Role of Two G-Protein α Subunits in Vegetative Growth, Cell Wall Integrity, and Virulence of the Entomopathogenic Fungus Metarhizium robertsii

Heterotrimeric G-proteins are crucial for fungal growth and differentiation. The α subunits of heterotrimeric G-proteins play an essential role in controlling signal transduction. However, the function of G-protein α subunits in entomopathogenic fungi remains poorly understood. Two group II Gα subunits (MrGPA2 and MrGPA4) were characterized in the entomopathogenic fungus, Metarhizium robertsii. Bioinformatics analysis showed that the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Both green fluorescent protein (GFP)-tagged MrGPA2 and MrGPA4 were localized at the cytoplasm. Furthermore, ∆MrGpa2∆MrGpa4 double mutants showed remarkably reduced vegetative growth compared to the wild-type and single-mutant strains, which was accompanied by the downregulation of several growth-related genes, such as ssk2, pbs2, stuA, hog1, and ac. Only the ∆MrGpa2∆MrGpa4 double mutant was sensitive to Congo red stress. The insect bioassay demonstrated significantly attenuated virulence for the ∆MrGpa2∆MrGpa4 double mutant compared to the wild-type and single-mutant strains. Further analysis indicated that double deletion of MrGpa2 and MrGpa4 had no effect on appressorium formation but suppressed the expression levels of several virulence-related genes in the insect hemocoel. These findings demonstrate that MrGpa2 and MrGpa4 exhibit functional redundancy and contribute to the vegetative growth, stress tolerance, and pest control potential in M. robertsii.


Introduction
Insect-pathogenic fungi, as a biological control agent that can cause the death of many insects, represent an environmentally friendly alternative to existing insecticides [1]. However, their use has been limited because of their slow killing speed, low sporulation rate, and sensitivity to high temperatures and UV stress. To kill pests, fungal spores first adhere to the pest cuticle. Then, the conidium germinates and penetrates the pest cuticle via mechanical pressure and cuticle-degrading enzymes. At that time, fungal pathogens must dodge acute immune responses to colonize the pest hemocoel. Finally, hyphae multiply within and emerge from the dead pest, and the newly created conidia disperse and infect additional insects if the conditions are favorable [2,3]. An understanding of the molecular mechanisms of its infection, disease development, and stress tolerance is necessary for the commercial application of fungal pathogens.
Heterotrimeric G-proteins, which are critical signaling proteins involved in receptormediated signal transduction, are present in all eukaryotic cells [4]. The G-proteins consisting of α, β, and γ subunits are coupled to the G-protein-coupled receptor (GPCR) for transferring environmental signals [5]. G-proteins are crucial for the regulation of cell function, division, and differentiation. In filamentous fungi, they contribute to hyphal growth, conidiation, conidial germination, stress resistance, and virulence [6][7][8][9].
G-protein α subunits can be classified into four groups (Gα i , Gα s , Gα q , and Gα 12 ) in mammalian species, and only two kinds of Gα subunits (canonical Gα and Extra-large Gα s ) exist in plants [4]. In yeast, two Gα subunits (GPA1 and GPA2) have been identified [10]. In particular, Gα subunits of the filamentous fungi are classified into three major groups: group I, group II, and group III [11]. The groups I and III Gα proteins contain a consensus myristylation site (MGXXXS) in the N terminus and a pertussis toxin-labeling site (CXXX) at the C termini of group I Gα proteins, but group II Gα proteins do not contain either site [11]. Based on their different structures, the functions of group II Gα proteins are diverse. The role of group II Gα proteins has been characterized in some fungi. For instance, the deletion of GNA-2 in Neurospora crassa intensified a reduction in mass accumulation on poor carbon sources in ∆gna-1 or ∆gna-3 strains [12]. In Magnaporthe grisea, disruption of magC resulted in a reduction in conidia [8]. ∆bcg-2 of Botrytis cinerea was only slightly reduced in pathogenicity [13]. Disruption of fga3 in Fusarium oxysporum f. sp. cucumerinum led to higher thermal resistance, possibly through the cAMP-dependent protein kinase A pathway [14]. However, the functions of the group II Gα proteins in entomopathogenic fungi are still unknown.

