Two secretory T2 RNases contribute to virulence of an insect fungal pathogen due to cytotoxicity and activation of host immune response

doi:10.21203/rs.3.rs-2462532/v1

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Two secretory T2 RNases contribute to virulence of an insect fungal pathogen due to cytotoxicity and activation of host immune response

https://doi.org/10.21203/rs.3.rs-2462532/v1

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The RNase T2 family are ubiquitously distributed in all kingdoms and involved in a variety of physical functions. RNase T2 members are secreted by several pathogens or parasites during infection, playing various roles in pathogen-host interaction. However, functions of those members in biocontrol microbes targeting their hosts are still unknown. Here, two secretory RNase T2 member, BbRNT2 and BbTrv, were found to be important virulence factors in an insect fungal pathogen, Beauveria bassiana. Although no significant effect on fungal virulence was cause by disruption of either BbRNT2 or BbTrv, significantly decreased virulence was examined in double gene disruption mutants. Overexpression of a single gene or both genes dramatically enhanced virulence, while few fungal cells proliferated in hemocoel, accompanying with dramatical reduction in hemocytes as compared to the wild type strain treatments. These results appeared to be due to cytotoxicity of the secreted proteins or excessive activation of the insect immune response. In vitro assays using yeast-expressed proteins verified the cytotoxicity of BbRNT2 and BbTrv against insect cells, including hemocytes from Galleria mellonella larvae and sf9 cells from Spodoptera frugiperda. Those effects of the two proteins depended on their RNases enzyme activities and glycosylation modification. Moreover, the two ribonucleases could also trigger humoral immune responses, with stimulation of reactive oxygen species levels, as well as phenol oxidase, lysozyme and nitric oxide synthase activities. These data demonstrate important roles of two secretory RNase T2 members in virulence of insect fungal pathogens, displaying prospects for improvement of biocontrol agents.

secretory RNase T2

virulence

cytotoxicity

insect fungal pathogen

Contribution of secretory T2 RNases in fungal virulence was characterized in an insect fungal pathogen

Two secretory T2 RNases acted as important virulence factors of an insect fungal pathogen

Overexpression of gene(s) significantly enhanced fungal virulence

Two secretory T2 RNases had cytotoxicity against insects and triggered immune responses

The study demonstrated a prospect of the secretory T2 RNases for improvement of biocontrol agents for pest control

Ribonucleases (RNases) are a family of transferase-type RNases that degrade single-stranded RNA through a 2', 3' cyclic phosphate intermediate, forming nucleotides with a terminal 3’-phosphate as products (EC 3.1.27) (Irie, 1999; Deshpande and Shankar, 2002). These enzymes have been classified into three different groups according to their distribution, pH, and base specificity: RNase A, RNase T1, and RNase T2 families. RNase T1 enzymes are only present in bacterial and fungal organisms with the alkaline (pH 7–8) enzyme activities. RNase A family mainly exist in animals and have weakly acidic activities with pH optima between 6.5 and 7.0. The RNase T2 family members are acidic endonucleases (pH 4–5) without base specificity, which are ubiquitously distributed in all kingdoms and involved in a variety of physical and cellular functions (Luhtala and Parker, 2010; MacIntosh, 2011). Most RNase T2 family members are secreted from cells, or localized in internal compartments such as the lysosome or vacuole, implying their functional diversity (Irie, 1999; Deshpande and Shankar, 2002; Hillwiga et al., 2011; MacIntosh, 2011). Plant RNase T2 enzymes are localized in the endoplasmic reticulum (ER), ER-derived structures, and vacuoles and play a conserved role in eukaryotes in scavenging phosphates/nucleic acids in those organelles (Hillwiga et al., 2011; Floyd et al., 2015; Morriss et al., 2017; Gho et al. 2020; MacIntosh and Castandet, 2020). Moreover, some RNase T2 members are involved in plant defense responses, probably due to their antimicrobial effects (Galiana et al., 1997; Kawano et al., 2002; MacIntosh et al., 2010; Singh et al., 2020). Others, termed S-RNases, participate in gametophytic self-incompatibility in several plant families by preventing self-fertilization (Kao and Tsukamoto, 2004; Liang et al., 2017; Qin et al., 2018). Involvement of RNase T2 enzymes in the innate immune response has also been demonstrated in the invertebrate leech Hirudo verbana (Annelida, Hirudinea) and the Mediterranean mussel Mytilus galloprovincialis. In the leach, the granulocyte-released HvRNASET2 triggers bacterial clumps formation and actively recruits phagocytic macrophages to elicit a rapid and effective eradication of the infecting microorganisms (Baranzini et al., 2020). In the Mediterranean mussel, the ribonuclease RNASET2 is involved in the inflammatory response that participates in recruitment and activation of macrophages in response challenge of bacterial pathogens (Parisi et al., 2022). The human RNase T2 proteins have been demonstrated to be involved in neurodevelopment, and tumor progression and growth. Involvement of RNase T2 in neurodevelopment may be due to their enzyme activities that clear RNAs to modulate the immune response, whereas the participation in tumor progression and growth control seems to be independent of their catalytic activities (Luhtalaand Parker, 2010; Acquati et al., 2011, 2013; Patel et al., 2012).

