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

ABSTRACT 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. IMPORTANCE Fungal 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.

mediated networks that regulate insect hemocoel colonization in those fungal species are not well characterized.
In this study, we characterized a Zn(II) 2 Cys 6 TF, BbHCR1, in B. bassiana, which regulated insect hemocoel colonization by regulating fungal dimorphic transition and development via targeting asexual developmental regulatory factor genes, brlA and abaA, and virulence-associated genes, as well as indirect control of the biosynthesis of immunosuppressive metabolites, oosporein and beauverolide.The regulatory function of BbHCR1 was collaboratively regulated by Fus3-and Hog1-MAP kinases via phosphor ylation.These data illustrated a new TF-mediated regulatory network that controls B. bassiana colonization of insect hemocoel and virulence.

Identification of TFs involved in B. bassiana colonization of insect body cavity
The gene expression data of 559 B. bassiana TFs were retrieved from the whole genome expression database that was constructed from 76 deep-sequenced RNA-seq samples covering fungal cells derived from cells in penetration of (insect) cuticle (PC), in vivo blastospores (hyphal bodies [HB]), liquid hyphae (LH), and aerial hyphae (AH) of B. bassiana (24).Co-expression analysis revealed that those TF genes with similar expres sion patterns were enriched into six clusters (Cls1-Cls6) (Fig. S1).Cls1, Cls2, and Cls4 represented TF genes that were highly expressed in HB, AH, and PC, respectively (Fig. 1A).TF genes in Cls3 were slightly downregulated in HB but slightly upregulated in AH, while those TF genes in Cls5 and Cls6 were downregulated in PC and HB, respectively (Fig. 1A).Although the transcripts of Cls1 TF genes were detected in PC, HB, LH, and AH, the relative expression levels were dramatically increased in HB (average LogCLR = 3.01 ± 0.48) as compared with the other three types of cells (PC 0.10 ± 0.07, LH 0.09 ± 0.50, and AH −0.01 ± 0.23).The Cls1 harbored 11 TFs, including 7 Zn(II) 2 Cys 6 , 3 CCHC-type zinc finger proteins, and 1 Myb TF.Expression patterns of those 11 TF genes were verified by RT-qPCR in B. bassiana Bb0062 strain.The results indicated that eight genes were highly active in HB, while other two and one genes were highly expressed in LH and PC, respectively (Fig. 1B).These data suggested that those eight TFs might regulate B. bassiana colonization of insect hemocoel.
One of those eight TFs with the tag code BBA_04034, designed as BbHCR1 (B.bassiana hemocoel colonization-associated regulator 1), consists of 928 amino acids containing two putative GAL4-like DNA-binding domains at residues 66-108 and 127-171 and a fungal-specific transcription factor domain at residues 332-757, with one predicted nuclear localization signal, PFRRGRS, at residues 798-804 (25).BLASTp analysis revealed that the homologs of BbHCR1 are commonly present in fungal species from yeast to filamentous fungi, whereas phylogenetic separation could be seen with hosts, i.e., insects and fungi (e.g., the hyperparasite fungus Trichoderma atroviride), plants, and mammals, despite the yeast Lipomyces starkeyi forming a separate branch (Fig. S2).Transcriptional activation assays revealed that yeast cells expressing BbHCR1 cDNA grew as well on an auxotrophic medium as the positive control yeast cells expressed a GAL4 activation domain with a blue colony (Fig. 2A).GFP fluorescence signals of BbHCR1::GFP cells (HB) were overlapped with a red fluorescent protein (RFP)-tagged H1 (histone), suggesting localization in the nucleus (Fig. 2B).These results indicated that BbHCR1 was a typical TF.
RT-qPCR analysis revealed that expression of BbHCR1 was significantly elevated in hemolymph-derived cells, HB, at 48-60 h after the injection of conidia, but dramatically decreased after 72 h of inoculation when fungal cells were switched from HB to hyphae.These results were consistent with the GFP fluorescence observation of PBbHCR1::GFP cells, in which the GFP gene was controlled by the BbHCR1 promoter (Fig. 2C and D), suggesting involvement in the early colonization of insect hemocoel.Moreover, BbHCR1 expression was slightly increased in LH and induced by low oxygen (LO) and oxidative [menadione (MND)] stresses (Fig. 2E and F).
It was noticed that the BbHCR1 OE -infected larvae appeared to have a melanization reaction at 36 h post-injection of conidia and turned black at 48 h, all of which were 12 h ahead of WT.More BbHCR1 OE hyphae penetrated outward from the cadaver for growth 96 h post-inoculation (hpi), whereas little penetrated hyphae of control strains (WT and Comp) were seen on the larvae surface.However, the melanization reaction of the ΔBbHCR1-inoculated larvae was delayed by 24 h at least as compared to the control strains, and little hyphae penetrated outward from the cadaver mouthparts and excretory channels were seen until 120 hpi.At 144 hpi, hyphae of the ΔBbHCR1 grown on the cadaver surface were obviously less than control strains, but the BbHCR1 OE growth was more than control strains (Fig. 3C).These data were consistent with their changes in virulence.

BbHCR1 is involved in insect hemocoel colonization
To probe the fungal development in the insect hemocoel, G. mellonella larvae were bled after the injection of conidia for microscopy examination.BbHCR1 OE germinated conidia (the germ tubes) more easily escaped hemocyte encapsulation at 24 hpi and freely grew as compared to control strains, which evaded the hemocyte encapsulation after 36 h of inoculation.Although few fungal cells were seen to evade the encapsulation at 36 h after the inoculation of ΔBbHCR1 conidia, some cells grew freely at 48 hpi and developed numerous in vivo blastospores from 60 to 72 hpi (Fig. 4A).However, compared to control strains that formed stick-shaped HB (in vivo blastospores) from 48 to 60 hpi, BbHCR1 OE -germinated conidia formed HB at 36 hpi and rapidly developed to hyphae at 48 hpi, with hardly any fungal cells seen in the hemolymph at 60 hpi (Fig. 4A).Changes in free-floating fungal cells in the hemolymph from 36 to 60 hpi were verified by qPCR analysis by amplification of the 18S rRNA sequence (5) (Fig. 4B).The similar morphological switches were also seen in vitro cultures (in 1/4 SDY broth) (Fig. S6A).The examination of the tissue section revealed that more fungal hyphae of BbHCR1 OE were distributed in larval tissues, e.g., fat bodies and subcutaneous tissues, than control strains at 60 hpi (Fig. 4C).These results suggested the role of BbHCR1 in regulating the morphological switch from in vivo blastospores to hyphae during hemocoel colonization.Moreover, reduced/increased growth of ΔBbHCR1/BbHCR1 OE cells was also seen in broth with oxidative (75 µM MND) or/and low oxygen (1 mM CoCl 2 , a hypoxia-mimicking agent) agents (Fig. S6), which mimicked stress niches in fungal infected insect hemocoel (27,28), suggesting linkage of BbHCR1-mediated fungal development to adaptation to infected insect hemocoel niches.

