A fungal pathogen deploys a small silencing RNA that attenuates mosquito immunity and facilitates infection

Abstract

Insecticidal fungi represent a promising alternative to chemical pesticides for disease vector control. Here, we show that the pathogenic fungus Beauveria bassiana exports a microRNA-like RNA (bba-milR1) that hijacks the host RNA-interference machinery in mosquito cells by binding to Argonaute 1 (AGO1). bba-milR1 is highly expressed during fungal penetration of the mosquito integument, and suppresses host immunity by silencing expression of the mosquito Toll receptor ligand Spätzle 4 (Spz4). Later, upon entering the hemocoel, bba-milR1 expression is decreased, which avoids induction of the host proteinase CLIPB9 that activates the melanization response. Thus, our results indicate that the pathogen deploys a cross-kingdom small-RNA effector that attenuates host immunity and facilitates infection.

Fig. 1

Roles of B. bassiana bba-milR1 on fungal pathogenicity against mosquitoes. a bba-milR1 cannot be detected in B. bassiana Dicer2 deletion mutant (ΔDcl2) and Dicer1/Dicer2 double-mutant (ΔDcl1/Dcl2), as revealed by RT-PCR. RNA was extracted from fungal mycelium. b Survival of adult female A. stephensi mosquitoes infected with the wild-type (WT) ARSEF252 and two mutant strains milR1-KO 1# and milR1-KO 2# following topical application of a spore suspension (10⁷ conidia/ml). Mosquitoes sprayed with sterile 0.01% Triton X-100 were used as a negative control (Triton). c Survival of female adult mosquitoes following topical application of a suspension of 10⁷ conidia/ml of WT and two overexpression strains milR1-OV 1# and milR1-OV 2#. The statistical significance of survival curves was analyzed with the Log-rank test. d Expression of bba-milR1 during B. bassiana ARSEF252 infecting A. stephensi. RNA was extracted from fungus-infected mosquitoes. The B. bassiana U6 small nuclear RNA (U6) was used as an internal reference in qRT-PCR assays. The expression values are normalized to time 0. The data represent three biological repeats with three technical replicates and are shown as mean ± s.e.m. Source data are provided as a Source Data file

Fig. 2

bba-milR1 enters host cells and binds to mosquito Argonaute 1 (AGO1). a bba-milR1 enters C6/36 mosquito cells via vesicles that were present outside the cells, on the cell surface or in the cytoplasm. Cy3-fluorescein-labeled bba-milR1 (2 μM) was added to C6/36 cells, incubated for 24 h, then washed with PBS, fixed in 4% paraformaldehyde, and stained with DAPI. DIC, differential interference contrast microscopy; red, Cy3; blue, DAPI. Scale bars, 5 μm. b bba-milR1 is translocated into adult mosquito fat body cells. Cy3-labeled bba-milR1 (20 μM) or PBS was injected into the hemocoel of A, stephensi female mosquitoes. The fat body was dissected 24 h later, washed with PBS, fixed in 4% paraformaldehyde, followed by nucleic acid staining with DAPI. Scale bars in DIC and merge: 25 μm; scale bars in zoom in: 5 μm. Images were acquired by confocal microscopy. c RT-PCR detection of AsAgo1-bound sRNAs. Fungal milRNAs are detected in the AsAGO1-RIP (RNA immunoprecipitation) fraction from the B. bassiana-infected A. stephensi mosquitoes that were collected at 36hpi, 60 hpi and 84 hpi, mixed and homogenized in ice-cold RIP lysis buffer, but not in the uninfected mosquitoes. AGO1-bound sRNAs were pulled down from fat body homogenates with antibody AsAGO1-linked magnetic beads. RNA was then released from AGO1-RIP fraction after digestion with protease K. Uninfected mosquitoes mixed with B. bassiana ARSEF252 mycelia were used as a control to rule out any nonspecific association between AGO1 and milRNAs during the experimental process. The AGO1-associated sRNAs were detected by RT-PCR. As a positive control, two A. stephensi miRNAs ast-miR10-5p and ast-miR2940-3p were detected in AsAGO1-RIP samples. Source data are provided as a Source Data file