Fungal Strains and Culture
The wild-type (WT) strain M. robertsii ARSEF 23 (ATCC No.MYA-3075) was isolated from Conoderus sp. in North Carolina, USA, in 1961. The fungus and all of the mutant strains were maintained on potato dextrose agar (PDA, 20% potato, 2% glucose, and 2% agar, w/v) at 25 • C under dark conditions for 10 days [15]. After 10 days of incubation, conidia were harvested by vortex-mixing in 0.05% (v/v) Tween-80, filtrated through sterile non-woven fabric, and washed with sterile water.

Sequence Analysis
To determine the phylogenetic relationship among MrGPA2, MrGPA4, and their orthologues, the amino acid sequences of those functionally characterized GPA subunits of different fungi were downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, accessed on 20 September 2021). A neighbor-joining tree was constructed using the program MEGAX (https://www.megasoftware.net/, accessed on 20 September 2021).

Subcellular Localization of MrGPA2 and MrGPA4
To investigate the subcellular localization of MrGPA2 and MrGPA4, two genes were fused with a GFP tag and inserted into the plasmid pDHt-SK-bar (kindly provided by Dr. Chengshu Wang; the vector conferred resistance against glufosinateammonium) containing a strong promoter and a terminator, which were transformed into M. robertsii and screened as previously described [16]. The subcellular localization of MrGPA2-GFP and MrGPA4-GFP fusion proteins in conidia and mycelia were visualized using confocal microscopy (LSCM, Zeiss LSM980, Zeiss Gruppe, Oberkochen, Baden-Wurttemberg, German).

Gene Deletion and Complementation
Targeted gene disruption of MrGpa2 and MrGpa4 was performed by homologous recombination via Agrobacterium tumefaciens transformation as previously described [9]. In brief, the 5 and 3 flanking regions of MrGpa2 and MrGpa4 were inserted into the pDHt-SK-bar, and then the vectors pDHt-MrGpa2-bar and pDHt-MrGpa4-bar were obtained for fungal transformation. For double-gene deletion, the 5 and 3 flanking regions of MrGpa4 were inserted into the pDHt-SK-ben vector (containing benomyl resistance gene), and the product was transformed into ∆MrGpa2 strain and screened as previously mentioned.
For mutant complementation, the entire MrGpa2 and MrGpa4 genes were amplified together with their upstream and downstream regions and then inserted into the vector pDHt-SK-ben to produce the vectors pDHt-cpMrGpa2-ben and pDHt-cpMrGpa4-ben for fungal transformation. Next, all of transformants were verified by DNA sequencing. All the primers used in this study are listed in Supplementary Materials, Table S1.

Phenotype Assays
To assess conidiation capacity, 30 µL of conidial suspension (1 × 10 6 conidia/mL) of WT, ∆MrGpa2, ∆MrGpa4, ∆MrGpa2∆MrGpa4, cp∆MrGpa2, and cp∆MrGpa4 strains was evenly spread on PDA medium and incubated for 14 days at 25 • C. These samples were then placed in 30 mL of 0.05% Tween 80 to release conidia by vortex-mixing, and these conidial suspensions were used for assessment of conidial concentration with a hemocytometer, followed by conversion of the number of conidia per square centimeter of the colony.
To analyze the chemical tolerance, conidial suspensions (1 µL; 1 × 10 7 conidia/mL) of strains were cultured on PDA plates supplemented with NaCl (0.5 M), H 2 O 2 (2 mM), or Congo red (2 mg/mL) in the dark at 25 • C for 10 days. The colony diameter was measured, and the relative inhibition rate was calculated.
To assay the heat tolerance, experiments were conducted on PDA plates. Conidial suspensions (5 × 10 6 conidia/mL) of strains were incubated in a water bath at 42 • C or 28 • C (as control) for 1 h, and then 10 µL of the suspension was incubated at 25 • C in the presence of the medium. Germination was observed with Olympus BX 51(Tokyo, Japan)) at 12, 16, 20, and 24 h. Conidia were considered germinated when the length of the germ tube projecting from it reached the half length of the conidia. A total of 300 hundred conidia were counted per plate, and the relative germination rates were calculated to allow comparisons of the number of germinated conidia with and without heat stress.
To evaluate fungal tolerance to ultraviolet B (UV-B) light, heat tolerance assays were conducted as previously mentioned. These plates with conidia were exposed to an irradiance of 312 nm wavelength at 100 µJ cm −2 using HL-2000 Hybrilinker (UVP, Upland, CA, USA). Relative percent germination was assessed using the aforementioned methods.
For virulence assays, the tests were performed using Galleria mellonella larvae as previously described [9]. Briefly, conidial suspensions (1 × 10 7 conidia/mL) harvested from a 10-day-old PDA culture were collected, and the larvae were separately immersed in each conidial suspension for 90 s, followed by incubation at 25 • C. All treated cohorts were performed in triplicate, with 18 larvae in each group, and monitored every 12 h to record survival, using the SPSS software to calculate the median lethal time (LT 50 ) by probit analysis.
For appressorium formation assay, 1 mL of conidial suspension (1 × 10 6 conidia/mL) in MMGly (minimal medium amended with 1% glycerol) was spread on a sterile plastic Petri dish (3.5 cm diameter), followed by 24 h incubation at 25 • C for 24 h. The appressorium formation rates were assessed as described previously [17].

