Botrytis cinerea G Protein β Subunit Bcgb1 Controls Growth, Development and Virulence by Regulating cAMP Signaling and MAPK Signaling

Botrytis cinerea is a necrotrophic phytopathogenic fungus that causes gray mold disease in many crops. To better understand the role of G protein signaling in the development and virulence of this fungus, the G protein β subunit gene Bcgb1 was knocked out in this study. The ΔBcgb1 mutants showed reduced mycelial growth rate, but increased aerial hyphae and mycelial biomass, lack of conidiation, failed to form sclerotia, increased resistance to cell wall and oxidative stresses, delayed formation of infection cushions, and decreased virulence. Deletion of Bcgb1 resulted in a significant reduction in the expression of several genes involved in cAMP signaling, and caused a notable increase in intracellular cAMP levels, suggesting that G protein β subunit Bcgb1 plays an important role in cAMP signaling. Furthermore, phosphorylation levels of MAP kinases (Bmp1 and Bmp3) were increased in the ΔBcgb1 mutants. Yeast two-hybrid assays showed that Bcgb1 interacts with MAPK (Bmp1 and Bmp3) cascade proteins (BcSte11, BcBck1, BcMkk1, and BcSte50), and the Bmp1-regulated gene Bcgas2 was up-regulated in the ΔBcgb1 mutant. These results indicated that Gβ protein Bcgb1 is involved in the MAPK signaling pathway in B. cinerea. In summary, our results revealed that Gβ protein Bcgb1 controls development and virulence through both the cAMP and MAPK (Bmp1 and Bmp3) signaling pathways in B. cinerea.


Introduction
Botrytis cinerea is an important phytopathogenic fungus and the causal agent of gray mold disease in more than 1400 plant species. It is responsible for significant economic losses in many important vegetables, fruits, and ornamentals [1]. The cost of controlling gray mold disease in the world has been estimated at over €1 billion per year. Due to its scientific and economic importance, B. cinerea is considered as the second most important fungal pathogen and the necrotrophic model fungus [2]. In the life cycle of B. cinerea, there are four different structures, including conidia, mycelia, sclerotia, and ascospores. Since sexual ascospores rarely occur in nature, the main source of the initial inoculum in the field is asexual conidia that formed from germinating sclerotia or hyphae, or survived in the last season [3]. Sclerotia are the melanized dormancy structures that can survive in adverse environment. When favorable conditions appear in spring, sclerotia will germinate to produce hyphae and conidia as the source of initial infection. Therefore, sclerotia and conidia play pivotal roles in the epidemic and life cycle of B. cinerea [3].
Heterotrimeric G proteins, which consist of Gα, Gβ, and Gγ subunits, transmit a variety of extracellular signals received by membrane-spanning G protein coupled receptors (GPCR) to intracellular effectors of eukaryotic cells [4]. When GPCR senses external signal stimulation, it triggers GDP-GTP exchange in Gα, leading to the dissociation of G protein complex as

Fungal Strains and Culture Conditions
The wild-type strain B05.10 and its derived strains, including Bcgb1 gene knockout mutants (∆Bcgb1-8, ∆Bcgb1-43, and ∆Bcgb1-64), were cultivated on potato dextrose agar (PDA) [17] at 20 • C. The Bcgb1 gene knockout mutants were maintained on PDA amended with 100 µg·mL −1 hygromycin B (Calbiochem, San Diego, CA, USA). For growth experiments, the mutants and B05.10 were grown on PDA at 20 • C. Each plate was inoculated with a 5 mm-diameter mycelial agar plug taken from the edge of a 2-day-old colony. To characterize the growth rate, sclerotia formation, and infection cushion formation, a different strain was cultured in constant darkness. To characterize the sporulation, strains were grown under a 12 h light/dark cycle. To test the mycelial biomass, 10 mycelial plugs (5 mm) of each strain were inoculated into an Erlenmeyer flask (250 mL) containing 100 mL potato dextrose broth (PDB) [17], with three flasks for each strain, and the flasks were shakeincubated at 20 • C and 150 rpm for 2 days. Mycelial biomass of each strain was harvested by paper-filtering, dried at 55 • C for 12 h, and weighed. To evaluate the response of Bcgb1 knockout mutants to abiotic stress, the wild-type strain and Bcgb1 knockout mutants were cultured on PDA medium amended with 1 M NaCl, 1 M KCl, 1 M sucrose, 1 M sorbitol, 0.1 mg/mL SDS, 0.3 mg/mL Congo Red (CR), 0.2 mg/mL CalcoFluor White (CFW), and 5mM H 2 O 2 . The colony diameters were measured at 72 h to calculate the relative mycelial growth rate of each strain. Each experiment was repeated three times.

