An entomopathogen-based small-RNA delivery system promotes the biocontrol efficacy against termites

Entomopathogenic fungus-derived microRNA-like RNAs (milRNAs) have been determined as novel virulence factors that exhibit cross-kingdom RNAi effects on insect hosts. However, which factors and how they function in social insects are still largely unknown. Using the pathogen Metarhizium anisopliae and its social host Reticulitermes aculabialis, we investigated how pathogen-derived small RNAs mediate cross-kingdom RNAi to promote infection in social insect colonies. By using sRNA-seq, we identified 27 milRNAs outside the fungal cells. Among them, Ma-milR-4 was significantly increased in the response to termite physiological defense and markedly increased the infection mortality of termites. The experiment of Cy3-labeled Ma-milR-4 localization showed that Ma-milR-4 could be translocated into host cells and fungal infection could contribute to this translocation. mRNA-seq and RT-qPCR revealed that Ma-milR-4 downregulated a wide range of genes that were associated with termite disease defenses. RNAi-mediated silencing of the target genes leucine-rich repeat protein (LRR) and T-complex protein 1 subunit β (TCPB) made termites more vulnerable to the pathogen infection. These findings demonstrate that M. anisopliae is competent for cross-kingdom RNAi, which inspired the development of this entomopathogen-based RNAi delivery platform. We further generated the engineered Metarhizium fungus that produce specific short hairpin RNA (shRNA) against the termite relish, a well-known gene in antifungal defense. We found that the engineered fungus exhibited significant silencing effects on termite relish and markedly increased host susceptibility to the pathogen infection. Our study determines an entomopathogen-based small-RNA delivery system to increase the fungal efficacy, which can be used to facilitate termite biological control.