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The white collar proteins WC1 and WC2 interact with each other to form a white collar complex acting as a well-known transcription regulator required for the operation of the circadian clock in Neurospora, but their roles in insect-pathogenic fungal lifecycles remain poorly understood. Here, we report that WC1 and WC2 orthologs co-regulate the conidiation capacity and conidial resistance to solar ultraviolet-B (UVB) irradiation in Beauveria bassiana, after their high activities in the photorepair of UVB-induced DNA damages were elucidated previously in the insect mycopathogen, which features non-rhythmic conidiation and high conidiation capacity. The conidial yield, UVB resistance, and photoreactivation rate of UVB-impaired conidia were greatly reduced in the null mutants of wc1 and wc2 compared to their control strains. However, many other lifecycle-related phenotypes, except the antioxidant response, were rarely affected in the two mutants. Transcriptomic analysis revealed largely overlapping roles for WC1 and WC2 in regulating the fungal gene networks. Most of the differentially expressed genes identified from the null mutants of wc1 (1380) and wc2 (1001) were co-downregulated (536) or co-upregulated (256) at similar levels, including several codownregulated genes required for aerial conidiation and DNA photorepair. These findings expand a molecular basis underlying the fungal adaptation to solar UV irradiation and offer a novel insight into the genome-wide co-regulatory roles of WC1 and WC2 in B. bassiana's asexual development and in vivo photoreactivation against solar UV damage. [ABSTRACT FROM AUTHOR]

The fluffy genes flbA–flbE are well-known players in the upstream developmental activation pathway that activates the key gene brlA of central developmental pathway (CDP) to initiate conidiation in Aspergillus nidulans. Here, we report insignificant roles of their orthologs in radial growth of Beauveria bassiana under normal culture conditions and different stresses although flbA and flbD were involved in respective responses to heat shock and H2O2. Aerial conidiation level was lowered in the deletion mutants of flbB and flbE (~15%) less than of flbA and flbC (~30%), in which the key CDP genes brlA and abaA were repressed consistently during normal incubation. The CDP-controlled blastospore production in submerged cultures mimicking insect hemolymph was abolished in the flbA mutant with brlA and abaA being sharply repressed, and decreased by 55% in the flbC mutant with only abaA being downregulated. The fungal virulence against a model insect was attenuated in the absence of flbA more than of flbC irrespective of normal cuticle infection or cuticle-bypassing infection (intrahemocoel injection). These findings unravel more important role of flbA than of flbC, but null roles of flbB/D/E, in B. bassiana's insect–pathogenic lifecycle and a scenario distinctive from that in A.nidulans. [ABSTRACT FROM AUTHOR]

The fluffy genes flbA–flbE are well-known players in the upstream developmental activation pathway that activates the key gene brlA of central developmental pathway (CDP) to initiate conidiation in Aspergillus nidulans. Here, we report insignificant roles of their orthologs in radial growth of Beauveria bassiana under normal culture conditions and different stresses although flbA and flbD were involved in respective responses to heat shock and H2O2. Aerial conidiation level was lowered in the deletion mutants of flbB and flbE (~15%) less than of flbA and flbC (~30%), in which the key CDP genes brlA and abaA were repressed consistently during normal incubation. The CDP-controlled blastospore production in submerged cultures mimicking insect hemolymph was abolished in the flbA mutant with brlA and abaA being sharply repressed, and decreased by 55% in the flbC mutant with only abaA being downregulated. The fungal virulence against a model insect was attenuated in the absence of flbA more than of flbC irrespective of normal cuticle infection or cuticle-bypassing infection (intrahemocoel injection). These findings unravel more important role of flbA than of flbC, but null roles of flbB/D/E, in B. bassiana’s insect–pathogenic lifecycle and a scenario distinctive from that in A.nidulans.

ABSTRACT Ssr4 serves as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but remains functionally uncharacterized due to its essentiality for yeast viability. Here, we report pleiotropic effects of the deletion of the ssr4 ortholog nonessential for cell viability in Beauveria bassiana, an asexual insect mycopathogen. The deletion of ssr4 resulted in severe growth defects on different carbon/nitrogen sources, increased hyphal hydrophilicity, blocked hyphal differentiation, and 98% reduced conidiation capacity compared to a wild-type standard. The limited Δssr4 conidia featured an impaired coat with disordered or obscure hydrophobin rodlet bundles, decreased hydrophobicity, increased size, and lost insect pathogenicity via normal cuticle infection and 90% of virulence via intrahemocoel injection. The expression of genes required for hydrophobin biosynthesis and assembly of the rodlet layer was drastically repressed in more hydrophilic Δssr4 cells. Transcriptomic analysis revealed 2,517 genes differentially expressed in the Δssr4 mutant, including 1,505 downregulated genes and 1,012 upregulated genes. The proteins encoded by hundreds of repressed genes were involved in metabolism and/or transport of carbohydrates, amino acids, and lipids, inorganic ion transport and energy production or conversion, including dozens involved in DNA replication, transcription, translation, and posttranslational modifications. However, purified Ssr4 samples showed no DNA-binding activity, implying that the role of Ssr4 in genome-wide gene regulation could rely upon its acting as a cosubunit of the two complexes. These findings provide the first insight into an essential role of Ssr4 in the asexual cycle in vitro and in vivo of B. bassiana and highlights its importance for the filamentous fungal lifestyle. IMPORTANCE Ssr4 is known to serve as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but has not been functionally characterized in fungi. This study unveils for the first time the pleiotropic effects caused by deletion of ssr4 and its role in mediating global gene expression in a fungal insect pathogen. Our findings confirm an essential role of Ssr4 in hydrophobin biosynthesis and assembly required for growth, differentiation, and development of aerial hyphae for conidiation and conidial adhesion to insect surface and its essentiality for insect pathogenicity and virulence-related cellular events. Importantly, Ssr4 can regulate nearly one-fourth of all genes in the fungal genome in direct and indirect manners, including dozens involved in gene activity and hundreds involved in metabolism and/or transport of carbohydrates, amino acids, lipids, and/or inorganic ions. These findings highlight a significance of Ssr4 for filamentous fungal lifestyle.