Secretory RNase T2 enzymes have been characterized in several pathogens and parasites and they play an important role in interactions with their hosts. One of the envelope glycoproteins, RNase T2 (termed E^rns), in the classic swine fever virus (CSFV) and bovine viral diarrhoea virus (BVDV), is secreted by infected cells, which shows cytotoxic effects against the lymphocytes of various host species, suggesting a role in inhibition of the host immune response (Hulst et al., 1994; Bruschke et al., 1997). The parasites Schistosoma mansoni and S. japonicum (commonly referred to as blood flukes) lays their eggs in the mammalian hosts during infection. The eggs secrete an RNase T2 family member (termed Omega-1 or SjCP1412) to drive polarization of the host Th2 immune response that aids in the egg’s excretion and transmission to a molluscan intermediate host (Pearce, 2005; Ke et al., 2017). An emerging bacterial pathogen Acinetobacter baumannii secretes an RNase T2 family member that is involved in motility and the ability of the pathogen to colonize inanimate surfaces (Jacobs et al., 2014). The human fungal pathogen Aspergillus fumigatus produces an RNase T2 during infection, which acts as an immune regulatory factor in allergic bronchopulmonary aspergillosis (ABPA), an allergic immunological response, primarily through augmentation of M2 macrophages polarization and thus induction of a Th2 immune response (Chen et al., 2020). In the plant fungal pathogen Ustilago maydis, two T2 ribonucleases Nuc1 and Nuc2 are secreted during infection and contribute to fungal virulence, as well as the completion of the pathogenic lifecycle. These effects may be due to their enzyme function in utilization of the extracellular RNA (presenting in maize apoplast) as a phosphate source at least (Mukherjee et al., 2020). A secretory RNase T2, FoRnt2, of Fusarium oxysporum, is localized in both the cytoplasm and nucleus in plant cells and contributes to fungal virulence possibly through the degradation of host RNA (Qian et al., 2022).

RNase T2 members are highly conserved in fungal species including biocontrol fungi, such as Metarhizium robertsii and Beauveria bassiana (Qian et al., 2022). However, the details of those enzymes in biocontrol fungi targeting their hosts are still unclear. B. bassiana is an economically important insect fungal pathogen that has been developed as a biocontrol agent for control of a variety of insect pests (Wang and Feng, 2014). In our previous study, two RNase T2 members were isolated as secreted proteins from B. bassiana (Yang et al., 2017) and they shared only 28.9% identity in amino acid sequences. In this study, we characterized roles of the two proteins in the interaction of B. bassiana with insects. Our data demonstrated that both proteins are important fungal virulence factors via their cytotoxicity to insect cells, including somatic cells and hemocytes, and activation of the immune response, all of which depended on their enzyme activities. Loss of one or two genes led to a significant reduction in virulence, whereas, overexpression strains (one or two genes) showed dramatically increased virulence, suggesting potential values of the two proteins or genes in biocontrol of insect pests.

Fungal and bacterial strains and insects

Wild type Beauveria bassiana strain Bb2860 (CGMCC 7.34) and its derived genetically modified strains were routinely cultured as described previously (He et al., 2015). Pichia pastoris GS115 (Invitrogen, USA) was used for expression of BbRNT2 and BbTrv and their mutated proteins as described previously (Lu et al., 2021). Escherichia coli DH5ɑ and Agrobacterium tumefaciens were employed for DNA manipulations and fungal transformations, respectively.

Silkworm (Bombyx mori) larvae were used for preparation of insect hemolymph and cuticle extracts as described previously (Deng et al., 2022). Last-instar larvae of Galleria mellonella, were used for bioassay and investigation of insect immune response. The G. mellonella hemocytes cultured in Schneider’s Insect Medium (Sigma, UK) and Spodoptera frugiperda ovarian epithelial cells, sf9 cells cultured in cultured in Sf-900™ II SFM medium (Invitrogen, USA), were used for examination of cytotoxicity.

Molecular Manipulation

All the primers used for gene manipulation are listed in Table S1. Single gene (BbRNT2 or BbTrv) disruption was performed using the herbicide phosphinothricin resistance bar gene with homologous recombination as described previously (He et al., 2015). For disruption of double genes, the BbTrv homologous recombination cassette was generated using the herbicide sulfonylurea resistance sur gene based on pK2-sur (Wang et al., 2018), which was introduced into the BbRNT2 disruption strain (ΔBbRNT2) using A. tumefaciens-mediated method (Ma et al., 2009). To reverse complement the single gene disrupted mutants, wild type genes, BbRNT2 and BbTrv, were separately cloned into the pK2-sur vector, which was then introduced into their gene disruption mutants, ΔBbRNT2 and ΔBbTrv.