Quantitative RT-PCR (RT-qPCR)
To analyze the transcriptional profiling of phenotype-related genes, WT, ∆MrGpa2∆MrGpa4, controls, and two single-mutant strains were cultured on PDA plates for 10 days, or G. mellonella larvae were infected by six strains for 48 h. Then, the mycelia or larval hemocoel were harvested and milled in liquid nitrogen for RNA extraction. Total RNA was isolated using the TRIzol reagent (Invitrogen, Foster City, CA, USA). cDNA was used as a template for RT-qPCR using a HiScript III 1st strand cDNA synthesis kit (Vazyme, Nanjing, China). Three biological replicates were analyzed for each treatment. qRT-PCR analysis was performed using the CFBR96TM Real-Time PCR System (Bio-Rad, Hercules, CA, USA) and AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The primers used are listed in Supplementary Material, Table S2. The gpd gene (MAA_07675, encoding glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control, and relative gene expression was determined by normalizing the expression of each gene to GAPDH. The data were analyzed using the 2 −∆∆Ct method [18].

Statistical Analysis
Each experiment was performed in triplicate. All data were analyzed using GraphPad Prism version 7.0 and SPSS v23.0 software (SPSS Inc., Chicago, IL, USA). Kolmogorov-Smirnov test and Levene's test were used to confirm that the data followed normality and homoscedasticity, respectively. Then, data (mean ± SE) from different experimental groups were analyzed using Student's t-test, one-or two-way analysis of variance (ANOVA) followed by a least significant difference (LSD) test. p < 0.05 and p < 0.01 were considered significant and extremely significant, respectively. * p < 0.05, ** p < 0.01.
The Gα sequences from the filamentous fungi were selected for phylogenetic analysis using Saccharomyces cerevisiae as an outgroup and were divided into three main clades including the group I (MrGPA1 M. robertsii), group II (MrGPA2 and MrGPA4 M. robertsii), and group III (MrGPA3 M. robertsii) clans ( Figure 1B). Moreover, these MrGPA2 and MrGPA4 homologs were grouped together, and the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Intriguingly, phylogenetic analysis indicated that M. robertsii MrGPA4 homologs were only found in members of the genus Metarhizium and its sister genus Pochonia, whereas M. robertsii MrGPA2 homologs existed in filamentous fungi. Based on the relationship between the two GPAs and their distribution in filamentous fungi, MrGPA4 is likely an additional copy of MrGPA2.
MrGPA4 was closer than that of other MrGPAs. Intriguingly, phylogenetic analysis indicated that M. robertsii MrGPA4 homologs were only found in members of the genus Metarhizium and its sister genus Pochonia, whereas M. robertsii MrGPA2 homologs existed in filamentous fungi. Based on the relationship between the two GPAs and their distribution in filamentous fungi, MrGPA4 is likely an additional copy of MrGPA2.