BbHCR1 targets fungal development and secondary metabolite synthesis genes at the early colonization of insect hemocoel
To reveal the underlying mechanism of BbHCR1 regulating the morphological switch from blastospores (HB) to hyphae and evasion of immune response in the early coloniza tion of insect hemocoel, RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) were performed to identify the BbHCR1 target genes.Compared to RNA-seq from normal cultures (1/4 SDY for 3 days), 197 and 210 genes were specifically upregulated and downregulated in the hemolymph-derived ΔBbHCR1 cells (HB, 48 h after injection of conidia), respectively, while 62 and 21 genes were specifically upregulated and downregulated in in vivo BbHCR1 OE cells as compared to those of WT, respectively (Fig. S7B).Among those differently expressed genes (DEGs) in in vivo cells, 28 DEGs displayed opposite expression patterns in the ΔBbHCR1 and BbHCR1 OE cells, including 11 and 17 DEGs upregulated and downregulated in the ΔBbHCR1 cells but downregulated and upregulated in the BbHCR1 OE cells, respectively (Fig. S8A).Those DEGs were enriched in nutrient utilization, secondary metabolism, fungal development, and other functions.Those nutrient utilization-involved genes encode hydrolases, such as proteases, peptidases, and glucanases, all of which were significantly upregulated in ΔBbHCR1 but downregulated in BbHCR1 OE (Fig. S8B).Whereas those secondary metabo lism genes were involved in the biosynthesis of oosporein (encoding Ops1, Ops4, Ops6, and Ops7) (29), beauverolide (encoding besA and besB) (30), and other metabolites, which were significantly downregulated in ΔBbHCR1 but upregulated in BbHCR1 OE cells (Fig. S8B).In those fungal development genes, two cell adhesion-associated genes (BBA_03909 and BBA_09863) and one conidial cell wall protein gene (BBA_07138) were significantly downregulated in ΔBbHCR1 but upregulated in BbHCR1 OE cells, whereas one cell cycle gene (BBA_02863) and one central development pathway (CDP) regulator abaA gene (BBA_00300) were significantly upregulated in ΔBbHCR1 but downregulated in BbHCR1 OE cells (Fig. S8B).We further probed the other CDP regulator genes and found that brlA was also significantly upregulated ΔBbHCR1 (log 2 FC = 1.7) but slightly downre gulated in BbHCR1 OE cells (log 2 FC = −0.9)(P value = 0.07).All those DEGs were associated with cellular events in the early colonization of insect hemocoel.
To probe the targets of BbHCR1, ChIP-seq was performed using a ΔBbHCR1 strain constitutively expressing 13 Myc-tagged BbHCR1 (probed with an anti-Myc antibody) grown in 1/4 SDY.A total of 521 unique ChIP-seq peaks were mapped to 175 different target genes (within 2.0 kb of an ORF start codon) (Fig. 5A).MEME analysis indicated the putative DNA-binding sequence for BbHCR1 to be VYHGYHGYB, with lowest P value (1.9e-1) and frequency (75/175) (Fig. 5B).Comparison of the ChIP-seq with the RNA-seq data sets (DEGs specifically expressed in in vivo cells) indicated the expression of 14 (8% of the total ChIP set) genes as BbHCR1 dependent, including five/seven upregula ted/downregulated genes in ΔBbHCR1 cells and three/one upregulated/downregulated genes in BbHCR1 OE cells, respectively, in which abaA (BBA_00300) and a metallopro tease-like protein gene (BBA_02374) were significantly upregulated/downregulated in ΔBbHCR1 but downregulated/upregulated in BbHCR1 OE cells, respectively.Two targets, BBA_07528 and BBA_01961, encoding two hypothetical (uncharacterized) proteins containing predicted N-terminal signal sequences, designated as HP1 and HP2, were significantly downregulated (log 2 FC = −3.0)/upregulated(log 2 FC = 2.2) in ΔBbHCR1 cells but slightly upregulated (log 2 FC = 0.84)/downregulated (log 2 FC = −0.65) in BbHCR1 OE cells, respectively (Fig. 5C).Promoter element scanning revealed that one binding motif for BbHCR1 presented at −3,979 to −3,667 bp upstream of brlA-coding region (present in the ChIP-seq data).It is demonstrated that a heterotrimeric transcription factor CCAATbinding complex regulates asexual reproduction (conidiation) of A. fumigatus by binding to the promoter region of brlA at −4,421 to −3,175 bp upstream of start site "ATG" and promoter regions of other fungal development genes (fluG, flbD, and flbC) (31), suggesting promoter region of brlA is over 4,000 bp.Thus, we considered that brlA was also a putative target of BbHCR1.Another target gene involved in fungal development and differentiation (BBA_01320, coding membrane fusion mating protein FIG1) (32) was significantly downregulated in the ΔBbHCR1 strain.Other targets associated with transcription and signaling transduction, secondary metabolism, and uncharacterized proteins displayed opposite expression patterns between ΔBbHCR1 and BbHCR1 OE strains (Fig. 5C).Binding of purified BbHCR1 to the promoter regions of four identified putative target genes, including brlA, abaA, HP1, and HP2, was verified by electrophoretic mobility shift assay (EMSA) using respective promoter fragments from each gene and the purified protein.We scanned the promoter region of each target and found that three, five, seven, and two BbHCR1-binding motifs were present in probes of brlA (472 bp from −3,901 to −3,430 bp), abaA (322 bp from −656 to −335 bp), HP1 (328 bp from −800 to −473 bp), and HP2 (369 bp from −370 to −2 bp), respectively, which was in line with the several positive signals in EMSA assays (Fig. 5D).