Disruption of Bcgb1
The Bcgb1 gene was disrupted using the split marker method [18]. The disruption strategy for Bcgb1 is showed in Figure S1. The 5 and 3 flanking sequences of Bcgb1 were amplified with the primers listed in Table S1 and then fused with part of the hygromycin fragment. Two split-marker DNA fragments were transformed into protoplasts of the WT strain B05.10 using the PEG-mediated transformed technique [19]. The hyphal tips of the deletion transformants were screened on PDA plates containing hygromycin B (100 µg mL −1 ) three times and verified by PCR. Single spore isolation was performed to obtain the homokaryotic deletion mutants. Three Bcgb1 deletion mutants, ∆Bcgb1-8, ∆Bcgb1-43, and ∆Bcgb1-64, were further confirmed by Southern blot analysis using the right flank of the Bcgb1 gene as a probe. Southern blot analysis was performed by the Gene Images TM AlkPhos Direct TM labeling and detection kit from GE Healthcare (Amersham Biosciences, Buckinghamshire, UK).

Extraction of DNA and RNA
Strains of B. cinerea were grown on PDA medium at 20 • C under darkness for 2 days. Genomic DNA of B. cinerea was extracted from the mycelia using the CTAB method [20]. Total RNA was extracted from mycelium samples of B. cinerea using the RNAiso Plus reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions.

Pathogenicity and Penetration Assays
A pathogenicity test was performed with 5-week-old tobacco (Nicotiana benthamiana) leaves using 5 mm mycelial plugs from wild-type, ∆Bcgb1-8, and ∆Bcgb1-43 mutant strains grown on PDA. Infected leaves were incubated at 20 • C under darkness with 100% relative humidity. The lesion diameters were measured at 72 h post inoculation.
Infection cushions were observed on onion epidermis as per a previous study [21]. Mycelial plugs (5 mm) of each strain were inoculated on onion epidermis and incubated at 20 • C under darkness. The epidermis was sampled and then stained with cotton blue before microscopic examination at 12 h and 24 h post inoculation, respectively. Each experiment was repeated three times.

Quantification of Intracellular cAMP
Mycelia were harvested from two-day-old PDB [17] liquid cultures, frozen in liquid nitrogen, and lyophilized for 20 h. For every 10 mg of lyophilized mycelium, 1 mL of 0.1 M HCl was added. Samples were centrifuged at 12,000 rpm for 15 min. The supernatant was used to determine cAMP concentration via the Monoclonal Anti-cAMP Antibody Based Direct cAMP ELISA Kit (NewEast Biosciences, Malvern, PA, USA) following the manufacturer's instructions.

Reverse Transcription and Fluorescence Quantitative PCR (RT-qPCR)
The cDNA was synthesized via the PrimeScript TM RT reagent kit (TaKaRa, Dalian, China) according to instructions from the manufacturer. An RT-qPCR was carried out in a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) with TB Green ® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Dalian, China). The B. cinerea actin gene BcactA (Bcin16g02020) was used as internal control. The relative expression of each gene was evaluated using the ∆∆ CT method [22]. All primers used for the RT-qPCR analyses are listed in Table S1. The RT-qPCR assay was repeated three times, each with three biological replicates.

Assays for Bmp1 and Bmp3 Phosphorylation
Total proteins were isolated from two-day-old mycelia with the protein lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 1% each of protease inhibitor cocktail, phosphatase inhibitor cocktail 2, and phosphatase inhibitor cocktail 3 (Sigma-Adrich, St. Louis, MO, USA) as previously described [23]. Then, the total proteins were separated by 10% SDS-PAGE and then transferred to PVDF (polyvinylidene difluoride) membranes (Bio-Rad, Hercules, CA, USA). Phosphorylation of the Bmp1 and Bmp3 MAP kinases was detected by using the phospho-p44/42 MAPK antibody (Cell Signaling Technology, Boston, MA, USA). The total Bmp1 and Bmp3 was detected with anti-MAPK ERK 1/2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The anti-GAPDH was used as a loading control.

