Comparative Transcriptomics of the Entomopathogenic Fungus Beauveria bassiana Grown on Aerial Surface and in Liquid Environment.

Beauveria bassiana, the causative agent of arthropod, proliferates in the host hemolymph (liquid environment) and shits to saprotrophic growth on the host cadaver (aerial surface). In this study, we used transcriptomic analysis to compare the gene expression modes between these two growth phases. Of 10,366 total predicted genes in B. bassiana, 10,026 and 9985 genes were expressed in aerial (AM) and submerged (SM) mycelia, respectively, with 9853 genes overlapped. Comparative analysis between two transcriptomes indicated that there were 1041 up-regulated genes in AM library when compared with SM library, and 1995 genes were down-regulated, in particular, there were 7085 genes without significant change in expression between two transcriptomes. Furthermore, of 25 amidase genes (AMD), BbAMD5 has high expression level in both transcriptomes, and its protein product was associated with cell wall in aerial and submerged mycelia. Disruption of BbAMD5 significantly reduced mycelial hydrophobicity, hydrophobin translocation, and conidiation on aerial plate. Functional analysis also indicated that BbAmd5 was involved in B. bassiana blastospore formation in broth, but dispensable for fungal virulence. This study revealed the high similarity in global expression mode between mycelia grown under two cultivation conditions.

Cla4A, a Novel Regulator of Gene Expression Networks Required for Asexual and Insect-Pathogenic Lifecycles of Beauveria bassiana.

Cla4, an orthologous p21-activated kinase crucial for non-entomopathogenic fungal lifestyles, has two paralogs (Cla4A/B) functionally unknown in hypocrealean entomopathogens. Here, we report a regulatory role of Cla4A in gene expression networks of Beauveria bassiana required for asexual and entomopathogenic lifecycles while Cla4B is functionally redundant. The deletion of cla4A resulted in severe growth defects, reduced stress tolerance, delayed conidiation, altered conidiation mode, impaired conidial quality, and abolished pathogenicity through cuticular penetration, contrasting with no phenotype affected by cla4B deletion. In ∆cla4A, 5288 dysregulated genes were associated with phenotypic defects, which were restored by targeted gene complementation. Among those, 3699 genes were downregulated, including more than 1300 abolished at the transcriptomic level. Hundreds of those downregulated genes were involved in the regulation of transcription, translation, and post-translational modifications and the organization and function of the nuclear chromosome, chromatin, and protein-DNA complex. DNA-binding elements in promoter regions of 130 dysregulated genes were predicted to be targeted by Cla4A domains. Samples of purified Cla4A extract were proven to bind promoter DNAs of 12 predicted genes involved in multiple stress-responsive pathways. Therefore, Cla4A acts as a novel regulator of genomic expression and stability and mediates gene expression networks required for insect-pathogenic fungal adaptations to the host and environment.

Comprehensive Insights into the Remarkable Function and Regulatory Mechanism of FluG during Asexual Development in Beauveria bassiana.

Asexual development is the main propagation and transmission mode of Beauveria bassiana and the basis of its pathogenicity. The regulation mechanism of conidiation and the key gene resources for utilization are key links to improving the conidia yield and quality of Beauveria bassiana. Their clarification may promote the industrialization of fungal pesticides. Here, we compared the regulation of morphology, resistance to external stress, virulence, and nutrient utilization capacity between the upstream developmental regulatory gene fluG and the key genes brlA, abaA, and wetA in the central growth and development pathway. The results showed that the ΔbrlA and ΔabaA mutants completely lost the capacity to conidiate and that the ΔwetA mutant had seriously reduced conidiation capacity. Although the deletion of fluG did not reduce the conidiation ability as much as deletions of brlA, abaA, and wetA, it significantly reduced the fungal response to external stress, virulence, and nutrient utilization, while the deletion of the three other genes had little effect. Via transcriptome analysis and screening the yeast nuclear system library, we found that the differentially expressed genes in the ΔfluG mutants were concentrated in the signaling pathways of ABC transporters, propionate metabolism, tryptophan metabolism, DNA replication, mismatch repair, and fatty acid metabolism. FluG directly acted on 40 proteins that were involved in various signaling pathways such as metabolism, oxidative stress, and cell homeostasis. The analysis indicated that the regulatory function of fluG was mainly involved in DNA replication, cell homeostasis, fungal growth and metabolism, and the response to external stress. Our results revealed the biological function of fluG in asexual development and the responses to several environmental stresses as well as its influence on the asexual development regulatory network in B. bassiana.

Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

Diversity study of Beauveria bassiana species for finding the most virulent strain to manage Bemisia tabaci in cotton.

Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

Essential roles of Rad6 in conidial property, stress tolerance, and pathogenicity of Beauveria bassiana.

Rad6 functions as a ubiquitin-conjugating protein that regulates cellular processes in many fungal species. However, its role in filamentous entomopathogenic fungi remains poorly understood. This study characterizes Rad6 in Beauveria bassiana, a filamentous fungus widely employed as a critical fungicide globally. The results demonstrate a significant association between Rad6 and conidial properties, heat shock response, and UV-B tolerance. Concurrently, the mutant strain exhibited heightened sensitivity to oxidative stress, cell wall interfering agents, DNA damage stress, and prolonged heat shock. Furthermore, the absence of Rad6 significantly extended the median lethal time (LT50) of Galleria mellonella infected by B. bassiana. This delay could be attributed to reduced Pr1 proteases and extracellular cuticle-degrading enzymes, diminished dimorphic transition rates, and dysregulated antioxidant enzymes. Additionally, the absence of Rad6 had a more pronounced effect on genetic information processing, metabolism, and cellular processes under normal conditions. However, its impact was limited to metabolism in oxidative stress. This study offers a comprehensive understanding of the pivotal roles of Rad6 in conidial and hyphal stress tolerance, environmental adaptation, and the pathogenesis of Beauveria bassiana.

The fungal protease BbAorsin contributes to growth, conidiation, germination, virulence, and antiphytopathogenic activities in Beauveria bassiana (Hypocreales: Cordycipitaceae).

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most destructive agricultural pests. The entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) is a biopesticide widely used for biocontrol of various pests. Secreted fungal proteases are critical for insect cuticle destruction and successful infection. We have previously shown that the serine protease BbAorsin in B. bassiana has entomopathogenic and antiphytopathogenic activities. However, the contribution of BbAorsin to fungal growth, conidiation, germination, virulence and antiphytopathogenic activities remains unclear. In this study, the deletion (ΔBbAorsin), complementation (Comp), and overexpression (BbAorsinOE) strains of B. bassiana were generated for comparative studies. The results showed that ΔBbAorsin exhibited slower growth, reduced conidiation, lower germination rate, and longer germination time compared to WT and Comp. In contrast, BbAorsinOE showed higher growth rate, increased conidiation, higher germination rate and shorter germination time. Injection of BbAorsinOE showed the highest virulence against S. frugiperda larvae, while injection of ΔBbAorsin showed the lowest virulence. Feeding BbAorsinOE resulted in lower pupation and adult eclosion rates and malformed adults. 16S rRNA sequencing revealed no changes in the gut microbiota after feeding either WT or BbAorsinOE. However, BbAorsinOE caused a disrupted midgut, leakage of gut microbiota into the hemolymph, and upregulation of apoptosis and immunity-related genes. BbAorsin can disrupt the cell wall of the phytopathogen Fusarium graminearum and alleviate symptoms in wheat seedlings and cherry tomatoes infected with F. graminearum. These results highlight the importance of BbAorsin for B. bassiana and its potential as a multifunctional biopesticide.

An efficient visual screening of gene knockout mutants in the insect pathogenic fungus Beauveria bassiana.

Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.

Recombinant Beauveria bassiana expressing Bacillus thuringiensis toxin Cyt1Aa: a promising approach for enhancing Aedes mosquito control.

The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb-Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana. The virulence of Bb-Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT50) for A. aegypti larvae infected with Bb-Cyt1Aa decreased by 33.3% at a concentration of 1 × 108 conidia/mL and by 22.2% at 1 × 107 conidia/mL. The LT50 for A. aegypti adults infected with Bb-Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 108 conidia/mL and by 33.3% at 1 × 107 conidia/mL. Likewise, the LT50 for A. aegypti adults infected with Bb-Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb-Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus, when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control. IMPORTANCE: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus, thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control.

A transcription factor-mediated regulatory network controls fungal pathogen colonization of insect body cavities.

Successful host tissue colonization is crucial for fungal pathogens to cause mycosis and complete the infection cycle, in which fungal cells undergo a series of morphological transition-included cellular events to combat with hosts. However, many transcription factors (TFs) and their mediated networks regulating fungal pathogen colonization of host tissue are not well characterized. Here, a TF (BbHCR1)-mediated regulatory network was identified in an insect pathogenic fungus, Beauveria bassiana, that controlled insect hemocoel colonization. BbHCR1 was highly expressed in fungal cells after reaching insect hemocoel and controlled the yeast (in vivo blastospores)-to-hyphal morphological switch, evasion of immune defense response, and fungal virulence. Comparative analysis of RNA sequencing and chromatin immunoprecipitation sequencing identified a core set of BbHCR1 target genes during hemocoel colonization, in which abaA and brlA were targeted to limit the rapid switch from blastospores to hyphae and fungal virulence. Two targets encoding hypothetical proteins, HP1 and HP2, were activated and repressed by BbHCR1, respectively, which acted as a virulence factor and repressor, respectively, suggesting that BbHCR1 activated virulence factors but repressed virulence repressors during the colonization of insect hemocoel. BbHCR1 tuned the expression of two dominant hemocoel colonization-involved metabolite biosynthetic gene clusters, which linked its regulatory role in evasion of immune response. Those functions of BbHCR1 were found to be collaboratively regulated by Fus3- and Hog1-MAP kinases via phosphorylation. These findings have drawn a regulatory network in which Fus3- and Hog1-MAP kinases phosphorylate BbHCR1, which in turn controls the colonization of insect body cavities by regulating fungal morphological transition and virulence-implicated genes.IMPORTANCEFungal pathogens adopt a series of tactics for successful colonization in host tissues, which include morphological transition and the generation of toxic and immunosuppressive molecules. However, many transcription factors (TFs) and their linked pathways that regulate tissue colonization are not well characterized. Here, we identified a TF (BbHCR1)-mediated regulatory network that controls the insect fungal pathogen, Beauveria bassiana, colonization of insect hemocoel. During these processes, BbHCR1 targeted the fungal central development pathway for the control of yeast (blastospores)-to-hyphae morphological transition, activated virulence factors, repressed virulence repressors, and tuned the expression of two dominant hemocoel colonization-involved immunosuppressive and immunostimulatory metabolite biosynthetic gene clusters. The BbHCR1 regulatory function was governed by Fus3- and Hog1-MAP kinases. These findings led to a new regulatory network composed of Fus3- and Hog1-MAP kinases and BbHCR1 that control insect body cavity colonization by regulating fungal morphological transition and virulence-implicated genes.

Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana.

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

Molecular taxonomic characterization and infra-specific diversity of entomopathogenic Beauveria bassiana fungi from Argentina.

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

Clonal genomic population structure of Beauveria brongniartii and Beauveria pseudobassiana: Pathogens of the common European cockchafer (Melolontha melolontha L.).