MrGPA2 and MrGPA4 Are Located in Cytoplasm
To assess the subcellular localization of MrGPA2 and MrGPA4, we generated MrGpa2gfp and MrGpa4-gfp strains for fluorescence observation (Supplementary Figure S1A-C). As shown in the LSCM images in Figure 2, the green fluorescence signals of MrGPA2 and MrGPA4 or 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei were detected, but they were only observed in the cytoplasm, either in hyphae or in conidia, which both exhibited the cytoplasmic localization of MrGPA2 and MrGPA4.

MrGPA2 and MrGPA4 Are Located in Cytoplasm
To assess the subcellular localization of MrGPA2 and MrGPA4, we generated MrGpa2-gfp and MrGpa4-gfp strains for fluorescence observation (Supplementary Figure S1A-C). As shown in the LSCM images in Figure 2, the green fluorescence signals of MrGPA2 and MrGPA4 or 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei were detected, but they were only observed in the cytoplasm, either in hyphae or in conidia, which both exhibited the cytoplasmic localization of MrGPA2 and MrGPA4.

Gene Knockout and Complementation
To further investigate the function of MrGpa2 and MrGpa4 in M. robertsii, ∆MrGpa4, ∆MrGpa4, double-knockout mutants ∆MrGpa2∆MrGpa4 were generated and detected by

Gene Knockout and Complementation
To further investigate the function of MrGpa2 and MrGpa4 in M. robertsii, ∆MrGpa4, ∆MrGpa4, double-knockout mutants ∆MrGpa2∆MrGpa4 were generated and detected by PCR amplification. (Supplementary Figure S1A,B). PCR analysis revealed that the one 286-bp fragment corresponding to the partial MrGpa2 gene sequence was not detected in the ∆MrGpa2 and ∆MrGpa2∆MrGpa4 strains, and the one 212-bp fragment corresponding to the partial MrGpa4 gene sequence was not found in the ∆MrGpa4 or ∆MrGpa2∆MrGpa4 strains. In addition, a partial 434-bp bar gene fragment was present in all transformants, and a partial 1993-bp ben gene fragment was detected in ∆MrGpa2∆MrGpa4, cp∆MrGpa2, and cp∆MrGpa4. These observations indicated the presence of deletion mutants (∆MrGpa4, ∆MrGpa2, and ∆MrGpa2∆MrGpa4) and complementation strains (cp∆MrGpa2 and cp∆MrGpa4).

∆MrGpa2∆MrGpa4 Strain Leads to a Decrease in Vegetative Growth but Had No Effect on Conidiation
We assessed the effect of MrGPAs' disruption on vegetative growth. The single mutants of MrGpa2 or MrGpa4 had no influence in vegetative growth, but the double mutants affected vegetative growth. The growth rate of the ∆MrGpa2∆MrGpa4 mutant was significantly reduced by 26% (F 5, 23 = 41.2, p < 0.01), 16% (F 5, 23 = 15.5, p < 0.01), and 18% (F 5, 23 = 36.9, p < 0.01) in PDA, SDAY, and 1/4 SDAY plates, respectively, compared to the WT strain ( Figure 3A,B). Figure S1A,B). PCR analysis revealed that the one 286-bp fragment corresponding to the partial MrGpa2 gene sequence was not detected in the ∆MrGpa2 and ∆MrGpa2∆MrGpa4 strains, and the one 212-bp fragment corresponding to the partial MrGpa4 gene sequence was not found in the ∆MrGpa4 or ∆MrGpa2∆MrGpa4 strains. In addition, a partial 434-bp bar gene fragment was present in all transformants, and a partial 1993-bp ben gene fragment was detected in ∆MrGpa2∆MrGpa4, cp∆MrGpa2, and cp∆MrGpa4. These observations indicated the presence of deletion mutants (∆MrGpa4, ∆MrGpa2, and ∆MrGpa2∆MrGpa4) and complementation strains (cp∆MrGpa2 and cp∆MrGpa4).
In addition, the conidial yield was also assessed for the three mutants, which con firmed that the ∆MrGpa2, ∆MrGpa4, and ∆MrGpa2∆MrGpa4 strains were not required fo conidial yield ( Figure 5).