Yeast Two-Hybrid Assays
The Matchmaker TM Gold yeast two-hybrid system (Clontech, Mountain View, CA, USA) was used to analyze the protein-protein interactions. The full-length cDNA of Bcgb1 (Bcin08g01420) was cloned into pGADT7 vector. Full-length cDNAs of BcSte11 (Bcin03g02630), BcSte7 (Bcin04g05630), BcBck1 (Bcin02g06590), BcMkk1 (Bcin03g07190), and BcSte50 (Bcin08g03660) were cloned into pGBKT7 vector. A pair of plasmids (pGBKT7-53 and pGADT7-T) served as a positive control and a pair of plasmids (pGBKT7-Lam and pGADT7-T) was used as a negative control. The resulting prey and bait constructs were co-transformed in pairs into yeast strain Y2H following the manufacturer's instructions. Transformants were grown on SD-Leu-Trp at 30 • C for 3 days, and then transferred to SD-His-Leu-Trp. The resulting yeast cells were further tested for β-galactosidase activities. The primers used in this experiment are listed in Table S1.

Bcgb1 Is Required for Hyphal Growth, Conidiation, Sclerotia Formation
To determine the role of Bcgb1 in hyphal growth, conidiation, and sclerotia formation, two ∆Bcgb1 mutants (∆Bcgb1-8 and ∆Bcgb1-43) grown on PDA were compared with the wild-type strain B05.10. Colonies of ∆Bcgb1 mutants showed a fluffy, dense aspect; a decreased colony diameter; and dramatically increased aerial hyphae compared to the wild type (Figure 2A,B). Microscopic analysis showed that the ∆Bcgb1 mutants produced more branches at the tip of the hyphae than that of the wild type ( Figure 2A). After 15 days of incubation on PDA, the wild-type strain produced a large number of conidia and formed sclerotia. However, the ∆Bcgb1 mutants were unable to produce conidia and sclerotia ( Figure 2A). In comparison with the wild type, the mycelial growth rate of the ∆Bcgb1 mutants was significantly reduced ( Figure 2C), but the mycelial biomass was increased ( Figure 2D). These results indicate that Bcgb1 plays an important role in hyphal growth, conidiation, and sclerotia formation.  duced more branches at the tip of the hyphae than that of the wild type ( Figure 2A). After 15 days of incubation on PDA, the wild-type strain produced a large number of conidia and formed sclerotia. However, the ΔBcgb1 mutants were unable to produce conidia and sclerotia ( Figure 2A). In comparison with the wild type, the mycelial growth rate of the ΔBcgb1 mutants was significantly reduced ( Figure 2C), but the mycelial biomass was increased ( Figure 2D). These results indicate that Bcgb1 plays an important role in hyphal growth, conidiation, and sclerotia formation.

Bcgb1 Is Involved in Response to Cell Wall and Oxidative Stresses
To investigate functions of Bcgb1 in cell-wall integrity, we examined the sensitivity of the ΔBcgb1 mutants to osmotic stress agents NaCl, KCl, sucrose, and sorbitol; cell-wall disturbing agents SDS, CR, and CFW; and oxidative stress H2O2. Our results show that there was no significant difference in relative growth rate between the ΔBcgb1 mutants and wild type when cultured on PDA containing NaCl, KCl, sucrose, sorbitol, and CR ( Figure 3). However, the relative growth rate of the ΔBcgb1 mutants significantly increased when cultured on PDA containing SDS, CFW, and H2O2 ( Figure 3A,B). These results indicate that Bcgb1 plays a role in response to cell-wall and oxidative stresses.

Bcgb1 Is Involved in Response to Cell Wall and Oxidative Stresses
To investigate functions of Bcgb1 in cell-wall integrity, we examined the sensitivity of the ∆Bcgb1 mutants to osmotic stress agents NaCl, KCl, sucrose, and sorbitol; cell-wall disturbing agents SDS, CR, and CFW; and oxidative stress H 2 O 2 . Our results show that there was no significant difference in relative growth rate between the ∆Bcgb1 mutants and wild type when cultured on PDA containing NaCl, KCl, sucrose, sorbitol, and CR ( Figure 3). However, the relative growth rate of the ∆Bcgb1 mutants significantly increased when cultured on PDA containing SDS, CFW, and H 2 O 2 ( Figure 3A,B). These results indicate that Bcgb1 plays a role in response to cell-wall and oxidative stresses.