For overexpression of the genes, cDNAs of BbRNT2 and BbTrv were cloned into pBARGPE1 under the control of Aspergillus nidulans gpd promoter, PgpdA. The resultant vectors were separately transformed into B. bassiana wild type strain as described previously (Ying and Feng, 2006), generating BbRNT2^OE and BbTrv^OE strains. To simultaneously overexpress the two genes, the Pgpd::BbTrv::trpC element was amplified from the BbTrv^OE vector and inserted into pK2-sur, which was introduced into BbRNT2^OE strain as described previously (Ma et al., 2009), forming the DOE strain.

To detect BbRNT2 and BbTrv expression, eGFP was separately fused to the promoters of the two genes. Briefly, the promoter sequences (855 bp for BbRNT2 and 1940 bp for BbTrv) were amplified with the primers shown in Table S1 and inserted into pK2surPB3-GFP (Lu et al., 2021) to replace the PB3 promoter. To examine protein distribution in fungal cells, eGFP was fused to the 3'-end of the gene. Briefly, the full genes of BbRNT2 and BbTrv were separately amplified using the primers (Table S1) and cloned into pK2surPB3-GFP to replace the PB3 promoter. All the resultant vectors were separately introduced into B. bassiana wild type strain using the A. tumefaciens-mediated method (Ma et al., 2009).

All the transformants were selected using the phosphinothricin (bar) or/and sulfonylurea (sur) resistance markers (Lu et al., 2021) and then verified using PCR, RT-PCR (gene disruption and reverse complement strains) or RT-qPCR (overexpression strains).

Rt-pcr, Rt-qpcr And Blotting

RT-PCR and RT-qPCR were performed as described previously (Lu et al., 2021). Loss or restoration of gene transcription in the target gene disruption mutants or its complementation strains was verified using RT-PCR analysis from 1/4 SDY cultures for 48 h at 26°C with 18S rRNA (BBA_07911) as a reference gene. RT-qPCR was used to evaluate transcription patterns of BbRNT2 and BbTrv in the wild type and overexpression strains, which was performed using a quantitative real-time PCR kit (Bio-Rad, CA) and 18S rRNA as the reference gene for normalization of candidate gene expression levels as described previously (Luo et al., 2012; He et al., 2015). Transcription patterns of BbRNT2 and BbTrv in the wild type strain under different nutrient and oxidative stress conditions were evaluated as described previously (Lu et al., 2021). Transcription levels in the overexpression strains as compared to the wild type strain were detected from 1/4 SDY cultures for 72 h at 26°C. Primer sequences for all the genes examined are given in Table S1.

Western blotting analysis was performed according to the manufacturer’s instructions (Clarity Max Western ECL Substrate; Bio-Rad, Philadelphia, PA) and routine methods as described previously (Lu et al., 2021). Relative amount of the proteins was measured by densitometric analyses of bands using the ImageJ software (Jensen, 2013).

Yeast–expression And Enzyme Assays

BbRNT2, BbTrv and their active site-mutated proteins were expressed in P. pastoris GS115 as described previously (Lu et al., 2021; Deng et al., 2022). To generate mutated proteins, primers were designed to introduce the mutated sites, in which the active sites 89H (histidine), 92W (tryptophan), 169H (histidine), 170E (glutamic acid), 73K (lysine) and 174H (histidine) in BbRNT2 and 69H (histidine), 72W (tryptophan), 126H (histidine), 127E (glutamic acid), 130K (lysine) and 131H (histidine) in BbTrv were all mutated to A (alanine). Briefly, cDNA fragments of BbRNT2 were separately amplified with the primer pairs BbRNT2-F1/R1, BbRNT2-F2/R2 and BbRNT2-F3/R3, and then fused together by overlap PCR, termed iBbRNT2. Similarly, cDNA fragments of BbTrv were amplified with the primer pairs BbTrv-F1/R1, BbTrv-F2/R2 and BbTrv-F3/R3, and then fused by overlap PCR, and designated iBbTrv. All the primers are shown in Table S1.

The expressed proteins were purified using a MagneHis Protein Purification System (Promega) according to the manufacturer’s instructions. Protein concentrations were determined using BCA kits (Generay Biotech, China). Ribonuclease activities of the proteins were assayed using total RNA from G. mellonella larvae and B. bassiana. Briefly, 1 µg of RNA was dissolved in 18 µL 50 mM Tris-HCl buffer (pH 7.0) and reacted at 37^oC for 30 min after addition of 2 µL 2 mg/mL protein. The reaction mixture was separated in 1% agarose gel, and stained with 1/10000 (v/v) Gelred (Biomed, China) and viewed under a Gel imager as described previously (Ke et al., 2017).

Insect Bioassays And Detection Of The Immune Response

Insect bioassays were performed using last-instar larvae of G. mellonella via two methods as described previously (Zhao et al., 2019): 1) inoculation via the larval cuticle (natural route of infection) by spraying 1 mL conidia suspension (5×10⁷ spores/ mL); 2) injection of 2 µL conidia suspensions (5×10⁷ spores/ mL) via the second proleg (by-passing the cuticle). The controls in both assays were treated with 0.05% (v/v) Tween-80. Mortality was recorded every 12 h for assay 1) and 6 h for assay 2). Each treatment was performed with three replicates of 30 insects each, and the experiments were repeated thrice.