The white collar proteins WC1 and WC2 interact with each other to form a white collar complex acting as a well-known transcription regulator required for the operation of the circadian clock in Neurospora , but their roles in insect-pathogenic fungal lifecycles remain poorly understood. Here, we report that WC1 and WC2 orthologs co-regulate the conidiation capacity and conidial resistance to solar ultraviolet-B (UVB) irradiation in Beauveria bassiana , after their high activities in the photorepair of UVB-induced DNA damages were elucidated previously in the insect mycopathogen, which features non-rhythmic conidiation and high conidiation capacity. The conidial yield, UVB resistance, and photoreactivation rate of UVB-impaired conidia were greatly reduced in the null mutants of wc1 and wc2 compared to their control strains. However, many other lifecycle-related phenotypes, except the antioxidant response, were rarely affected in the two mutants. Transcriptomic analysis revealed largely overlapping roles for WC1 and WC2 in regulating the fungal gene networks. Most of the differentially expressed genes identified from the null mutants of wc1 (1380) and wc2 (1001) were co-downregulated (536) or co-upregulated (256) at similar levels, including several co-downregulated genes required for aerial conidiation and DNA photorepair. These findings expand a molecular basis underlying the fungal adaptation to solar UV irradiation and offer a novel insight into the genome-wide co-regulatory roles of WC1 and WC2 in B. bassiana 's asexual development and in vivo photoreactivation against solar UV damage.

Ssr4 serves as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but remains functionally uncharacterized due to its essentiality for yeast viability. Here, we report pleiotropic effects of the deletion of the ssr4 ortholog nonessential for cell viability in Beauveria bassiana , an asexual insect mycopathogen. The deletion of ssr4 resulted in severe growth defects on different carbon/nitrogen sources, increased hyphal hydrophilicity, blocked hyphal differentiation, and 98% reduced conidiation capacity compared to a wild-type standard. The limited Δ ssr4 conidia featured an impaired coat with disordered or obscure hydrophobin rodlet bundles, decreased hydrophobicity, increased size, and lost insect pathogenicity via normal cuticle infection and 90% of virulence via intrahemocoel injection. The expression of genes required for hydrophobin biosynthesis and assembly of the rodlet layer was drastically repressed in more hydrophilic Δ ssr4 cells. Transcriptomic analysis revealed 2,517 genes differentially expressed in the Δ ssr4 mutant, including 1,505 downregulated genes and 1,012 upregulated genes. The proteins encoded by hundreds of repressed genes were involved in metabolism and/or transport of carbohydrates, amino acids, and lipids, inorganic ion transport and energy production or conversion, including dozens involved in DNA replication, transcription, translation, and posttranslational modifications. However, purified Ssr4 samples showed no DNA-binding activity, implying that the role of Ssr4 in genome-wide gene regulation could rely upon its acting as a cosubunit of the two complexes. These findings provide the first insight into an essential role of Ssr4 in the asexual cycle in vitro and in vivo of B. bassiana and highlights its importance for the filamentous fungal lifestyle. IMPORTANCE Ssr4 is known to serve as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but has not been functionally characterized in fungi. This study unveils for the first time the pleiotropic effects caused by deletion of ssr4 and its role in mediating global gene expression in a fungal insect pathogen. Our findings confirm an essential role of Ssr4 in hydrophobin biosynthesis and assembly required for growth, differentiation, and development of aerial hyphae for conidiation and conidial adhesion to insect surface and its essentiality for insect pathogenicity and virulence-related cellular events. Importantly, Ssr4 can regulate nearly one-fourth of all genes in the fungal genome in direct and indirect manners, including dozens involved in gene activity and hundreds involved in metabolism and/or transport of carbohydrates, amino acids, lipids, and/or inorganic ions. These findings highlight a significance of Ssr4 for filamentous fungal lifestyle., (Copyright © 2020 Shao et al.)