Bcgb1 Is Important for Virulence in B. cinerea
To analyze the role of Bcgb1 in pathogenicity, unwounded and wounded tobacco leaves were inoculated with the mycelial agar plugs of ∆Bcgb1 mutants. The ∆Bcgb1 mutants showed significantly reduced virulence in tobacco leaves ( Figure 4A). At 72 hpi, the lesion size of ∆Bcgb1 mutants on both unwounded and wounded leaves decreased by more than 50% compared with that of the wild type ( Figure 4B). To determine the virulence defects of ∆Bcgb1 mutants in detail, we performed a penetration assay on onion epidermis. As show in Figure 4C, the wild-type strain formed numerous infection cushions and successfully penetrated onion cells at 12 hpi and 24 hpi. However, the average number of infection cushions of ∆Bcgb1 mutants was much less than that of the wild type ( Figure 4D). This revealed that the ∆Bcgb1 mutants delayed the formation of infection cushions to penetrate plant cells, resulting in the decrease of virulence. These results show that Bcgb1 is important for infection cushion formation and virulence.

Bcgb1 Is Involved in the Regulation of Intracellular cAMP Levels
To test whether deletion of Bcgb1 affects the cAMP levels in B. cinerea, the intracellular cAMP levels were measured in the hyphae stage of the ∆Bcgb1 mutants and wild type. The cAMP levels of two ∆Bcgb1 mutants were drastically increased about fourfold and sixfold, respectively, compared to the wild type ( Figure 5A).

Bcgb1 Is Important for Virulence in B. cinerea
To analyze the role of Bcgb1 in pathogenicity, unwounded and wounded tobacco leaves were inoculated with the mycelial agar plugs of ΔBcgb1 mutants. The ΔBcgb1 mutants showed significantly reduced virulence in tobacco leaves ( Figure 4A). At 72 hpi, the lesion size of ΔBcgb1 mutants on both unwounded and wounded leaves decreased by more than 50% compared with that of the wild type ( Figure 4B). To determine the virulence defects of ΔBcgb1 mutants in detail, we performed a penetration assay on onion epidermis. As show in Figure 4C, the wild-type strain formed numerous infection cushions and successfully penetrated onion cells at 12 hpi and 24 hpi. However, the average number of infection cushions of ΔBcgb1 mutants was much less than that of the wild type (Figure 4D). This revealed that the ΔBcgb1 mutants delayed the formation of infection cushions to penetrate plant cells, resulting in the decrease of virulence. These results show that Bcgb1 is important for infection cushion formation and virulence.

Bcgb1 Is Involved in the Regulation of Intracellular cAMP Levels
To test whether deletion of Bcgb1 affects the cAMP levels in B. cinerea, the intracellular cAMP levels were measured in the hyphae stage of the ΔBcgb1 mutants and wild type. The cAMP levels of two ΔBcgb1 mutants were drastically increased about fourfold and sixfold, respectively, compared to the wild type ( Figure 5A).
Due to the cAMP levels having increased in ΔBcgb1 mutants, we further examined The cAMP levels of two ΔBcgb1 mutants were drastically increased about fourfold and sixfold, respectively, compared to the wild type ( Figure 5A). Due to the cAMP levels having increased in ΔBcgb1 mutants, we further examined the transcript levels of the cAMP signaling pathway-related genes, such as the adenylate cyclase gene Bac, two phosphodiesterase genes (BcPde1 and BcPde2), and three cAMP-dependent protein kinase (PKA) encoding genes (BcPka1, BcPka2, and BcPkaR). Interestingly, the expression of these six genes (Bac, BcPde1, BcPde2, BcPka1, BcPka2, and BcPkaR) was all significantly reduced in the ΔBcgb1 mutants ( Figure 5B,C). These results indicate that Bcgb1 is required for maintaining normal cAMP levels in B. cinerea.  Due to the cAMP levels having increased in ∆Bcgb1 mutants, we further examined the transcript levels of the cAMP signaling pathway-related genes, such as the adenylate cyclase gene Bac, two phosphodiesterase genes (BcPde1 and BcPde2), and three cAMPdependent protein kinase (PKA) encoding genes (BcPka1, BcPka2, and BcPkaR). Interestingly, the expression of these six genes (Bac, BcPde1, BcPde2, BcPka1, BcPka2, and BcPkaR) was all significantly reduced in the ∆Bcgb1 mutants ( Figure 5B,C). These results indicate that Bcgb1 is required for maintaining normal cAMP levels in B. cinerea.