To detect the immune response after inoculation, blackened nodules on the cuticle were observed and counted after inoculation. Moreover, infected larvae were bled to examine the interaction of fungal cells and hemocytes. The numbers of fungal cells (hyphal bodies) and hemocytes were counted.

Assays For Cytotoxicity Against Insect Cells

Insect hemocytes were collected from the blood of healthy G. mellonella larvae after surface sterilization by immersion in 75% ethanol for 5 min. Hemocytes were immediately harvested in 200 µL Schneider’s insect medium (Sigma, UK) containing 0.6 mg/mL L-cysteine and cultured at 28°C. Hemocytes were sampled for observation under a microscope at intervals of 15 min after addition of the expressed proteins at a final concentration of 34 µg/mL. Alternatively, S. frugiperda sf9 cells cultured in Sf-900™ II SFM medium (Invitrogen, USA) were adjusted to 0.4×10⁶ cells/mL and used for assessment of cytotoxicity of yeast-expressed BbRNT2 and BbTrv at a final concentration of 34 µg/mL. Cells treated with 0.1 M PBS (pH 8.0) at the same volume were used as controls.

Cell viability was assayed using a Cell Counting Kit 8 (CCK-8, Sangon Biotech, China) that uses a water-soluble tetrazolium salt to quantify the number of live cells by producing an orange formazan dye upon bio-reduction in the presence of an electron carrier. Briefly, 5×10⁴ cells/mL cells were treated with protein at a final concentration of 34 µg/mL. At intervals of 15 min, 100 µL cells were sampled and labeled using 10 µL CCK-8 solution, and were immediately used to test absorbance at 450 nm using a microplate reader. Cells treated with 1× PBS were used for controls. Cell viability was calculated by the following formula: At/Ac ×100, where At and Ac were the absorbance values of protein-treated cells and controls, respectively. The experiments were repeated three times.

Assays for phenoloxidase (PO) activities, reactive oxygen species (ROS), nitric oxide synthase (NOS) and lysozyme activities

Insect phenoloxidase (PO) activity weas assayed according to a method described previously (Yang et al., 2014). Reactive oxygen species (ROS) in haemolymph were evaluated by examination of H₂O₂ levels using a Hydrogen Peroxide Colorimetric/Fluorometric Assay Kit (BioVision, USA) according to the manufacturer’s instructions as described previously (Pan et al., 2012). Nitric oxide synthase (NOS) activity was measured using a T-NOS assay kit (Njjcbio, China) according to the manufacturer’s instructions. Lysozyme activity was assayed using the fluorogenic substrate 4-methylumbelliferyl-β-D-N, N’, N”-tetraacetylchitotriose as described previously (Fruttero et al., 2016). The reaction mixture was assayed at 355 and 460 nm as the respective excitation and emission wavelengths using a plate reader, respectively. Enzyme activity was expressed as relative fluorescence units (RFU)/µg protein/20 min.

Data analysis

The survival data in insect bioassays were plotted as Kaplan–Meyer curves, and a log-rank test was used to analyze the differences between groups. The median lethal time (LT₅₀) was calculated using SPSS 17.0. The differences between two groups and between more than two groups were tested using the Student’s t test (t-test) and one-way analysis of variance (ANOVAs) with a subsequent LSD test, respectively. All data are shown as the results obtained from three independent experiments with standard deviations (SD) of the mean. The values of P < 0.05 were considered statistically significant.

BbTrv and BbRNT2 have RNase T2 features

In our previous study, two secretory proteins, annotated as ribonuclease T2 family protein (tag locus: BBA_00428) and ribonuclease Trv (tag locus: BBA_00766), are isolated from cell surface/ secretory protein pools of conidia (from SDAY) and/or blastospores (from SDY), respectively (Yang et al., 2017). SMART program analysis revealed that both proteins contained a ribonuclease T2 domain, harboring two conserved motifs, CASI and CASII, vectoring three histidine active sites, which were named BbRNT2 (BBA_00428) and BbTrv (BBA_00766), respectively. BbRNT2 consists of 328 and amino acids containing four putative N-glycosylation sites and seven O-glycosylation sites, with predicted signal peptide sequences in residues 1–19 in the N-terminal region, while BbTrv contains 257 amino acids possessing a secretion signal peptide domain in residues 1–17 in the N-terminal region that contains two putative N-glycosylation sites and two O-glycosylation sites (Fig. 1a). The two proteins shared only 28.9% of identity in their amino acid sequences. Sequence alignment of the ribonuclease T2 domain of BbRNT2 and BbTrv with T2 family ribonuclease proteins from other fungal species showed that all contained two highly conserved CASI and CASII motifs, which contained six putative active sites, including three histidine sites (MacIntosh 2011) (Fig. 1b). Phylogenetic tree analysis revealed that BbRNT2 and BbTrv were classified into two main ribonuclease T2 member clades. Many fungal species, including insect fungal pathogens and biocontrol fungi, such as B. brongniartii, Cordyceps sp, Akanthomyces lecanii, Trichoderma sp, and Metarhizium sp, possess both BbRNT2 and BbTrv homologs. Yeast RNY1, plant fungal pathogens Fusarium oxysporum FoRnt2 (Qian et al., 2022) and Ustilago maydis Nuc1 and Nuc2 (Mukherjee et al., 2020) were classified into the BbTrv clade, whereas member of Colletotrichum viniferum was classified into the BbRNT2 clade (Fig. 1c).