BbHCR1 targets brlA and abaA and virulence factor genes to control morpho logical transition in the insect hemocoel and virulence
To reveal linkage of BbHCR1-regulated morphological switch from in vivo blastospores to hyphae with its targets, brlA and abaA, ΔbrlA::ΔBbHCR1 or ΔabaA::ΔBbHCR1 mutants, and brlA OE ::BbHCR1 OE or abaA OE ::BbHCR1 OE strains were generated by the disruption of brlA or abaA in ΔBbHCR1 background and overexpression of brlA or abaA in BbHCR1 OE background, respectively.Since the resultant ΔbrlA::ΔBbHCR1 and ΔabaA::ΔBbHCR1 lost capacities to sporulate either on agar or in broth, their hyphal fragments (20 mg/mL fresh hyphae) were used for insect bioassay by microinjection into the larvae hemo coel.As compared to the ΔBbHCR1 strain whose virulence was significantly lower than WT, dramatically decreased virulence was detected in the ΔbrlA::ΔBbHCR1 and ΔabaA::ΔBbHCR1 strains, in which the former virulence was significantly lower than the latter (Fig. 6A).Unlike the ΔBbHCR1 hyphal fragments that formed in vivo blas tospores at 72 hpi, hyphal fragments of ΔbrlA::ΔBbHCR1 and ΔabaA::ΔBbHCR1 were encapsulated by hemocytes with a melanization reaction until 96 hpi (Fig. 6B).With respect to brlA OE ::BbHCR1 OE and abaA OE ::BbHCR1 OE strains, topical cuticle inoculation and intrahemocoel injection were performed for bioassays.Although the virulence of the two overexpression strains was increased as compared to WT, the brlA OE ::BbHCR1 OE was less virulent than the BbHCR1 OE strain (P < 0.01 and P < 0.05 in topical cuticle inoculation and intrahemocoel injection bioassays, respectively), while abaA OE ::BbHCR1 OE displayed slightly decreased virulence in topical inoculation bioassay (P < 0.05) but not in intrahemocoel injection bioassay as compared to the BbHCR1 OE strain (Fig. 6C).However, the morphological switch of BbHCR1 OE cells from in vivo blastospores to hyphae was restored to wild type by overexpression of brlA or abaA in BbHCR1 OE strain (Fig. 6D and E).These results suggested that BbHCR1 targeted brlA and abaA to control the morphological transition from blastospores to hyphae in the infected insect blood.hemocoel colonization, with the former being significantly repressed in ΔBbHCR cells but slightly upregulated in BbHCR1 OE cells, while the latter being significantly upregu lated in the ΔBbHCR cells but slightly decreased in BbHCR1 OE cells, were selected as representatives to be characterized.The structures of the two proteins are shown in Fig. S9A.Both HP1 and HP2 were highly expressed in WT HB (in vivo blastospores, 48 h of inoculation) (Fig. S9B).As expected, disruption of HP1 (ΔHP1) significantly decreased B. bassiana virulence, while significantly decreased virulence was examined in the HP2 overexpression strain (HP2 OE ), which resulted in increased LT 50 values for ΔHP1/HP2 OE strains in both bioassays (Fig. S9C through F).No obvious difference in cuticle penetra tion was assayed between HP1 and HP2 disruption or overexpression strains and WT (Fig. S5).These results suggested that BbHCR1 might activate virulence genes (e.g., HP1) but repress virulence-repressor-associated genes (e.g., HP2) during insect hemocoel colonization.

BbHCR1 is phosphorylated by Fus3-and Hog1-MAP kinases
Bioinformatic analysis showed the putative phosphorylation sites of Fus3-MAP kinase (S815) and Hog1-/Fus3-MAP kinase (T865) present in BbHCR1 (Fig. 7A).Yeast two-hybrid tests verified the possible interaction between BbHCR1 with Hog1-MAP kinase (Bbhog1) (33) or Fus3-MAP kinase (Bbmpk1) (34) (Fig. 7B).To reveal the contribution of the predicted phosphorylation sites to BbHCR1 function, serine at position 815 or/and threonine at 865 were mutated to alanine and generated BbHCR1 S815A , BbHCR1 T865A , and BbHCR1 S815A-T865A , which were separately introduced into ΔBbHCR1 strain.Bioassays via hemocoel injection of conidia revealed that the introduction of BbHCR1 S815A seemed to restore ΔBbHCR1 to the wild type, while the introduction of BbHCR1 T865A restored ΔBbHCR1 virulence only by 4.7% (P < 0.01).However, the introduction of BbHCR1 S815A-T865A hardly restored the ΔBbHCR1 virulence (Fig. 7C).These results suggested that BbHCR1 phosphorylation by Fus3-MAP kinase (Bbmpk1) and Hog-MAP kinase (BbHog1) was crucial for its function in fungal virulence, and the contribution of the phosphorylation at site T865 was larger than at site S815.
To verify the effects of Fus3-and Hog1-MAP kinases on the phosphorylation of BbHCR1, PB3::BbHCR1::13myc was separately introduced into wild type, ΔBbmpk1 (34) and ΔBbHog1 (33) strains.Phosphorylation levels of BbHCR1 were examined using Western blots with anti-Myc and horseradish peroxidase (HRP)-conjugated streptavidin following separation of fungal proteins by Phos-tag SDS-PAGE under the normal (1/4 SDY), oxidative (75 µM MND), or low oxygen (1 mM CoCl 2 ) conditions.BbHCR1 protein was probed using SDS-PAGE and Western blots.Proteins blotted with anti-β-tubulin antibody were used as the internal standard.The BbHCR1 phosphorylation levels were slightly decreased in the ΔBbmpk1 strain under the normal condition (by 18.7%) but observably decreased (by 36.2%-52.6%)under the stress conditions as compared to those in WT (Fig. 7D).Whereas dramatically decreased BbHCR1 phosphorylation levels were detected in ΔBbHog1 strain as compared to WT either under normal or the stressed conditions (decreased by 50%-72.9%)(Fig. 7E).These results further confirmed the contribution of Fus3-and Hog1-MAP kinases to the phosphorylation of BbHCR1.