Deletion of Both MrGpa2 and MrGpa4 Enhanced Cell Wall Integrity
To examine whether the ∆MrGpa2, ∆MrGpa4, and ∆MrGpa2∆MrGpa4 strains showed an altered response to chemical stress, mycelial growth was observed on a PDA medium containing NaCl, H2O2, or Congo red ( Figure 6A,B). On PDA plates containing Congo red the ∆MrGpa2∆MrGpa4 strain showed altered mycelial diameter and a 78.3% reduction i the inhibition ratio compared to that of the WT strain (F5, 21 = 33.9, p < 0.01), which indicate the enhanced cell wall integrity of the double mutant. However, no effects on osmotic o antioxidant stress were observed in the ∆MrGpa2, ∆MrGpa4, or ∆MrGpa2∆MrGpa4 strains Furthermore, to confirm the role of MrGpa2 and MrGpa4 in heat and UV-B tolerance conidial germination rates were calculated at 12, 16, 20, and 24 h after exposure to heat o UV-B irradiation stress ( Figure 6C). Compared with the WT and CP strains, the conidia germination rates in the three mutants were not significantly different.

Deletion of Both MrGpa2 and MrGpa4 Enhanced Cell Wall Integrity
To examine whether the ∆MrGpa2, ∆MrGpa4, and ∆MrGpa2∆MrGpa4 strains showed an altered response to chemical stress, mycelial growth was observed on a PDA medium containing NaCl, H 2 O 2 , or Congo red ( Figure 6A,B). On PDA plates containing Congo red, the ∆MrGpa2∆MrGpa4 strain showed altered mycelial diameter and a 78.3% reduction in the inhibition ratio compared to that of the WT strain (F 5, 21 = 33.9, p < 0.01), which indicates the enhanced cell wall integrity of the double mutant. However, no effects on osmotic or antioxidant stress were observed in the ∆MrGpa2, ∆MrGpa4, or ∆MrGpa2∆MrGpa4 strains.
Furthermore, to confirm the role of MrGpa2 and MrGpa4 in heat and UV-B tolerance, conidial germination rates were calculated at 12, 16, 20, and 24 h after exposure to heat or UV-B irradiation stress ( Figure 6C). Compared with the WT and CP strains, the conidial germination rates in the three mutants were not significantly different.
Furthermore, the appressorium formation rates were not remarkably different among the WT, ∆MrGpa2∆MrGpa4, control, or the two single-mutant strains, suggesting that MrGpa2 and MrGpa4 have no effect on fungal appressorium formation, which is required for cuticle penetration ( Figure 7C). Furthermore, the appressorium formation rates were not remarkably different among the WT, ∆MrGpa2∆MrGpa4, control, or the two single-mutant strains, suggesting that MrGpa2 and MrGpa4 have no effect on fungal appressorium formation, which is required for cuticle penetration ( Figure 7C).

Discussion
As previously reported, G-proteins regulate a variety of fungal physiological processes, including conidiation, vegetative growth, stress tolerance, appressoria formation, and virulence [5,9,25]. In this study, we identified two group II Gα proteins, MrGPA2 and MrGPA4, in M. robertsii, both of which are involved in the vegetative growth, cell wall integrity, and virulence of M. robertsii, although the two single mutants did not display any defect in phenotypic characteristics.
Fungal Gα-proteins are classified into three major groups: group I, group II, and group III. In contrast to yeast, which contains two Gα proteins, most characterized filamentous fungi possess three Gα proteins that are members of distinct groups, including Magnaporthe grisea, Neurospora crassa, and Fusarium oxysporum f. sp. cucumerinum [4,7,8,12,14]. However, four Gα proteins have been identified in the genus Metarhizium and its sister genus Pochonia. Further analysis showed that group II consisted of MrGPA4 and MrGPA2. In this study, we found that MrGPA2 and MrGPA4 were functionally redundant in M. robertsii.
To transmit extracellular signals to intracellular effectors, G-proteins are localized to the plasma membrane [26]. However, in recent years, G-proteins have been identified in