To reveal the enzyme activities of BbTrv and BbRNT2, the two proteins were separately expressed in Pichia pastoris, in which the putative N-glycosylation sites were verified according to changes in the molecular weight that caused by addition of the deglycosylation enzyme, Endo H (specific for N-glycosylation) (Fig. 1d). Total RNA isolated from B. bassiana and the insect Galleria mellonella were used to test enzyme activities. Both BbTrv and BbRNT2 displayed high RNA degradation activities. However, those enzymes treated by Endo H lost enzyme activity (Fig. 1e), suggesting that glycosylation of the proteins is crucial for their enzyme activities. To examine the effects of putative active sites on enzyme activities, those predicted active sites were all mutated to A (alanine) and expressed in P. pastoris, termed iBbRNT2 and iBbTrv. These proteins also lost RNA degradation activities (Fig. 1e).

BbTrv and BbRNT2 were induced by insect hemolymph and oxidative stress

To reveal expression patterns of BbTrv and BbRNT2, RT-qPCR analysis and detection of GFP fluorescence in B. bassiana, in which GFP was separately controlled by BbRNT2 and BbTrv promotors (termed PBbRNT2::GFP and PBbRNT2::GFP), were performed. RT-qPCR analysis indicated that both genes displayed similar transcription patterns, being slightly upregulated in fungal cultures from BS (basic salt broth), PDB (potato dextrose broth), 1/4 SDY (1:4-diluted Sabouraud dextrose broth supplemented with 1% (wt/vol) yeast) and SC (silkworm cuticle-contained broth) as compared to those from CZB (Czapek-Dox Broth) (~ 0.6-2.7-fold), and dramatically upregulated by ~ 5.0-8.3-fold in SH (silkworm hemolymph-contained broth) cultures. Moreover, transcription of both genes was significantly induced by the oxidizing agents, H₂O₂, MND (menadione) and tBHP (tert-Butyl hydroperoxide) (~ 3-7.9-fold) (Fig. 2a). RT-qPCR data were confirmed by detection of GFP fluorescence in fungal cells of the PBbRNT2::GFP and PBbRNT2::GFP strains (Fig. 2b). These results suggested roles of the two proteins in the pathogen-insect interactions and oxidative stress response.

Bbtrv And Bbrnt2 Distribute In Cytoplasm And Secreted In Response Insect-derived Nutrients

To examine distributions of the BbTrv and BbRNT2 in fungal cells and their secretion characteristics, the GFP gene was separately fused to 3'-ends of the two genes, termed BbTrv::GFP and BbRNT2::GFP. Obvious GFP fluorescence signals were seen in the cytoplasm of vegetative mycelium of BbTrv::GFP and BbRNT2::GFP strains, including aerial hyphae (AHY) and liquid hyphae (LHY), while only slightly weak fluorescence was detected in their blastospores and insect hemolymph-derived in vivo hyphal bodies, but and was absent in conidia (Fig. 2c). These results suggested that BbTrv and BbRNT2 distribute in the cytoplasm of B. bassiana cells, especially in cells of vegetative mycelium. Both BbTrv::GFP and BbRNT2::GFP proteins were detected in the broth of all cultures, including 1/4SDY, silkworm cuticle-contained broth (SC) and silkworm hemolymph-contained broth (SH), and in hemolymph of the infected insects (CS) for 72 h using Western blotting with an anti-GFP antibody following removal fungal cells. Compared to secretory BbTrv::GFP and BbRNT2::GFP in 1/4 SDY, more proteins were probed in SC, SH and CS, with increases by ~ 1.8-3.5-fold, 2.6-4.8-fold and 3.1-6.0-fold (relative gray values per 10 µg total proteins), respectively (Fig. 2d), suggesting possible roles of the two secretory proteins in utilization of insect-derived nutrients.