Beauveria brongniartii is a fungal pathogen that infects the beetle Melolontha melolontha, a significant agricultural pest in Europe. While research has primarily focused on the use of B. brongniartii for controlling M. melolontha, the genomic structure of the B. brongniartii population remains unknown. This includes whether its structure is influenced by its interaction with M. melolontha, the timing of beetle-swarming flights, geographical factors, or reproductive mode. To address this, we analysed genome-wide SNPs to infer the population genomics of Beauveria spp., which were isolated from infected M. melolontha adults in an Alpine region. Surprisingly, only one-third of the isolates were identified as B. brongniartii, while two-thirds were distributed among cryptic taxa within B. pseudobassiana, a fungal species not previously recognized as a pathogen of M. melolontha. Given the prevalence of B. pseudobassiana, we conducted analyses on both species. We found no spatial or temporal genomic patterns within either species and no correlation with the population structure of M. melolontha, suggesting that the dispersal of the fungi is independent of the beetle. Both species exhibited clonal population structures, with B. brongniartii fixed for one mating type and B. pseudobassiana displaying both mating types. This implies that factors other than mating compatibility limit sexual reproduction. We conclude that the population genomic structure of Beauveria spp. is primarily influenced by predominant asexual reproduction and dispersal.

The Bbotf1 Zn(â ¡)2Cys6 transcription factor contributes to antioxidant response, fatty acid assimilation, peroxisome proliferation and infection cycles in insect pathogenic fungus Beauveria bassiana.

The abilities to withstand oxidation and assimilate fatty acids are critical for successful infection by many pathogenic fungi. Here, we characterized a Zn(II)2Cys6 transcription factor Bbotf1 in the insect pathogenic fungus Beauveria bassiana, which links oxidative response and fatty acid assimilation via regulating peroxisome proliferation. The null mutant ΔBbotf1 showed impaired resistance to oxidants, accompanied by decreased activities of antioxidant enzymes including CATs, PODs and SODs, and down-regulated expression of many antioxidation-associated genes under oxidative stress condition. Meanwhile, Bbotf1 acts as an activator to regulate fatty acid assimilation, lipid and iron homeostasis as well as peroxisome proliferation and localization, and the expressions of some critical genes related to glyoxylate cycle and peroxins were down-regulated in ΔBbotf1 in presence of oleic acid. In addition, ΔBbotf1 was more sensitive to osmotic stressors, CFW, SDS and LDS. Insect bioassays revealed that insignificant changes in virulence were seen between the null mutant and parent strain when conidia produced on CZP plates were used for topical application. However, propagules recovered from cadavers killed by ΔBbotf1 exhibited impaired virulence as compared with counterparts of the parent strain. These data offer a novel insight into fine-tuned aspects of Bbotf1 concerning multi-stress responses, lipid catabolism and infection cycles.

Essential roles of ferric reductase-like proteins in growth, development, stress response, and virulence of the filamentous entomopathogenic fungus Beauveria bassiana.

In yeasts, ferric reductase catalyzes reduction of ferric ion to ferrous form, which is essential for the reductive iron assimilation system. However, the physiological roles of ferric reductases remain largely unknown in the filamentous fungi. In this study, genome-wide annotation revealed thirteen ferric reductase-like (Fre) proteins in the filamentous insect pathogenic fungus Beauveria bassiana, and all their functions were genetically characterized. Ferric reductase family proteins exhibit different sub-cellular distributions (e.g., cell periphery and vacuole), which was due to divergent domain architectures. Fre proteins had a synergistic effect on fungal virulence, which was ascribed to their distinct functions in different physiologies. Ten Fre proteins were not involved in reduction of ferric ion in submerged mycelia, but most proteins contributed to blastospore development. Only two Fre proteins significantly contributed to B. bassiana vegetative growth under the chemical-induced iron starvation, but most Fre proteins were involved in resistance to osmotic and oxidative stresses. Notably, a bZIP-type transcription factor HapX bound to the promoter regions of all FRE genes in B. bassiana, and displayed varying roles in the transcription activation of these genes. This study reveals the important role of BbFre family proteins in development, stress response, and insect pathogenicity, as well as their distinctive role in the absorption of ferric iron from the environment.