Bcgb1 Plays an Important Role in Two MAPK (Bmp1 and Bmp3) Signaling Pathways
To investigate whether Bcgb1 plays a role in the MAPK (Bmp1 and Bmp3) signaling pathway, we examined the phosphorylation levels of Bmp1 and Bmp3 in ∆Bcgb1 mutants with an anti-TpEY antibody. A Western blotting assay showed that ∆Bcgb1 mutants were increased in Bmp1 and Bmp3 phosphorylation compared with the wild type ( Figure 6A). To further explore the role of Bcgb1 in Bmp1 and Bmp3 phosphorylation, we examined the interaction of Bcgb1 with the components of two MAPK signaling cascades (BcSte11/BcSte7/Bmp1, BcBck1/BcMkk1/Bmp3, and the MAPK adapter protein BcSte50). The results of yeast twohybrid show that Bcgb1 directly interacted with both Bmp1 cascade protein (BcSte11) and Bmp3 cascade proteins (BcBck1 and BcMkk1). Moreover, Bcgb1 directly interacted with the MAPK adapter protein BcSte50 ( Figure 6B).
To test whether deletion of Bcgb1 altered expression of the downstream target genes of Bmp1, we measured the transcript level of a target gene, Bcgas2 [16], in the ∆Bcgb1 mutants and wild type. The results of the qRT-PCR show that the Bcgas2 transcript level was significantly increased in the ∆Bcgb1 mutants ( Figure 6C). Our findings suggest that Bcgb1 plays an important role in the MAPK (Bmp1 and Bmp3) signaling pathway.