Discussion
As previously reported, G-proteins regulate a variety of fungal physiological processes, including conidiation, vegetative growth, stress tolerance, appressoria formation, and virulence [5,9,25]. In this study, we identified two group II Gα proteins, MrGPA2 and MrGPA4, in M. robertsii, both of which are involved in the vegetative growth, cell wall integrity, and virulence of M. robertsii, although the two single mutants did not display any defect in phenotypic characteristics.
Fungal Gα-proteins are classified into three major groups: group I, group II, and group III. In contrast to yeast, which contains two Gα proteins, most characterized filamentous fungi possess three Gα proteins that are members of distinct groups, including Magnaporthe grisea, Neurospora crassa, and Fusarium oxysporum f. sp. cucumerinum [4,7,8,12,14]. However, four Gα proteins have been identified in the genus Metarhizium and its sister genus Pochonia. Further analysis showed that group II consisted of MrGPA4 and MrGPA2. In this study, we found that MrGPA2 and MrGPA4 were functionally redundant in M. robertsii.
To transmit extracellular signals to intracellular effectors, G-proteins are localized to the plasma membrane [26]. However, in recent years, G-proteins have been identified in many other intracellular organelles, such as the mitochondria, Golgi apparatus, endoplasmic reticulum, and nucleus [27]. In our previous Metarhizium GPA study, MrGPA1 and MrGPA3 were found to be localized in the mitochondria and vacuoles, respectively [9]. However, in this study, both MrGPA2 and MrGPA4 were found to be localized in the cytoplasm, unlike the S. cerevisiae GPA2 embedded in the plasma membrane [10]. We speculate that the function of MrGPA2 and MrGPA4 differs from that of other fungal GPAs.
In this study, the ∆MrGpa2∆MrGpa4 double mutant showed inhibited growth in PDA, SDAY, 1/4 SDAY, and CZAs plates. Further growth analyses indicated that the doubledeletion mutant was involved in carbon and nitrogen metabolism. In other filamentous fungi, the role of the group II Gα subunits is not related to growth, such as N. crassa, M. grisea, B. cinerea, and F. oxysporum f. sp. cucumerinum [11,13,14]. Thus, the relative importance of group II Gα subunits for colony growth may vary among fungal groups.
The LT 50 values of the ∆MrGpa2∆MrGpa4 strain after topical application and injection were higher than those of the WT strain. However, appressorium formation in the ∆MrGpa2∆MrGpa4 double mutant was unaffected. In addition, the double-deletion mutant was found to be involved in carbon and nitrogen metabolism. Thus, the double deletion of MrGpa2 and MrGpa4 led to a statistically significant increase in the survival rate of G. mellonella larvae, which was probably due to the slower growth of the ∆MrGpa2∆MrGpa4 strain in G. mellonella hemocoel. This hypothesis was subsequently confirmed by the decrease in the expression of atm1 in the ∆MrGpa2∆MrGpa4 strain. In addition, the decrease in virulence of the ∆MrGpa2∆MrGpa4 strain might be partly due to defects in colonization, owing to the decrease in the expression of mcl1 (evading insect immune responses) [20,24]. Similarly, the group II Gα subunit of ∆bcg-2 in B. cinerea is slightly reduced in pathogenicity, and BCG2 is also necessary for a normal colonization rate [13].
Gα-proteins are extremely important in heterotrimeric G-protein signaling and regulate the growth and differentiation of pathogenic fungi. In Magnaporthe grisea, Gα subunits MAGA and MAGB are directly modulated by RGS1, which physically interacts with GPCR PTH11 in vivo to activate MAPK cascades in appressorium formation [28]. The loss of G-protein-coupled receptor K (GPRK) causes defects in radial growth in M. robertsii [29]. In M. robertsii, the deletion of GPCR MrGpr8 significantly downregulates two Gα subunit genes during appressorium formation [30]. In this study, we found that the deletion of two Gα subunits (MrGpa2 and MrGpa4) resulted in the defect of fungal growth rather than appressorium formation. We also found that relative to the WT, the MAPK components ssk2, pbs2, and hog1 were significantly downregulated in ∆MrGpa2∆MrGpa4. Thus, Gα subunits act as downstream GPCRs and upstream components of MAPK during appressorium differentiation and fungal growth.
Overall, this study revealed that both MrGPA2 and MrGPA4, belonging to group II Gα, are functionally redundant and required for vegetative growth, cell wall integrity, and virulence.
These findings broaden our understanding of the functions of G-proteins in entomopathogenic fungi and provide new insights into the genetic improvement of M. robertsii for pest control.