The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana Vuillemin is an entomopathogenic fungus that has been developed as a biological insecticide. B. bassiana can be infected by single or multiple mycoviruses, most of which are double-stranded RNA (dsRNA) viruses, while infections with single-stranded RNA (ssRNA) viruses, especially negative single-stranded RNA (-ssRNA) viruses, have been observed less frequently. In the present study, we sequenced and analyzed the complete genomes of two new different mycoviruses coinfecting a single B. bassiana strain: a -ssRNA virus which we have named "Beauveria bassiana negative-strand RNA virus 1" (BbNSRV1), and a dsRNA virus, which we have named "Beauveria bassiana orthocurvulavirus 1" (BbOCuV1). The genome of BbNSRV1 consists of a single segment of negative-sense, single-stranded RNA with a length of 6169 nt, containing a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) with 1949 aa (220.1 kDa). BLASTx analysis showed that the RdRp had the highest sequence similarity (59.79%) to that of Plasmopara viticola lesion associated mononegaambi virus 2, a member of the family Mymonaviridae. This is the first report of a -ssRNA mycovirus infecting B. bassiana. The genome of BbOCuV1 consists of two dsRNA segments, 2164 bp and 1765 bp in length, respectively, with dsRNA1 encoding a protein with conserved RdRp motifs and 70.75% sequence identity to the putative RdRp of the taxonomically unassigned mycovirus Fusarium graminearum virus 5 (FgV5), and the dsRNA2 encoding a putative coat protein with sequence identity 64.26% to the corresponding protein of the FgV5. Phylogenetic analysis indicated that BbOCuV1 belongs to a taxonomically unassigned group of dsRNA mycoviruses related to members of the families Curvulaviridae and Partitiviridae. Hence, it might be the member of a new family that remains to be named and formally recognized.

High photoreactivation activities of Rad2 and Rad14 in recovering insecticidal Beauveria bassiana from solar UV damage.

Anti-ultraviolet (UV) roles of Rad2 and Rad14 depend on nucleotide excision repair (NER) of UV-induced DNA lesions in budding yeast but remain unexplored yet in filamentous fungi. Here, nucleus-specific Rad2 and Rad14 orthologs are shown to recover Beauveria bassiana, a main source of wide-spectrum mycoinsecticides, from solar UV damage through photorepair-depending photoreactivation. As a photorepair index, photoreactivation (germination) rates of lethal UVB dose-irradiated conidia via a 3- or 5-h light plus 9- or 7-h dark incubation at 25 °C were drastically reduced in the Δrad2 and Δrad14 mutants versus a wild-type strain. As an NER index, nighttime-mimicking 12-h dark reactivation rates of low UVB dose-impaired conidia decreased sharply compared to the corresponding photoreactivation rates in the presence or absence of either ortholog, indicating that its extant NER activity was limited to recovering light UVB damage in the field. The high photoreactivation activity of either Rad2 or Rad14 was derived from its tight link to a large protein complex formed by photolyase regulators and other anti-UV proteins through multiple protein-protein interactions revealed by yeast two-hybrid assays. Therefore, Rad2 and Rad14 recover B. bassiana from solar UV damage through photoreactiovation in vivo that depends primarily on photorepair, although they contribute little to the fungal lifecycle-related phenotypes. These findings unveil a novel scenario distinguished from the NER-depending anti-UV roles of Rad2 and Rad14 in the model yeast and broaden a biological basis crucial for rational application of fungal insecticides to improve pest control efficacy via feasible recovery of solar UV damage.

Two sirtuin proteins, Hst3 and Hst4, modulate asexual development, stress tolerance, and virulence by affecting global gene expression in Beauveria bassiana.