Deletion of Bcgb1 Affects the Expression of Sclerotia Formation-Related Genes
Because ∆Bcgb1 mutants lost the ability to form sclerotia, we examined whether Bcgb1 is involved in controlling the expression of sclerotia formation-related genes in B. cinerea. Twelve genes that were confirmed to be related to sclerotia formation were selected to detect the expression in the ∆Bcgb1 mutants and wild type by qRT-PCR (Figure 7). Three genes encoding the VELVET complex (BcLaeA1, BcVEL1, and BcVEL2) were differentially affected in the ∆Bcgb1 mutants. The expression of BcLaeA1 in ∆Bcgb1 mutants was similar to that in wild type. However, in ∆Bcgb1 mutants, the transcript level of BcVEL1 was downregulated, whereas BcVEL2 was up-regulated. The expression of two NADPH oxidases genes (BcNoxA and BcNoxD) was significantly reduced in ∆Bcgb1 mutants. Among six melanogenic genes, four genes (Bcbrn2, Bcscd1, Bcsmr1, and Bcpks12) were repressed in ∆Bcgb1 mutants. In contrast, other two melanogenic genes (Bcbrn1 and Bcpks13) were overexpressed in ∆Bcgb1 mutants. Furthermore, expression of the bZIP transcription factor gene BcAtf1, which is required for sclerotia formation, was decreased in ∆Bcgb1 mutants.
Taken together, the expression studies suggested that Bcgb1 plays an important role in regulation of sclerotia formation-related gene expression in B. cinerea. the interaction of Bcgb1 with the components of two MAPK signaling cascades (BcSte11/BcSte7/Bmp1, BcBck1/BcMkk1/Bmp3, and the MAPK adapter protein BcSte50). The results of yeast two-hybrid show that Bcgb1 directly interacted with both Bmp1 cascade protein (BcSte11) and Bmp3 cascade proteins (BcBck1 and BcMkk1). Moreover, Bcgb1 directly interacted with the MAPK adapter protein BcSte50 ( Figure 6B).
To test whether deletion of Bcgb1 altered expression of the downstream target genes of Bmp1, we measured the transcript level of a target gene, Bcgas2 [16], in the ΔBcgb1 mutants and wild type. The results of the qRT-PCR show that the Bcgas2 transcript level was significantly increased in the ΔBcgb1 mutants ( Figure 6C). Our findings suggest that Bcgb1 plays an important role in the MAPK (Bmp1 and Bmp3) signaling pathway.  NADPH oxidases genes (BcNoxA and BcNoxD) was significantly reduced in ΔBcgb1 mutants. Among six melanogenic genes, four genes (Bcbrn2, Bcscd1, Bcsmr1, and Bcpks12) were repressed in ΔBcgb1 mutants. In contrast, other two melanogenic genes (Bcbrn1 and Bcpks13) were overexpressed in ΔBcgb1 mutants. Furthermore, expression of the bZIP transcription factor gene BcAtf1, which is required for sclerotia formation, was decreased in ΔBcgb1 mutants. Taken together, the expression studies suggested that Bcgb1 plays an important role in regulation of sclerotia formation-related gene expression in B. cinerea.
Loss of Bcgb1 in B. cinerea caused mutants with a significant decrease in virulence. This is consistent with the function of the Gβ gene in most plant pathogenic fungi, except that the Gβ gene deletion mutants showed a slightly reduced in virulence in U. maydis [25] and F. verticillioides [27]. In B. cinerea, an infection cushion is a special infection structure that is necessary for successful infection of mycelia. The ΔBcgb1 mutant was defective in infection cushion formation, and was responsible for reduced virulence. Similarly, the Gβ gene played a critical role in the infection structure (appressorium) formation and pathogenicity in M. grisea [7] and C. heterostrophus [26].
Deletion of Bcgb1 resulted in altered colony morphology and decreased mycelial growth rate, but increased aerial hyphae and mycelia biomass. Alteration of colony morphology was also presented in the Gβ deletion mutant of F. oxysporum [9] and V. dahliae [12]. In Aspergillus nidulans, the Gβ deletion mutant ΔsfaD showed a significant reduction in mycelial mass, although the growth rate was similar to wild type [29]. Similar to the ΔBcgb1 mutant, more aerial hyphae were also found in the Gβ mutant of M. grisea [7]. In contrast, the Gβ gene cpgb-1 was required for normal aerial hyphae formation in the chestnut blight fungus C. parasitica [24]. In F. verticillioides, the Gβ gene gbb1 was dispensable
Loss of Bcgb1 in B. cinerea caused mutants with a significant decrease in virulence. This is consistent with the function of the Gβ gene in most plant pathogenic fungi, except that the Gβ gene deletion mutants showed a slightly reduced in virulence in U. maydis [25] and F. verticillioides [27]. In B. cinerea, an infection cushion is a special infection structure that is necessary for successful infection of mycelia. The ∆Bcgb1 mutant was defective in infection cushion formation, and was responsible for reduced virulence. Similarly, the Gβ gene played a critical role in the infection structure (appressorium) formation and pathogenicity in M. grisea [7] and C. heterostrophus [26].
Deletion of Bcgb1 resulted in altered colony morphology and decreased mycelial growth rate, but increased aerial hyphae and mycelia biomass. Alteration of colony morphology was also presented in the Gβ deletion mutant of F. oxysporum [9] and V. dahliae [12]. In Aspergillus nidulans, the Gβ deletion mutant ∆sfaD showed a significant reduction in mycelial mass, although the growth rate was similar to wild type [29]. Similar to the ∆Bcgb1 mutant, more aerial hyphae were also found in the Gβ mutant of M. grisea [7]. In contrast, the Gβ gene cpgb-1 was required for normal aerial hyphae formation in the chestnut blight fungus C. parasitica [24]. In F. verticillioides, the Gβ gene gbb1 was dispensable for mycelial growth and mycelial mass but important for mycotoxin fumonisin B 1 production [27]. These results indicate that Gβ in filamentous fungi plays different roles in mycelial growth.