Fig. 3

bba-milR1 targets the mosquito genes Spz4 and CLIPB9. a The sequences of bba-milR1 target sites in the coding regions of Spz4 and CLIPB9 genes of A. stephensi mosquito. Red characters are seed regions of bba-milR1. Black bold characters are target sequences. The mutated forms of target genes are shown in blue characters. b bba-milR1 suppresses Spz4 expression. c bba-milR1 induces CLIPB9 expression. The interactions between bba-milR1 and the target sites of Spz4 and CLIPB9 in A. stephensi were determined by dual-luciferase reporter assays in HEK293T cells that were co-transfected with bba-milR1 mimics and psiCHECK2 vectors containing wild or mutant target sequences of Spz4 or CLIPB9 genes. d The transcript level of Spz4 is suppressed in mosquitoes injected with bba-milR1 agomir (200 nM). The mosquitoes injected with control agomir (200 nM) served as a negative control. Gene expression was quantified by qRT-PCR. e The transcript level of CLIPB9 is upregulated in mosquitoes injected with bba-milR1 agomir compared with the control. Values are mean ± s.e.m. The expression values are normalized to control. The experiments were repeated three times with similar results. **P < 0.01, ***P < 0.001, ns, not significant (Student’s t test). Source data are provided as a Source Data file

Fig. 4

bba-milR1 attenuates host immunity by suppressing Toll receptor ligand Spätzle 4. a The relative transcript levels of Spz4, cecropin 1 (CEC1), and defensin 1 (DEF1) in mosquitoes at 48 h post infection (hpi) with WT and bba-milR1-overexpressing strain milR1-OV 1#. The expression values are normalized to WT. b Effect of Spz4 silencing on the expression of CEC1 and DEF1. The expression values are normalized to dsGFP. c The relative transcript levels of Spz4, CEC1, and DEF1 in the mosquitoes injected with bba-milR1 agomir (200 nM) or control agomir (200 nM). The expression values are normalized to control. d Effect of Spz4 silencing on fungal hyphal body formation in the mosquito hemocoel at 60 hpi. Black arrows point to hyphal bodies, and the red arrow points to a mosquito hemocyte. Scale bars, 10 μm. e qPCR-based quantification of fungal load in mosquitoes injected with dsGFP or dsSpz4 at 60 hpi. Fungal levels are expressed as that of fungal 18S rRNA relative to A. stephensi ribosomal protein S7 (AsS7) DNA. f Effect of Spz4 silencing on the survival of mosquitoes following topical application of a suspension of 10⁷ conidia/ml of B. bassiana ARSEF252 (Log-rank test). Values are mean ± s.e.m. The experiments were repeated three times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001. P-value < 0.05 was regarded as statistically significant (Student’s t test). Source data are provided as a Source Data file

Fig. 5

The interaction of bba-milR1 with the target gene CLIPB9. a Diagram of the mosquito melanization response. Clip domain serine proteinase: CLIPB9; Serine protease inhibitor: SRPN2; prophenoloxidase: PPO; phenoloxidase: PO. b The relative transcript levels of CLIPB9 in mosquitoes at 36 h post infection with WT, milR1-OV 1#, or milR1-KO 1# strains. There was no significant difference in CLIPB9 expression among the groups at this time point (Student’s t test). c The relative transcript levels of CLIPB9 in mosquitoes at 84 h post infection with WT, milR1-OV 1#, or milR1-KO 1# strains. The expression values are normalized to WT. d Infection of milR1-OV1# and milR1-KO 1# results in significant increase and decrease in A. stephensi hemolymph PO activity compared with WT at 84 h post infection, respectively. e Effect of CLIPB9 silencing on fungal hyphal body formation in the mosquito hemocoel at 60 hpi. The black arrows and red arrow indicate the hyphal bodies and mosquito hemocyte, respectively. Scale bars, 10 μm. f qPCR-based quantification of fungal load in mosquitoes injected with dsGFP or dsCLIPB9 at 60 hpi. Fungal levels are expressed as that of fungal 18S rRNA relative to A. stephensi ribosomal protein S7 (AsS7) DNA. g Effect of CLIPB9 silencing on the survival of mosquitoes following topical application of a suspension of 10⁷ conidia/ml of B. bassiana ARSEF252 (Log-rank test). Values are mean ± s.e.m. Similar results were obtained in three biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001. P-value < 0.05 was regarded as statistically significant (Student’s t test). Source data are provided as a Source Data file

Fig. 6

Model of Beauveria bassiana deploying bba-milR1 to modulate mosquito immunity. During early stages of infection, B. bassiana bba-milR1 is highly expressed, and translocated into the mosquito cells to attenuate mosquito immune responses by suppressing the expression of the key activator gene Spz4. At this early stage in the integument, the bba-milR1 is not accessible to circulating hemocytes, the site of CLIPB9 gene expression. During later infection stages when the fungus enters the hemocoel, B. bassiana markedly decreases the expression of bba-milR1 to elaborately avoid induction of CLIPB9 and activation of melanization via conversion of prophenoloxidase (PPO) to phenoloxidase (PO) (see Fig. 5a)