DISCUSSION
Insect pathogenic fungi switch from invasive hyphae to in vivo blastospores (hyphal bodies) in the insect hemocoel after penetration of the cuticle, which aids fungal cells to escape the insect immune defense response and colonize insect body cavities.Although some molecules have been characterized in fungal pathogen colonization of insect hemocoel, their regulatory mechanisms are largely unknown (10,16).Here, we characterized a highly expressed TF (BbHCR1) in B. bassiana during the early colonization of insect hemocoel, which regulated the fungal colonization of insect hemocoel.During this process, BbHCR1 targeted CDP activator genes, brlA or abaA, to control morphologi cal transition from in vivo blastospores to hyphae and some virulence-associated genes, and indirectly controlled oosporein and beauverolide biosynthetic gene clusters that are involved in the colonization of insect body cavities (29,35).Moreover, BbHCR1 was collaboratively regulated by Fus3-and Hog1-MAP kinases via phosphorylation, which was crucial for its regulatory function in insect hemocoel colonization and fungal virulence.These results demonstrated a new regulatory network that controls fungal colonization of insect body cavities.
Significantly decreased/increased virulence was assayed in ΔBbHCR1/BbHCR1 OE strains by either topical inoculation (cuticle infection) or injection of conidia into the insect hemocoel (cuticle-bypassing infection) as compared to the wild-type strain; however, no obvious difference was examined in their abilities to penetrate the cuticle using the cicada hind wings to mimic the insect cuticle.These results suggested that the altered virulence of ΔBbHCR1 and BbHCR1 OE might be due to their different abilities to colonize insect hemocoel.Examination of fungal development revealed that the ΔBbHCR1 strain generated massive in vivo blastospores in insect hemocoel with few hyphae, but in vivo blastospores formed from BbHCR1 OE -geminated conidia (germ tubes) rapidly switched to hyphae, suggesting involvement of BbHCR1 in the regulation of morphological transition from blastospores to hyphae in vivo.A similar morphological transition was seen in in vitro broth.However, reduced/increased conidia production was examined on rich-nutrient 1/4 SDAY in ΔBbHCR1/BbHCR1 OE strains, suggesting that BbHCR1 played a distinct role in the regulation of conidiation and blastospore gener ation.The filamentous fungal conidiation is programmed by a CDP, which comprises the regulators followed by BrlA, AbaA, and WetA that activate target genes to com plete conidia formation and maturation (36), all of which are essential for sporulation (conidiation and blastospore generation) and important for hemocoel colonization after the injection of hyphal fragments in B. bassiana (35).Our RNA-seq and ChIP-seq data revealed that BbHCR1 acted as a negative regulator of brlA and abaA during hemocoel colonization since the two genes were significantly upregulated in the ΔBbHCR1 mutant but downregulated in the overexpression cells.The regulatory role of BbHCR1 in brlA and abaA expression in insect hemocoel colonization was verified by the disruption of brlA or abaA in the ΔBbHCR1 strain and overexpression of them in the BbHCR1 OE strain.Virulence of double mutation (ΔbrlA::ΔBbHCR1 or ΔabaA::ΔBbHCR1) was further decreased as compared to ΔBbHCR1.Although the two double overexpression mutants (brlA OE ::BbHCR1 OE and brlA OE ::BbHCR1 OE ) were more virulent than WT and a difference in virulence was examined in the two strains as compared to the BbHCR1 OE strain, overexpression of brlA or abaA in the BbHCR1 OE background restored the morphological transition of BbHCR1 OE to the wild type that produced numerous in vivo blastospores in insect hemocoel.Our RNA-seq data revealed that several fungal development genes, such as two cell adhesion-associated genes, one conidial cell wall protein gene, and one cell cycle gene displayed opposite expression patterns in insect hemolymph-derived ΔBbHCR1 and BbHCR1 OE cells.One fungal development and differentiation gene-coding membrane fusion mating protein, FIG1 (32), was identified as a BbHCR1 target, which was significantly downregulated in ΔBbHCR1 cells.These genes might also contribute to BbHCR1-controlled fungal development in insect hemocoel.
Insect fungal pathogens infect their hosts by direct penetration of the cuticle.After reaching hemocoel, penetrated hyphae differentiate into in vivo blastospores that evade the insect immune system and proliferate in the hemocoel (37,38).However, the ΔBbHCR1 strain formed numerous blastospores in the insect hemocoel with few hyphae, and unexpectedly, its virulence was significantly decreased.The opposite phenomena were detected in the BbHCR1 OE strain.These results suggested that other virulenceor/and immune-evasion-involved factors might be regulated by BbHCR1 to colonize the insect hemocoel besides control of the morphological transition.Our ChIP-seq and RNA-seq data revealed that four secretory protein genes, encoding a metalloproteaselike protein (BBA_02374) and three hypothetical proteins (BBA_07528 [HP1], BBA_01961 [HP2], and BBA_02460), were identified as BbHCR1 targets, in which BBA_02374 and HP1 were significantly downregulated in ΔBbHCR1 cells but upregulated in BbHCR1 OE cells, while other two genes were upregulated in ΔBbHCR1 cells but downregulated in BbHCR1 OE cells, suggesting these proteins might play opposite roles in fungal coloni zation of insect hemocoel.Two of the four genes, HP1 and HP2, were selected as representatives and characterized in this study.As expected, disruption of HP1 significantly decreased B. bassiana virulence, while overexpression of HP2 led to a decrease in fungal virulence.Although the underlying mechanisms were unclear and needed to be revealed in future work, these results suggested that BbHCR1 might activate virulence genes (e.g., HP1) but repress virulence repressor-associated genes (e.g., HP2) to help colonize insect hemocoel.While the contribution of other BbHCR1 