Overexpression of BbTrv and BbRNT2 increase fungal growth and tolerance to oxidative stress

To evaluate the functions of BbTrv and BbRNT2 in fungal virulence, a single gene disruption (ΔBbRNT2 or ΔBbTrv) and double genes (DKO) disruption mutants, reverse complementation strains (BbRNT2^RC or BbTrv^RC), and strains overexpressing a single gene (BbRNT2^OE or BbTrv^OE) and the both genes (DOE) were generated (Fig. S1). Significantly increased colony growth was examined on 1/4 SDAY and Czapek agar (CZA) in those overexpression strains, BbRNT2^OE, BbTrv^OE and DOE strains as compared to the wild type strain. However, no effects on colony growth were seen by disruption of single gene (ΔBbRNT2 or ΔBbTrv) or two genes (DKO) (Fig. 3a and b). However, no obvious difference in growth was examined on K₂HPO₄-free CZA in all the overexpression strains and other strains, suggesting roles of BbTrv and BbRNT2 in phosphate utilization (Fig. 3a and b). To examine sensitivity to oxidative stresses, all strains were cultured on CZA containing 2.64 mM H₂O₂ or 60 µM menadione (MND). All of overexpression strains displayed increased tolerance to both oxidant agents, with generated relative growth inhibition (RGI) rates decreased by 45.2% -51.9% for H₂O₂-stress and 46.9% -53.1% for MND as compared to the wild type strain (P < 0.01) (Fig. 3c and d). However, increased sensitivities were detected for the DKO strain, with produced RGI rates increased by 22.1% for H₂O₂-stress and 32% for MND as compared to the wild type strain (P < 0.05). Whereas, no obvious effect was noticed for single gene knockout mutant, ΔBbRNT2 or ΔBbTrv (Fig. 3c and d). Unless otherwise noted, in all phenotypes examined, the complemented strains, BbRNT2^RC and BbTrv^RC, were identical to the wild type strain.

Bbtrv And Bbrnt2 Contribute To Fungal Virulence

Insect bioassays were performed using last-star larvae of G. mellonella using either topical inoculation by spraying conidia suspension (via the cuticle) or micro-injection of the conidia suspension into the insect hemocoel (by-passing the cuticle). There was no obvious difference in fungal virulence between ΔBbRNT2, ΔBbTrv and the wild type strains in both assays, however, a significant decrease in mortality of infected insects was examined after disruption of both BbRNT2 and BbTrv (DKO) as compared to the wild type strain. More rapid death of infected insects was seen for the overexpression strains, but no significant difference was seen in the mortality caused by BbRNT2^OE, BbTrv^OE or DOE strains (Fig. 4a and b). In topical bioassays, LT₅₀ (the mean lethal time) increased from 129.8 ± 1.3 h for the wild type strain to 149.4 ± 0.7 h for DKO (P < 0.01), whereas decreased to 119.1 ± 0.8 h for BbRNT2^OE, 121.8 ± 2.1 h for BbTrv^OE and 115.9 ± 0.8 h for DOE at a concertation of 5×10⁷ conidia/mL. Similar results were also examined in intra-hemocoel injection bioassays (Fig. S2).

More blackened nodules occurred on the cuticle of larvae infected by overexpression strains as compared to insects inoculated with other strains in both assays. Although larvae killed by any one of the three overexpression strains were pigmented finally, pigmentation was significantly delayed as compared to those treated by the wild type and gene disruption strains after topical inoculation (Fig. 4c). However, only blackened cadavers were caused after micro-injection inoculation of the overexpression strains (Fig. 4d). Although the three overexpression strains killed insects more rapidly than other strains, little mycelia penetrated outwards from the insect cadaver for growth and sporulation as compared to other strains at 192 h after inoculation in topical bioassays and 144 h in intra-hemocoel injection bioassays (Fig. 4c and d), suggesting impaired abilities to penetrate the cuticle outwards.

To understand the relationship of fungal virulence and fungal development within the insect haemocoel, a subset of insects in the intra-hemocoel injection bioassays were bled at 24, 36 and 48 h post-inoculation and examined under a microscope. The penetrated hyphae of all strains were attacked and melanized by hemocytes at 24 h, and escaped from hemocyte encapsulation after 36 h (Fig. 4g). However, count of hyphal bodies of all three overexpression strains at 48 h and 60 h were dramatically lower than those of wild type and other strains (Fig. 4h). It is interesting to note that the hemocyte numbers of insects inoculated with the three overexpression strains were also significantly lower than in treatments with other strains at 36 and 48 h post inoculation, implying damage by the two secreted proteins to hemocytes. Whereas, counts of double gene disruption mutant (DKO) hyphal bodies were significantly lower than those of the wild type strain at examined hours, but numbers of hemocytes were high than in those treatments of the wild type strain (Fig. 4h), which was consistent with its reduced virulence.

