNAs were found to be differentially expressed during the early stage of infection with V. dahliae using sRNA sequencing. Among these, the expression levels of miR160, miR164, miR166, miR167, miR390 and miR156h were confirmed by northern blot. Reverse transcription quantitative real time polymerase chain reaction results showed that the induction of miRNAs (miR160, miR164, miR166 and miR167) upon V. dahliae infection downregulated the expression of their targeted genes (ARF10, NAC1, PHV and ARF6), respectively. In addition, we identified specific phased siRNAs generated from distinct regions of two libraries. Profiling of these miRNAs and sRNAs lay the foundation for further understanding and utilising the host-induced gene silencing strategy to control plant vascular pathogens.