targets (12 of 14) to fungal virulence needs to be determined in the additional work, these results explained the reduced/increased virulence of the ΔBbHCR1/BbHCR1 OE strains to some extent.B. bassiana produce secondary metabolites, oosporein and beauverolide, during the infection of insects.The former is synthesized after insect body cavity colonization, which contributes to fungal virulence via inhibiting prophenoloxidase activity that facilitates fungal cells to evade the host immune response (29) and to complete the infection cycle via limiting bacterial growth after host death that allows the fungus to maximally use host nutrients (39).The latter also contributes to fungal virulence due to its immunostimulatory and immunosuppressive properties (30).Thus, the two secondary metabolites play important roles in the fungal pathogen colonization of insect body cavities.Our RNA-seq data revealed that biosynthetic gene cluster or genes of oosporein (Ops1, Ops4, Ops6, and Ops7) (29) and beauverolide (besA and besB) (30) were significantly downregulated in ΔBbHCR1 but upregulated in BbHCR1 OE cells proliferated in the insect blood.These results account for the differences between BbHCR1 OE and ΔBbHCR1 in the evasion of insect immune responses to some extent (the former but not the latter easily evading insect immune defense responses), affecting their abilities to colonize insect hemocoel.
Our RNA-seq data revealed that some nutrient utilization-involved genes were significantly upregulated in the ΔBbHCR1 cells but downregulated in the BbHCR1 OE cells, which seemed to be in line with the reduced growth for the BbHCR1 OE strain but more aerial hyphae grown for the ΔBbHCR1 strain on agar plates.However, rapid growth for BbHCR1 OE cells but delayed growth for ΔBbHCR1 cells were examined in insect hemocoel, suggesting distinct regulatory roles of BbHCR1 in in vitro and in vivo growth and development.The contradictory phenomena in in vitro and in vivo growth of BbHCR1 OE and ΔBbHCR1 might be caused by their differences in the evasion of the insect immune defense responses examined.Moreover, increased and delayed growth was also examined in BbHCR1 OE and ΔBbHCR1 strains in broth containing oxidative agent or/and hypoxia stressor, mimicking stress niches in fungal-infected insect hemocoel (27,28), as compared to WT, respectively, which partially explained their different adaptations to the infected insect hemocoel and their potential role in the control of "oxidative burst" and low oxygen niches during host infection.
The Fus3-MAP kinase-mediated cascades have been shown to control the morpho logical transition of fungal pathogens, such as the infection structure (appressorium) formation or function, development of infective hyphae, dimorphism, and sporulation, despite the difference in fungal species (10,40).The pathway also mediates adaptation to oxidative stress derived from insect immune response-generated reactive oxygen species in some fungal species (41).The Hog1-MAP kinase pathway controls osmotic, oxidative, and thermal stress responses, as well as sporulation, viability, and fungal virulence (33), and has been implicated in mediating adaptation to low oxygen niches in fungal-infected insect hemocoel (28).Yeast two-hybrid assays revealed that BbHCR1 interacted with either BbHog1 (Hog1-MAP kinase) (33) or Bbmpk1 (Fus3-MAP kinase) (34), suggesting the involvement of the two kinases in the phosphorylation of BbHCR1.Bioinformatics analysis showed that two predicted phosphorylation sites, S815 and T865, were present in BbHCR1, which were predicted to be special for Fus3-MAP kinase and Fus3-/Hog1-MAP kinase, respectively.The effects of the two kinases on BbHCR1 phosphorylation level were verified using Western blotting assays, in which the phosphorylation levels were significantly decreased in the ΔBbmpk1 strain (inacti vation of Fus3-MAP kinase) under the low oxygen or oxidative stress conditions as compared to those under normal conditions, suggesting the phosphorylation was at least involved in the adaptation to stress niches in the fungal-infected insect hemocoel.Whereas phosphorylation levels of BbHCR1 were significantly decreased in the ΔBbHog1 strain (inactivation of Hog1-MAP kinase) under normal, low oxygen, or oxidative stress conditions, suggesting a crucial role of Hog1-MAP kinase in the control of BbHCR1 in fungal development in vitro and in vivo, as well as adaptation to stress niches in insect hemocoel.Although the introduction of the gene with the mutated phosphorylation site S815 (predicted for Fus3-MAP kinase) seemingly restored the ΔBbHCR1 virulence, mutation of the site T865 (predicted for either Fus3-or Hog1-MAP kinase) only slightly restored the ΔBbHCR1 virulence, suggesting that the contribution of phosphorylation at the latter site was greater than at the former site to the BbHCR1 function in fungal colonization of insect hemocoel and virulence.However, mutation of both the sites hardly restored the ΔBbHCR1 virulence.While it was unclear whether the site S865 was phosphorylated by either Fus3-MAP kinase or Hog1-MAP kinase as a prediction that needs to be determined in the additional work, these results suggested that Fus3-MAP kinase and Hog1-MAP kinase collaboratively regulated BbHCR1 by phosphorylating at the S815 and T865 sites.Moreover, how Fus3-MAP kinase or /and Hog1-MAP kinase affect(s) the function of BbHCR1 via phosphorylation should also be detailed in future work, i.e., controlling the TF internalization into nuclei, transcription activity, as well as binding affinity to different target genes.
Altogether, our results show a TF-mediated network that regulates the fungal colonization of insect body cavities, in which BbHCR1 is collaboratively controlled by Fus3-and Hog1-MAP kinases via phosphorylation, which in turn targets brlA and abaA and other likely targets for control of morphological transition and virulence factors by tuning the expression of hemocoel colonization-involved immunosuppressive metabo lite biosynthetic gene clusters.