Beauveria bassiana is a widely used entomopathogenic fungus in insect biological control applications. In this study, we investigated the role of two sirtuin homologs, BbHst3 and BbHst4, in the biological activities and pathogenicity of B. bassiana. Our results showed that deletion of BbHst3 and/or BbHst4 led to impaired sporulation, reduced (~50%) conidial production, and decreased tolerance to various stresses, including osmotic, oxidative, and cell wall-disturbing agents. Moreover, BbHst4 plays dominant roles in histone H3-K56 acetylation and DNA damage response, while BbHst3 is more responsible for maintaining cell wall integrity. Transcriptomic analyses revealed significant changes (>1,500 differentially expressed genes) in gene expression patterns in the mutant strains, particularly in genes related to secondary metabolism, detoxification, and transporters. Furthermore, the ΔBbHst3, ΔBbHst4, and ΔBbHst3ΔBbHst4 strains exhibited reduced virulence in insect bioassays, with decreased (~20%) abilities to kill insect hosts through topical application and intra-hemocoel injection. These findings highlight the crucial role of BbHst3 and BbHst4 in sporulation, DNA damage repair, cell wall integrity, and fungal infection in B. bassiana. Our study provides new insights into the regulatory mechanisms underlying the biological activities and pathogenicity of B. bassiana and emphasizes the potential of targeting sirtuins for improving the efficacy of fungal biocontrol agents.IMPORTANCESirtuins, as a class of histone deacetylases, have been shown to play important roles in various cellular processes in fungi, including asexual development, stress response, and pathogenicity. By investigating the functions of BbHst3 and BbHst4, we have uncovered their critical contributions to important phenotypes in Beauveria bassiana. Deletion of these sirtuin homologs led to reduced conidial yield, increased sensitivity to osmotic and oxidative stresses, impaired DNA damage repair processes, and decreased fungal virulence. Transcriptomic analyses showed differential expression of numerous genes involved in secondary metabolism, detoxification, transporters, and virulence-related factors, potentially uncovering new targets for manipulation and optimization of fungal biocontrol agents. Our study also emphasizes the significance of sirtuins as key regulators in fungal biology and highlights their potential as promising targets for the development of novel antifungal strategies.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa.

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Rad6, a ubiquitin conjugator required for insect-pathogenic lifestyle, UV damage repair, and genomic expression of Beauveria bassiana.

The E2 ubiquitin conjugator Rad6 is required for DNA damage bypass in budding yeast but remain functionally unknown in filamentous fungi. Here, we report pleiotropic effect of Rad6 ortholog in Beauveria bassiana, a wide-spectrum fungal insecticide. Global ubiquitination signal was greatly attenuated in the absence of rad6. The blocked ubiquitination led to severe growth defect, blocked asexual development, and abolished infectivity/insect pathogenicity, which correlated with compromised conidial quality (including viability, hydrophobicity, adherence to insect cuticle, and thermotolerance) and blocked secretion of cuticle-degrading enzymes including Pr1 family proteases. Importantly, Rad6 played much greater role in photoreactivation of UVB-impaired conidia by a 3- or 5-h light plus 9- or 7-h dark incubation than in dark reactivation of those impaired conidia by a 12-h dark incubation. The high activity of Rad6 in photoreactivation in vivo was derived from its link to a protein complex cored by the photolyase regulators WC1 and WC2 via the strong interactions of Rad6 with the E3 partner Rad18 and Rad18 with WC2 revealed in yeast two-hybrid assays. Transcriptomic analysis resulted in identification of 2700 differentially regulated genes involved in various function categories and metabolism pathways, indicating a regulatory role of Rad6-mediated ubiquitination in gene expression networks and genomic stability. Conclusively, Rad6 is required for asexual and insect-pathogenic lifecycles, solar UV damage repair, and genomic expression of B. bassiana. The primary dependence of its strong anti-UV role on photoreactivation in vivo unveils a scenario distinct from the core role of its yeast ortholog in DNA damage bypass.