Bbtrv And Bbrnt2 Display Cytotoxicity That Depend On Their Rnases Enzyme Activities And Glycosylation

Because overexpression of BbRNT2 or/and BbTrv resulted in significant increases in fungal virulence, but were accompanied by few fungal cells growing in the hemocoel and dramatic reduction in hemocyte counts in the infect insects, we speculated that secreted BbRNT2 and BbTrv from those strains were responsible for the cytotoxicity against hemocytes. To verify the speculation, yeast-expressed proteins (at a final concentration of 34 µg/mL) were separately added to the harvested G. mellonella hemocytes and cultured for different time. After culturing for 15–45 min with BbRNT2 or BbTrv, abnormal cell morphology was observed with increased numbers of broken hemocytes seen (Fig. 5a). Cell viability assays revealed that the percentages of surviving hemocytes decreased from 100% for control to 62–65% for 15 min, 56–58% for 30 min, 45–49% for 45 min, 37–40% for 60 min, 24–25% for 75 min, and 11–12% for 90 min for both proteins, suggesting the cytotoxicity of the two proteins. However, hemocyte viability was not affected when cultured with proteins with mutated active sites (iBbRNT2 and iBbTrv), although cell aggregation was seen in the treatments with iBbRNT2 for 30 min to 75 min (Fig. 5b), suggesting that the cytotoxicity of the two proteins depended on their RNases enzyme activities. Similar results were also demonstrated with ovarian epithelial cells of S. frugiperda, sf9 cells, representing insect somatic cells. Cell morphological changes and aggregation were observed after treatment with BbRNT2 and BbTrv for 30 min to 60 min, and the number of cells dramatically decreased after treatment for 30 min to 120 min, a result that was confirmed by cell viability assays (Fig. 5c and d). Moreover, BbRNT2 and BbTrv lost their cytotoxicity after removal of lost N-glycosylation modification after treatment with the deglycosylation enzyme Endo H (Fig. S3), suggesting that glycosylation sites are essential for their cytotoxicity.

Bbrnt2 And Bbtrv Trigger Humoral Immune Responses

To reveal additional roles of BbRNT2 and BbTrv besides their cytotoxicity, we examined the effects of the two proteins on the humoral immune response. Four µL yeast-expressed proteins (2 µg/mL) were separately injected into last-star larvae of G. mellonella. The treated insects were bled at intervals of 1 h during 6 h after injection to determine phenoloxidase (PO) activity, reactive oxygen species (ROS), lysozyme activity and nitric oxide synthase (NOS) activity. PO activities were increased from 1 to 3 h and decreased after 3 h of injection for all treatments, including the 1 × PBS (pH 8.0)-treated controls. However, the activities of BbRNT2 and BbTrv treatments were significantly higher than those of controls during the examined time points (Fig. 6a). ROS levels, lysozyme and NOS activities were also significantly stimulated by BbRNT2 and BbTrv, being ~ 34-56.2%, 38.7–59.1% and 42.8–97.6% higher than those of controls during the examined time points, respectively (P < 0.01 for each, Fig. 6b, c and d). In contrast, no obvious effects on PO activities, ROS levels, lysozyme and NOS activities were caused by iBbRNT2 and iBbTrv-treatments as compared to their controls (injection with 1 × PBS (pH 8.0) (Fig. 6). These results suggested that both BbRNT2 and BbTrv trigger humoral immune responses that depend on their RNases enzyme activities.

RNase T2 members are only ribonuclease family that is widely distributed in all kingdoms from viruses to animals (Luhtala and Parker, 2010; MacIntosh, 2011). Most of RNase T2 members are secreted proteins that participate in a variety of physical and cellular functions. In addition to their conserved function in ribosomal RNA (rRNA) and inorganic phosphate source recycling (Floyd et al., 2015; Morriss et al., 2017; Gho et al. 2020; MacIntosh and Castandet, 2020), some members are involved to plant self-incompatibility (Liang et al., 2017; Qin et al., 2018), defense responses (MacIntosh et al., 2010; Singh et al., 2020; Baranzini et al., 2020; Parisi et al., 2022) due to their selective cytotoxicity. Moreover, some viral T2 RNases act as cytotoxins to suppress the host immune system (Hulst et al., 1994; Bruschke et al., 1997). Several fungal pathogens including the phytopathogens U. maydis and F. oxysporum, and the human pathogen A. fumigatus secrete RNase T2 during infection that are required for full fungal virulence, which are seemly due to their enzyme activities in utilization of plant RNA presenting in plant apoplast, cytoplasm and nucleus (Mukherjee et al., 2020; Qian et al., 2022). The enzyme in A. fumigatus apparently modulates the human immune response (Chen et al., 2020). Our results demonstrated that two secretory RNase T2 members of the animal (insect) fungal pathogen, B. bassiana, contribute to virulence primarily via their cytotoxicity to host cells, as well as activation of insect humoral immune responses that depend on their RNases enzyme activities and glycosylation modification. However, the details of those enzymes entering host cells, as well as those reaching the plant nucleus from plant fungal pathogens, are still unclear and need to be revealed in the future work.

Several plant pathogens, including Blumeria graminis, Zymoseptoria tritici, Setosphaeria turcica, Fusarium graminearum and Verticillium dahlia, secrete ribonuclease (RNase) effectors, homologs of ribotoxins, induce plant cell death and interfere plant immune systems in various ways, although the underlying mechanisms of them acting on plant immune system are unclear (Kettles et al., 2018; Pennington et al., 2019; Yang et al., 2021; Tariqjaveed et al., 2021; Yin et al., 2022). Our data showed that the two T2 ribonucleases of B. bassiana triggered insect humoral immune responses. The possibility of them acting as effectors to activate immune responses could not been excluded. Further experiments should be designed to identify the possible target proteins and address the issues.