Transcription factor co-expression analysis
B. bassiana transcription factors were retrieved from Fungal Transcription Factor Database (http://ftfd.snu.ac.kr/download.php)and their gene expression data were extracted from the whole genome expression database that was constructed from 76 deep-sequenced samples covering the growth, development, stress responses, and infection during the life cycle of B. bassiana (24).The TF expression data were normal ized, and co-expression analysis was performed from the cuticle penetration, hyphal bodies, liquid hyphae, and aerial hyphae database (24).LogCLR and Pearson's correla tion coefficient (PCC) were calculated, and genes with similar expression profiles were clustered.The co-expression network was generated with PCC ≥ 0.6.

Fungal strains, culture, and gene manipulation
B. bassiana wild-type Bb0062 (CGMCC 7.34) and its derived genetically modified strains were cultured as described previously (5).Yeast Y2HGold was used for transcrip tion activation assay and yeast two-hybrid assays.Escherichia coli 5α were used for DNA manipulations, and Agrobacterium tumefaciens AGL1 were employed for fungal transformation.
Target gene (BbHCR1, HP1, or HP2) disruption was performed using A. tumefaciensmediated homologous recombination by replacing the partial gene coding region with the herbicide phosphinothricin resistance bar cassette as previously (5).For the disruption of BbHCR1-regulated gene (abaA or brlA) on BbHCR1 mutation background, partial coding region of the candidate gene was replaced by the herbicide sulfonylurea resistance gene sur cassette via A. tumefaciens-mediated homologous recombination (48), generating ΔabaA::ΔBbHCR1 and ΔbrlA::ΔBbHCR1.To reverse complement the mutation strain ΔBbHCR1, the wild-type BbHCR1 (6,711 bp) containing the promoter region were amplified and cloned into XbaI/HindIII sites of pK2-sur (47), which were introduced into ΔBbHCR1 mutant using A. tumefaciens-mediated transformation with the herbicide resistance sur gene as the selective marker (5).The correct integration event of the putative gene disruption mutants and reverse complement strains was verified via PCR, while loss or regain of gene transcription was verified by reverse transcription-PCR (RT-PCR).
For overexpression of the genes, coding regions of BbHCR1, HP1, HP2, abaA, and brlA were amplified from B. bassiana, respectively, and cloned into BamHI/EcoRV sites of pK2surPB3-GFP, replacing the GFP sequence and leading to the expression of those target genes controlled by the promoter PB3.The resultant vectors were separately transformed into B. bassiana WT (for BbHCR1, HP1, and HP2) and BbHCR1 OE (for abaA and brlA) strains via A. tumefaciens-mediated transformation (45), generating BbHCR1 OE , HP1 OE , HP2 OE , abaA OE ::BbHCR1 OE , and brlA OE ::BbHCR1 OE strains.

Prediction of phosphorylation sites and detection of phosphorylation levels
Phosphorylation sites of BbHCR1 were predicted using Scansite 4.0 (https://scan site4.mit.edu/#scanProtein).The interactions of BbHCR1 with the predicted kinases were verified using yeast two-hybrid tests.The amino acids of phosphorylation sites were mutated to alanine by introducing mutated sites in primers.The BbHCR1 (total 6,711 bp, containing the promoter region) with introduced mutated sites was amplified and inserted into XbaI/HindIII sites of pK2-sur (47), which were individually intro duced into ΔBbHCR1 using A. tumefaciens-mediated transformation with the herbi cide resistance sur gene as the selective marker (5), generating ΔBbHCR1::BbHCR1 S815A , ΔBbHCR1::BbHCR1 T865A , and ΔBbHCR1::BbHCR1 S815A-T865A .The contribution of phosphoryla tion sites to the BbHCR1 function was investigated by comparison of virulence between the reverse complement strains with the WT gene and the mutated site(s)-containing gene(s) and WT.All the primers are shown in Table S1.
For the detection of BbHCR1 and its phosphorylated protein, Western blotting was performed.Briefly, fungal cells harboring the BbHCR1::13 myc fusion gene (in WT, ΔBbhog1, and ΔBbmpk1) from 1/4 SDY were cultured at 26°C for 60 h (normal condi tion) or in the Czapek-Dox Broth (BD Difco) containing stressors (75 µM menadione or 1 mM CoCl 2 ) for 6 h at 26°C.Fungal cells were collected and immediately homogen ized in liquid nitrogen for protein extraction.Proteins were extracted from ca. 0.1 g samples using 600 µL of cell lysis buffer [0.4 M NH 4 (SO 4 ) 2 , 10 mM MgCl 2 , 10% glycerol, and 2 mM β-mercaptoethanol in 0.2 M Tris-HCl (pH 8.0)] by adding protease inhibi tors phenylmethanesulfonyl fluoride (0.2 mg/mL), cOmplete Tablets EDTA-free (Roche) (0.98 mg/mL), and protein phosphatase inhibitor, PhosSTOP (Roche) (4.7 mg/mL).Protein was quantified using the BCA Assay Kit (GENEray), and 60 µg proteins was separated by 12.5% SDS-PAGE and Phos-tag SDS-PAGE (including 50 µM Phos-tag [Wako Pure Chemical Industries] and 100 µM MnCl 2 ).The BbHCR1 and its phosphorylated protein were blotted with anti-myc tag mouse polyclonal antibody (Thermo Fisher Scientific Inc.) and HRP-conjugated rabbit anti-mouse secondary antibody (Thermo Fisher Scientific Inc.).Anti-β-tubulin antibody (Sigma) was used as the internal standard.The gray values of the protein bands were estimated in Western blots using ImageJ program (49), and the relative phosphorylation levels of BbHCR1 were estimated by comparison of phosphorylated protein bands (from Phos-tag SDS-PAGE) to total BbHCR1 protein bands (from SDS-PAGE).

