Yan Wang, Chunlai Cui,  Guandong Wang, Yifei Li, and  Sibao Wang

PNAS May 11, 2021; vol. 118 (19): e2023802118

Significance

The entomopathogenic fungus Beauveria bassiana can overcome insecticide resistance and represents a promising tool for controlling mosquitoes and other insect pests. Improvement of fungal efficacy requires better understanding of fungus–mosquito interactions. Here, we report on a new strategy of insect defense against fungal infection that employs translocation of miRNAs to silence virulence genes. Fungal infection activates production of Anopheles let-7 and miR-100 microRNAs (miRNAs). These miRNAs translocate into fungal hyphae to specifically silence the sec2p and C6TF fungal genes. Both genes are essential for fungal invasive growth and pathogenicity. Notably, virulence of a B. bassiana strain expressing both anti–let-7 and anti–miR-100 “sponge” RNAs is dramatically increased. This study may lead to new strategies for improved fungal-based vector control efficacy.

Abstract

Chemical insecticides remain the main strategy to combat mosquito-borne diseases, but the growing threat of insecticide resistance prompts the urgent need to develop alternative, ecofriendly, and sustainable vector control tools. Entomopathogenic fungi can overcome insecticide resistance and represent promising biocontrol tools for the control of mosquitoes. However, insects have evolved robust defense mechanisms against infection. Better understanding of mosquito defenses against fungal infection is critical for improvement of fungal efficacy. Here, we show that as the pathogenic fungus Beauveria bassiana penetrates into the host hemocoel, mosquitoes increase expression of the let-7 and miR-100 microRNAs (miRNAs). Both miRNAs translocate into fungal hyphae to specifically silence the virulence-related genes sec2p and C6TF, encoding a Rab guanine nucleotide exchange factor and a Zn(II)₂Cys₆ transcription factor, respectively. Inversely, expression of a let-7 sponge (anti–let-7) or a miR-100 sponge (anti–miR-100) in the fungus efficiently sequesters the corresponding translocated host miRNA. Notably, B. bassiana strains expressing anti–let-7 and anti–miR-100 are markedly more virulent to mosquitoes. Our findings reveal an insect defense strategy that employs miRNAs to induce cross-kingdom silencing of pathogen virulence-related genes, conferring resistance to infection.

See: https://www.pnas.org/content/118/19/e2023802118

Figure 1:

Deep sequencing identification of host miRNAs in fungal hyphae recovered from infected insect cadavers. (A) Schematic representation of the study design. Silkworms killed by B. bassiana were surface sterilized with 1% bleach and placed in petri dishes containing wet filter papers to facilitate fungal emergence. Four days later, fungal hyphae grown out of the Bb^cadavers were recovered carefully for RNA isolation. (B) RT-PCR detection confirmed that fungal mycelia recovered from Bb^cadavers were not contaminated with silkworm tissues. B. mori ribosomal protein 49 gene (Bmo-rp49) was used to detect the B. mori nucleic acids. B. bassiana actin (Bb-actin) was used to detect the B. bassiana nucleic acid. Bb^plate, fungal mycelia grown on plates; Bmo, noninfected B. mori silkworm. (C) Host miRNAs identified in fungal hyphae recovered from silkworm cadavers. (D) Mature let-7 and miR-100 sequences are highly conserved across insect species. aae, Aedes aegypti; aga, Anopheles gambiae; bmo, B. mori; and dme, Drosophila melanogaster. All miRNA sequences were obtained from the miRNA database.