In our study, disruption of a single gene, either BbRNT2 or BbTrv, did not affect B. bassiana virulence, whereas deletion of both genes led to a significant reduction in virulence. Overexpression of single gene or both genes significantly increased fungal virulence, although no significant difference was examined in insect mortality in overexpression of single gene and both genes. These data suggested that BbRNT2 and BbTrv have functional redundancy or complementation in fungal pathogenicity. Similar results are also showed in the plant fungal pathogen U. maydis, although overexpression of gene(s) do not affect the fungal virulence (Mukherjee et al., 2020). Our data showed overexpression of gene(s) increased colony growth on agar plates, which was seemingly associated with the function of ribonucleases in utilization of inorganic phosphate source in the media, because the effects disappeared when the fungal strains were cultured on inorganic phosphate-free media. Thus, the utilization of phosphate sources in the infected insect should be included. Although overexpression strains killed insects more rapidly than wild type strain, contrary to expectation, significantly decreased hyphae growing in the infected insect haemocoel was examined in all the overexpression strains as compared to the wild type strain. We speculated that overexpression strains might overproduce RNase T2, BbRNT2 or/and BbTrv that activated the immune response, and produce more cytotoxic molecules, such as reactive oxygen/nitrogen species (ROS/RNOS), NO and polyphenol oxidases (PPOs), and possibly produce antimicrobial peptides (Sugumaran, 2002; Cerenius and Söderhäll, 2004; González-Santoyo and Córdoba-Aguilar, 2012; Wojda, 2017), that inhibited hyphal growth. As a result, the overexpression strains displayed impairment of cuticle penetration outwards from the cadavers for growth and conidiation, implying the potential for development of fungal insecticides due to reduced environmental persistence as the genetically engineered fungi. A similar phenomenon has been also demonstrated in another genetically engineered insect fungal pathogen, Metarhizium anisopliae, constitutively expressing a cuticle degrading protease (Pr1) gene, that significantly increase virulence due to its toxic effects, but the fungus file to penetrate the cuticle outwards from the cadavers for conidiation (St Leger et al., 1996).

It was noticed that expression of BbRNT2 or BbTrv was dramatically induced by oxidative stress, which was consistent with the phenotypes of the overexpression strains that displayed increased tolerance to oxidative agents. However, the underlying mechanism is a puzzle. The role of secretory T2 RNase, Rny1, in response to stress, is also demonstrated in the yeast Saccharomyces cerevisiae, which is seemly associated with the permeability or stability of membranes (MacIntosh et al., 2001). Specific RNAs are implicated in binding to biological membranes and affect the membrane permeability to ions (Khvorova et al., 1999), implying a possibility that the yeast secretes RNY1 to degrade membrane-bound RNAs for regulation of membrane permeability in response to stress. Our results showed that both secretory BbRNT2 and BbTrv signals (labeled with GFP) were observed in the cytoplasm, but the possibility of distribution in the membrane could not be excluded. Further experiments should be designed to unravel the mystery.

Overall, our data demonstrated that two secretory RNase T2 members act as important virulence factors in insect fungal pathogens due to their cytotoxicity and activation of insect humoral immune responses. The results suggest prospects for improvement of biocontrol agents.

Funding

This work was supported by a grant from the National Nature Science Foundation of China (32072485).

Author contributions

YY, ZL and YZ designed the study. WD cultured the sf9 cells and provided protocols. YY, JD, HW, TL, YJ and XP performed the experiments and analyzed data. YY, WD, ZL, and YZ revised to the subsequent manuscript. All the authors have read and approved the manuscript.

Conflict of interest The authors declare that they do not have any conflicts of interest.

Consent to participate Consent was given by all the participants included in the study.

Consent for publication All of the authors consent to the publication of this manuscript in Journal of Pest Science.

Ethical approval This study does not contain any experiments using any animal species that require ethical approval.

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No competing interests reported.

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Two secretory T2 RNases contribute to virulence of an insect fungal pathogen due to cytotoxicity and activation of host immune response

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Abstract

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Key Message

Introduction

Materials And Methods

Fungal and bacterial strains and insects

Molecular Manipulation

Rt-pcr, Rt-qpcr And Blotting

Yeast–expression And Enzyme Assays

Insect Bioassays And Detection Of The Immune Response

Assays For Cytotoxicity Against Insect Cells

Data analysis

Results

BbTrv and BbRNT2 have RNase T2 features

Bbtrv And Bbrnt2 Distribute In Cytoplasm And Secreted In Response Insect-derived Nutrients

Bbtrv And Bbrnt2 Contribute To Fungal Virulence

Bbtrv And Bbrnt2 Display Cytotoxicity That Depend On Their Rnases Enzyme Activities And Glycosylation

Bbrnt2 And Bbtrv Trigger Humoral Immune Responses

Discussion

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