Gene expression analysis, transcription activation, and yeast two-hybrid assays
Gene expression analysis was performed using RT-PCR, RT-qPCR, and/ or by the detection of GFP fluorescence signals in fungal cells of the PBHCR1::GFP strain.For RT-PCR and RT-qPCR, 300 ng of total RNA was reverse-transcribed using an oligo(dT)-primed cDNA synthesis kit with gDNA Eraser (Aidlab, Beijing).The first-strand cDNA was used as the template for PCR.RT-qPCR analysis was conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing).RT-PCR and RT-qPCR were performed by using 18S rRNA (BBA_07911) as a reference gene as described previously (5).Loss or regain of target gene transcription in the gene disruption or complement strains was analyzed using RT-PCR from 1/4 SDY cultures for 60 h.The transcription level of BbHCR1 in the gene overexpression strains was evaluated using RT-qPCR from 1/4 SDY cultures for 60 h.Transcription levels of BbHCR1 in WT at different infection stages (in vivo), saprophytic hyphae (in vitro), and stressed by low oxygen or oxidative agents were evaluated using RT-qPCR and by the detection of GFP fluorescence signals in the PBHCR1::GFP cells.To prepare fungal cells during insect infection, 2 µL conidial suspension (10 7 conidia/mL) was inoculated in the last-instar Galleria mellonella larvae by injection at the second proleg.Fungal cells during insect infection were separately collected by blooding at 48 and 72 h post-injection and dissecting the tissues at 84 and 96 h after inoculation.The saprophytic hyphae (in vitro), including aerial and submerged hyphae, were prepared from 3-day 1/4 SDAY (agar) or 1/4 SDY (broth) with aeration at 26°C as described previously (5).The fungal cells stressed by LO or oxidative stresses were prepared by inoculation of 1/4 SDY cultures (for 60 h) in the Czapek-Dox Broth containing 1 mM CoCl 2 (a hypoxia-mimicking agent) (50,51) or oxidative stressors (75 µM MND) for 6 h.All primers for gene expression analysis are shown in Table S1.
For the transcriptional activation test, full-length BbHCR1 cDNA was amplified from WT and cloned into the EcoRI/BamHI sites of the yeast vector pGBKT7 (Clontech) under the GAL4 promoter to generate plasmid pGBKT7-BbHCR1, which was transformed into the yeast Y2HGold strain.The putative transformants were grown on the medium SD/-Trp and SD/-Trp with 0.35 µg/mL AbA (Aureobasidin A) and X-α-Gal.The positive control yeast was made by transforming with the pGBKT7 vector containing the GAL4 activation domain and the GAL4-binding domain, while the negative control yeast was transformed with the blank vector harboring the GAL4-binding domain.
Yeast two-hybrid assays were used to verify the interaction between BbHCR1 and the predicted kinase(s) that might phosphorylate BbHCR1.Briefly, cDNAs of the predicted kinases, Fus3-MAP kinase (Bbmpk1) (34) and Hog1-MAP kinase (Bbhog1) (33) genes, were separately amplified from B. bassiana and individually inserted into EcoRI/BamHI sites of the plasmid pGBKT7 as the prey carriers.The BbHCR1 cDNA was amplified and cloned into EcoRI/BamHI sites of the plasmid pGADT7 as the bait carrier.The resultant plasmids were co-transformed into Y2HGold cells.The resultant strains were verified by PCR and cultured on SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade auxotroph medium containing X-α-Gal and AbA (0.35 µg/mL), respectively.Y2HGold cells harboring pGADT7-T and pGBKT7-53 or pGADT7-T and pGBKT7-Lam were used as positive or negative control, respectively.All the primer pairs are shown in Table S1.

Insect bioassays and detection of fungal development in the insect hemocoel
Insect bioassays were performed using last-instar G. mellonella larvae either by topical application of conidia on the insect cuticle or direct injection of conidia into the insect hemocoel as described previously (5).For the topical application, 1 mL of conidial suspension (10 7 conidia/mL) was inoculated by spray on the larvae surface.For injection assays, 2 µL of conidial suspension (10 5 conidia/mL) was microinjected into the larvae via the second proleg.Since the capacities of ΔbrlA::ΔBbHCR1 and ΔabaA::ΔBbHCR1 strains to sporulate were completely lost, the mycelial fragments were prepared from their 1/4 SDY cultures for 3 days with ultrasonication (AMPL 25%, 6 s on and 6 s off for 10 min).The fragments were suspended in 0.05% Tween-80 and adjusted to a concentration of 20 mg fresh hyphae/mL, which were used for insect bioassay via injection (3 µL per larvae).Controls were treated with 0.05% Tween-80.All experiments were repeated three times with 90 larvae per replicate.Mortality was recorded every 12 h.
A subset of larvae in microinjection bioassays were bled at 24, 36, 48, 60, and 72 h post-treatment to microscopically monitor the fungal development.Fungal cells were quantified using qPCR analysis by amplification of the 18S rRNA sequence as previously described (5).For the tissue frozen section, the infected larvae were quickly frozen with liquid nitrogen at 60 h post-injection and sectioned with Microtome Cryostat (Thermo), in which the fungal cells in tissues were stained with lactic acid phenol cotton blue solution for microscopic observation.