The Gβ gene is required for sporulation in B. cinerea, which was also found in several fungi, such as C. parasitica [24], M. grisea [7], F. oxysporum [9], C. heterostrophus [26], and F. verticillioides [26]. However, the opposite results, that deletion of Gβ gene caused increased conidiation, were observed in A. nidulans [29] and V. dahliae [12]. In addition, the ∆Bcgb1 mutants failed to form sclerotia, but the Gβ mutants of V. dahliae enhanced sclerotia formation [12]. It is suggested that the role of Gβ in conidiation and sclerotia formation was opposite in B. cinerea and V. dahliae. The qRT-PCR results revealed that loss of Gβ affected the expression of sclerotia formation-related genes, indicating that Gβ is an upstream regulatory component of these genes.
In filamentous fungi, G proteins are involved in the regulation of cAMP signaling that controls multiple cellular processes, including growth, development, and virulence [4]. Deletion of the Gβ gene resulted in reduced intracellular cAMP levels in N. crassa [30], M. grisea [7], and F. oxysporum [9]. In addition, loss of Gβ caused a decreased in Gα protein levels in C. parasitica [31] and N. crassa [30]. Therefore, Gβ should maintain normal levels of Gα protein, which stimulates adenylate cyclase activity to form cAMP [32]. However, the intracellular cAMP levels were drastically increased in ∆Bcgb1 mutants ( Figure 5A), indicating that Gβ serves as an inhibitor to suppress the activity of Gα proteins in B. cinerea. The adenylate cyclase (cAMP biosynthesis) and phosphodiesterase (cAMP hydrolysis) are crucial regulators for maintaining the balance of intracellular cAMP levels [33]. In this study, expression of adenylate cyclases gene (Bac) and phosphodiesterases genes (BcPde1 and BcPde2) was significantly reduced in the ∆Bcgb1 mutants ( Figure 5B). The possible explanation is that Bcgb1 deletion inhibits the transcription of BcPde1 and BcPde2, resulting in increased cAMP levels that may feedback suppress the expression of Bac. Thus, the activities of adenylate cyclase and phosphodiesterase in ∆Bcgb1 mutants needs to be further investigated. Another cAMP signaling component is the cAMP-dependent protein kinase (PKA), consisting of two regulatory subunits and two catalytic subunits. In B. cinerea, BcPka1 and BcPka2 belong to a catalytic subunit, and BcPkaR is the regulatory subunit [34]. Deletion mutants of PKA (∆BcPka1, ∆BcPka2, and ∆BcPkaR) all showed significantly increased intracellular cAMP levels in mycelia, suggesting that the PKA (BcPka1, BcPka2, and BcPkaR) negatively regulates the intracellular cAMP levels in B. cinerea [34]. Similarly, a significant reduction in expression of three PKA genes (BcPka1, BcPka2, and BcPkaR) and increased intracellular cAMP levels were also observed in ∆Bcgb1 mutants ( Figure 5C).
In Saccaromyces cerevisiae yeast, the Gβ protein Ste4p is required to transfer the pheromone signal to activate the MAPK mating pathway [35]. However, deletion of Gβ gene fgb1 did not affect phosphorylation level of the MAP kinase Fmk1 in F. oxysporum [31]. Our results show that phosphorylation levels of MAP kinases (Bmp1 and Bmp3) were increased in ∆Bcgb1 mutants ( Figure 6A), supporting the hypothesis that Gβ regulates the MAPK signaling pathway downstream in Cryptococcus neoformans [36] and M. grisea [7]. Yeast two-hybrid assays showed that Gβ protein Bcgb1 directly interacted with MAPK cascade proteins (BcSte11, BcBck1, BcMkk1, and BcSte50) ( Figure 6B). This provides evidence that Gβ is involved in the MAPK signaling pathway. Additional evidence is that Bcgas2, the downstream regulated gene of Bmp1 [16], was up-regulated in the ∆Bcgb1 mutant. These results suggest that Gβ protein Bcgb1 plays an important role in the regulation of the MAPK signaling pathway in B. cinerea.
Previous studies have demonstrated that deletion of the MAP kinase Bmp1 causes defects in conidia germination, reduces mycelial growth, causes a failure to form sclerotia, and induces a loss of pathogenicity in B. cinerea [37]. Another MAP kinase, Bmp3, is important for growth, conidiation, sclerotia formation, and virulence [38]. Interestingly, the ∆Bcgb1 mutants showed similar defective phenotypes, but increased phosphorylation levels of Bmp1 and Bmp3. Maintenance of normal phosphorylation levels of MAPK is critical for the MAPK signaling pathway in eukaryotic cells. Our data indicate that Gβ protein Bcgb1 is required for maintaining normal phosphorylation levels of Bmp1 and Bmp3 in B. cinerea.
In conclusion, this study presents evidence that Bcgb1 not only plays an important role in the cAMP signaling pathway, but also regulates the MAPK signaling pathway. Bcgb1 may function in cross-talks between these signaling pathways. This might explain the defects of the ∆Bcgb1 mutant in mycelial growth, conidiation, sclerotia formation, and virulence. These data provide new insight into the multiple functions of the Gβ protein in filamentous fungi. Further studies are necessary to reveal the molecular mechanism of Gβ in regulating the cAMP signaling pathway and MAPK signaling pathway.  Table S1: Primers used in this study.