Fig 1.
Genomic organization of BbPmV4-2.
(A) Agarose gel electrophoresis of purified dsRNA extracted from B. bassiana strain Bb2151. Lane M, DNA molecular weight marker; Lane 1, dsRNAs of BbPmV4-2. (B) Schematic representation of the BbPmV4-2 genomic structure. ORFs (blue flanked by 5’ and 3’ untranslated regions (black lines)). (C) Schematic representation of the relationship between dsRNA 5 and the two defective dsRNAs 7 and 8. (D) Agarose gel electrophoresis of RT-PCR amplicons from purified dsRNA 5, dsRNA 7 and dsRNA 8, using sequence specific primers (D7-F/R and D8-F/R. Lane M, DNA molecular weight marker; Lane 1, RT-PCR using primers D7-F/R from dsRNA5; Lane 2, RT-PCR using primers D7-F/R from dsRNA 7 and dsRNA 8; Lane 3, RT-PCR using primers D8-F/R from dsRNA5; Lane 4, RT-PCR amplification using primers D8-F/R from dsRNA7 and dsRNA8). Primers used are listed in S1 Table.
Fig 2.
Impacts of BbPmV4-2 on heat and UV stress responses.
(A) Relative conidial germination rate and (B) microscopic images (scale bar 20 μm) of VF and VI strains on PDA plates 24 h after exposure to 42 °C for 60 min. (C) Relative conidial germination rate and (D) microscopy images (scale bar: 20 μm) of VF and VI strains on PDA plates 24 h after exposure to 100 mJ/cm2 UV for 10 sec. Relative transcript levels in VF and VI strains of (E) eight hsp genes and (F) twelve DNA damage repair genes, as measured by RT-qPCR. For each gene, expression in VF was set as 1 and relative expression in VI was calculated. Primers used are listed in S2 Table. Data were present as the mean ± standard deviation (SD) from three replications. Student’s t-test or ANOVA, * P < 0.05, ** P < 0.01 or *** P < 0.001.
Fig 3.
Impacts of BbPmV4-2 on virulence.
(A) Survival of G. mellonella larvae, (B) LT50 and (C) mycosis after cuticular infection with VF and VI strains. (D) Survival of G. mellonella larvae, (E) LT50 and (F) mycosis after injection with VF and VI strains. Control group (CK) larvae were treated with sterile water. (G) Conidial hydrophobicity rates of VF and VI strains. (H) Relative expression levels in VF and VI strains of five hydrophobicity genes and three conidial adherence genes, as measured by RT-qPCR. (I) Conidial adherence of VF and VI strains. (J) Growth of VF and VI strains on SDAY plates following penetration of cicada wings (scale bar 6 mm) and (K) quantification of the conidial penetration ability of VF and VI strains. (L) Relative expression levels in VF and VI strains of eight cuticle degradation and virulence genes, as measured by RT-qPCR. For each gene, expression in VF was set as 1 and relative expression in VI was calculated. Data were present as the mean ± standard deviation (SD) from three replications. Student’s t-test or ANOVA, * P < 0.05, ** P < 0.01 or *** P < 0.001.
Fig 4.
Impact of BbPmV4-2 ORFs 1-8 on B. bassiana.
(A) Survival of G. mellonella larvae and LT50 after cuticular infection with VF, VI, and ex-ORFs 1-8 strains. (B) Relative conidial germination rate of VF, VI and ex-ORFs 1-8 strains on PDA plates 24 h after exposure to 100 mJ/cm2 UV for 10 sec. (C) Relative conidial germination rate of VF, VI and ex-ORFs 1-8 strains on PDA plates 24 h after exposure to 42 °C for 60 min. (D) Colony morphology of VF, VI and ex-ORFs 1-8 strains (25 °C, 10 dpi). Scale bar = 6 mm. (E) Conidial yields of VF, VI and ex-ORFs 1-8 strains 10 dpi. Data were present as the mean ± standard deviation (SD) from three replications. Student’s t-test or ANOVA, Groups with no significant differences share the same letter, while those with significant differences are labeled with different letters.
Fig 5.
Interaction between P5 and BbGAP1, and between P5 and BbSDU1 in B. bassiana.
(A) Y2H analysis of the interaction between P5 and BbGAP1. (B) BiFC assays for detecting in vivo protein interactions (scale bar 1 μm). (C) Co-IP assay of P5 with BbGAP1. IP(HA), immunoprecipitation with FLAG and HA. (D) Y2H analysis of the interaction between P5 and BbSDU1. (E) BiFC assays for detecting in vivo protein interactions (scale bar 1 μm). (F) Co-IP assay of P5 with BbSDU1. IP(GFP), immunoprecipitation with FLAG and GFP. Primers used are listed in S4 Table.
Fig 6.
Impacts of BbGap1 on virulence and conidial adhesion, hydrophobicity, and penetration.
(A) Survival of larvae and (B) LT50 after cuticular infection with VF and ΔBbGap1 strains. CK larvae were treated with sterile water. (C) Conidial adherence of VF and ΔBbGap1 strains. (D) Conidial hydrophobicity rates of VF and ΔBbGap1 strains. (E) Relative expression levels in VF and ΔBbGap1 strains of five hydrophobicity genes and three conidial adherence genes, as measured by RT-qPCR. (F) Growth of VF and ΔBbGap1 strains on SDAY plates following penetration of cicada wings (scale bar 6 mm) and quantification of the conidial penetration ability of VF and ΔBbGap1 strains. (G) Relative expression levels in VF and ΔBbGap1 strains of eight cuticle degradation and virulence genes, as measured by RT-qPCR. For each gene, expression in VF was set as 1 and relative expression in VI was calculated. Data were present as the mean ± standard deviation (SD) from three replications. Student’s t-test or ANOVA, * P < 0.05, ** P < 0.01 or *** P < 0.001.
Fig 7.
Impacts of BbSdu1 on virulence and fitness of B.bassiana.
(A) Relative conidial germination rate of VF and ΔBbSdu1 strains on PDA plates 24 h after exposure to 42 °C for 60 min. (B) Relative conidial germination rate of VF and ΔBbSdu1 strains on PDA plates 24 h after exposure to 100 mJ/cm2 UV for 10 sec. (C) Conidial yields of VF and ΔBbSdu1 strains 10 dpi. (D) Survival of larvae and LT50 after cuticular infection with VF and ΔBbSdu1 strains. CK larvae were treated with sterile water. (E) Conidial adherence of VF and ΔBbSdu1 strains. (F) Conidial hydrophobicity rates of VF and ΔBbSdu1 strains. (G) Growth of VF and ΔBbSdu1 strains on SDAY plates following penetration of cicada wings (scale bar 6 mm) and (H) quantification of the conidial penetration ability of VF and ΔBbSdu1 strains. Data were present as the mean ± standard deviation (SD) from three replications. Student’s t-test or ANOVA, * P < 0.05, ** P < 0.01 or *** P < 0.001.
Fig 8.
Mechanistic model illustrating how mycovirus infection enhances the fitness of B. bassiana.
Each BbPmV4-2 genomic segment expresses viral proteins. The viral protein P3 interacts with B. bassiana proteins, upregulating genes involved in UV-B response and enhancing UV-B tolerance. The viral protein P5 interacts with B. bassiana proteins (BbGAP1 and BbSDU1), upregulating genes involved in UV-B and heat shock response, and virulence. This interaction enhances UV-B and heat shock tolerance, and increases the insecticidal ability. Although individual viral proteins were not shown to affect sporulation, BbPmV-4-2 infection as a whole contributes to increased sporulation.