RNA sequencing and ChIP sequencing
RNAs were isolated from WT, ΔBbHCR1, and BbHCR1 OE cells that were proliferated in the G. mellonella larvae hemocoel for 48 h after microinjection of 2 µL of conidial suspension (5 × 10 7 conidia /mL) or cultured in 1/4 SDY for 3 days and sequenced using the Illumina HiSeq 2000 platform (Novogene, Beijing, China).Briefly, the inoculated larvae were bled and mixed with the anticoagulant solution as described previously (38).The mixtures were resuspended in sterile water for breaking the hemocytes (due to low osmotic pressure) and centrifuged to remove hemocyte and hemolymph debris, which were repeated three times.After examination of fungal cells under a microscope, the pellets were used for RNA isolation.Reads were mapped to the B. bassiana 2860 genome (52) after the removal of adaptor tags, low-quality tags, and tags with only a single copy.Differentially expressed genes were identified between ΔBbHCR1 or BbHCR1 OE and WT RNA-seq libraries using the number of fragments per kilobase of exon region per million mappable reads (FPKM).A minimum of twofold expressional difference (i.e., log 2 FoldChange < −1.0 or > 1.0) in the paired libraries was used as a standard to judge each DEG at the false discovery rate of 0.05 or less (53).Sequencing was performed two times with different batches of fungal cells, and the reliability was assessed by Pearson's correlation coefficients (r) based on FPKM values.DEGs were classified and annotated using KEGG pathway enrichment analysis.
ChIP was performed using the B. bassiana PB3::BbHCR1::13myc strain, in which 13 Myc-tagged BbHCR1 controlled by the constitutive promoter PB3 was expressed in the ΔBbHCR1 strain.The resultant strain restored phenotypes of ΔBbHCR1 to wild type, including growth and virulence.ChIP was performed as described previously (54) using Pierce anti-c-myc magnetic beads (ThermoFisher) with fungal cells from 1/4 SDY cultures at 26°C for 60 h with aeration (200 rpm).The purified DNA from the immunocomplexes was used for ChIP sequencing with the Illumina HiSeq 2000 platform (Novogene, Beijing, China) for 76 bp paired-end sequencing.After cleaning contaminating adaptors using trimmomatic software (55), reads were aligned to the B. bassiana 2860 genome (52).Peaks with a P value of <0.005 were chosen as candidate binding sites and targeted genes were identified if peaks were located within their promoter regions (about 2.0 kb).The motifs within ChIP-seq peaks were analyzed using an online motif predictor, the Multiple Em for Motif Elicitation (https://meme-suite.org/meme/) tool (56).The identified target genes of BbHCR1 were verified using electrophoretic mobility shift assay.Briefly, the predicted DNA-binding domain of BbHCR1 (411 bp) was amplified and cloned into pET28a, which was introduced into E. coli BL21 to express His-tagged fusion protein following standard protocol.Proteins were purified using a Mag-Beads His-Tag Protein Purification Kit (Sangon, China).The promoter fragments containing predicted BbHCR1binding motifs of the target genes (472, 322, 328, and 369 bp for brlA, abaA, HP1, and HP2, respectively) were amplified (primers are shown in Table S1) and used for EMSA assays with His-tagged DNA-binding domain of BbHCR1.The ability of BbHCR1 to bind promoter regions of the target genes was detected in gels using the LightShift Chemilu minescent EMSA Kit (Thermo Fisher, USA) following the manufacturer's instructions.

FIG 2
FIG 2 Transcription factor characteristics and expression profiles of BbHCR1.(A) Transcriptional activation assays in yeast.Yeast strain Y2HGold expressing the GAL4 activation and the GAL4 DNA-binding domains (positive control), the blank vector with the GAL4 DNA-binding domain (negative control), or the GAL4 DNA binding and BbHCR1 (BD GAL4 -BbHCR1) were cultured on the medium SD/-Trp and SD/-Trp with 0.35 µg/mL AbA (Aureobasidin A) and X-α-Gal at 30°C for 3 days.(B) Subcellular localization of BbHCR1 in B. bassiana.Distribution of GFP fluorescence of BbHCR1::GFP hyphal bodies.Nucleus was labeled with RFP by fusion RFP at the C-terminal of histone (H1::RFP).(C and E) RT-qPCR analysis of BbHCR1 expression patterns in fungal cells proliferated in insect hemocoel after the injection of conidia at indicated hours in aerial hyphae and liquid hyphae (C) and under stressed conditions (E).Fungal cells were cultured in CZB (control) and CZB containing 1.0 M sorbitol, 75 µM menadione (MND), or 1 mM CoCl 2 for 6 h.(D and F) GFP fluorescence of PBbHCR1::GFP strain in panels C and E, in which GFP was driven by BbHCR1 promoter.Scale bar = 5 µm.

FIG 4
FIG 4 Fungal development and insect immune responses in hemocoel.(A) Microscopic images of fungal development and insect immune responses at the indicated time after injection.White arrows indicate fungal cells.Scale bar = 5 µm.(B) Quantification of the hyphal bodies from hemocoel at 36, 48, and 60 h after infection using qPCR analysis by amplification of the 18S rRNA sequence as detailed in Materials and Methods.*P < 0.05; **P < 0.01; and ***P < 0.001.(C) Frozen section of larvae at 60 h after the injection of conidia.Sample stained by lactic acid phenol cotton blue solution.SC, insect cuticle.FB, fat body.White arrows indicate fungal cells (FC).Scale bar = 20 µm.

FIG 5 FIG 6
FIG 5 Identification the BbHCR1 target genes by comparative analysis of ChIP-seq and RNA-seq.(A) Venn diagram of RNA-seq (|fold change| ≥ 2) and ChIP-seq.(B) MEME analysis indicating the likely binding sequence of BbHCR1.(C) Annotation of identified BbHCR1 gene targets and their expression patterns.Solid blue circles indicate proteins containing signaling peptides.(D) EMSA verification of four identified gene targets of BbHCR1.The promoter fragments containing predicted BbHCR1-binding motifs of four target genes, 472, 322, 328, and 369 bp for brlA, abaA, HP1, and HP2, respectively, were amplified and used for EMSA assays with His-tagged DNA-binding domain of BbHCR1 (15.4 kDa), which was expressed in Escherichia coli BL21 and purified.

FIG 7
FIG 7 BbHCR1 function in fungal virulence depends on phosphorylation by Fus3-MAP kinase (Bbmpk1) and Hog1-MAP kinase (Bbhog1).(A) Predicted phosphorylation sites of BbHCR1 for the possible kinases.(B) Yeast two-hybrid tests of the interaction of BbHCR1 and the predicted kinases.(C) Survival of G. mellonella larvae and the calculated LT 50 values following intrahemocoel injection (2 µL of 10 5 conidia/mL) of the indicated fungal conidia.(D and E) Assays for the phosphorylation of BbHCR1 in WT, ΔBbmpk1, and ΔBbhog1 strains.Phosphorylation of BbHCR1 was probed in fungal cells using the 13× Myc-tagged BbHCR1 and Western blotting via Phos-tag SDS-PAGE.BbHCR1 proteins were blotted with anti-myc antibody.Western blot with anti-β-tubulin antibody was used as the internal standard.Proteins were isolated from fungal cells grown under normal (CZB) and CZB containing stressors (75 µM menadione or 1 mM CoCl 2 ) at 26°C for 6 h.The relative amounts of the phosphorylated BbHCR1 as compared to their total protein levels for each sample were measured by densitometric analyses of bands using the ImageJ software.