BcMtg2 is required for multiple stress tolerance, vegetative development and virulence in Botrytis cinerea

In Saccharomyces cerevisiae, the Mtg2 gene encodes the Obg protein, which has an important function in assembling ribosomal subunits. However, little is known about the role of the Obg GTPase in filamentous fungi. In this study, we identified an Mtg2 ortholog, BcMtg2, in B. cinerea. The BcMtg2 deletion mutant showed a defect in spore production, conidial germination and sclerotial formation. Additionally, the mutant increased sensitivity to various environmental stresses. The BcMtg2 mutant exhibited dramatically decreased virulence on host plant tissues. BcMtg2 mutant showed increased sensitivity to osmotic and oxidative stresses, and to Congo red (cell wall stress agent). In the yeast complement assay, growth defects of yeast BY4741ΔMTG2 mutant were partly restored by genetic complementation of BcMtg2 under these environmental stresses. Additionally, compared with the parental strain and complement strain, the BcMtg2 deletion mutant displayed a minor glycerol response to osmosis stress. These defective phenotypes were recovered in the complement strain ΔBcMtg2C, which was created by adding the wild-type BcMtg2 gene to the ΔBcMtg2 mutant. The results of this study indicate that BcMtg2 has a necessary role in asexual development, environmental stress response and pathogenicity in B. cinerea.

in ribosomal assembly via binding the majority ribosomal subunit 19 . Thus, these studies have indicated that various Obg GTP proteins perform different functions in specific organisms. In our study, we analyzed the roles of the Mtg2 homologous gene BcMtg2 in Botrytis cinerea, this knowledge provides information for designing novel fungicides to control gray mold caused by B. cinerea and exploring the function of BcMtg2 in B. cinerea.
B. cinerea causes gray mold which is a widespread and serious plant disease throughout the world 20 . Most plant organs, including leaves, shoots, bulb, flowers, and root tubers, are sensitive to B. cinerea infection. In varied environmental conditions, B. cinerea uses various patterns to infect its multiple hosts. In addition, B. cinerea can survive as conidia, sclerotia and mycelia for a period of time 21 . Therefore, controlling gray mold presents many challenges. Understanding the biology and host-pathogen interactions of B. cinerea is valuable for the discovery of an efficient strategy to control gray mold. A whole-genome search uncovered that B. cinerea an orthologue of Mtg2 (hereafter named BcMgt2). Base on the previous research, we hypothesized that BcMtg2 might own important effect on vegetative differentiation and virulence in B. cinerea. To clarify this hypothesis, the major objective of this study was to analyse the genetic, biological and biochemical function of BcMtg2 using target gene deletion strategy. Our study revealed that BcMtg2 plays significant role in regulating the asexual reproduction, vegetative growth, various stresses response and virulence in B. cinerea.

Results
Identification of the BcMtg2 gene. Using the amino acid sequence of the S. cerevisiae Mtg2 protein, BcMtg2 was identified in B. cinerea using a BLASTP search in the Botrytis cinerea genome database (http://www. broad.mit.edu). The nucleotide sequence of BcMtg2 is 1653 bp in size. The protein encoded by BcMtg2 contains 550 amino acids. The predicted amino acid sequence indicated no introns in BcMtg2. An analysis of domains in the BcMtg2 protein by SMART showed that BcMtg2 includes two conserved domains: a Mg 2+ /GTP binding site in the N-terminus and a switch region in the C-terminus (Fig. S1).
BcMtg2 regulates vegetative growth and asexual reproduction in B. cinerea. On either complete medium (CM) or potato dextrose agar medium (PDA), the mycelial growth rate of the BcMtg2 deletion mutant (ΔBcMtg2) was similar to the parental strain B05.10 and the complement strain ΔBcMtg2C. However, on minimal medium (MM), ΔBcMtg2 grew more slowly than the wild-type strain B05.10 and the complement strain ΔBcMtg2C (Fig. 1A). In conidiation assay, ΔBcMtg2 produced significantly fewer conidia than B05.10 and ΔBcMtg2C on PDA plate cultivated for 10 days (Fig. 1B). These results showed that BcMtg2 was involved in conidiation in B. cinerea. The germination rate of conidia in ΔBcMtg2 was reduced by 56%, compared with B05.10 and ΔBcMtg2C incubated for 15 h on Water-Agar medium ( Fig. 2A). In addition, the germ tubes of the BcMtg2 deletion mutant were malformed (Fig. 2B). These results showed that BcMtg2 had an important role in mycelia growth and asexual reproduction in B. cinerea.

Sensitivity of B. cinerea response to various stresses is regulated by BcMtg2.
To explore the role of BcMtg2 in B. cinerea response to multiple stresses, we measured the sensitivity of the wild-type strain B05.10, the BcMtg2 deletion mutant, and the complemented strain ΔBcMtg2C to cell wall-damaging agents, osmotic stresses and oxidative pressure. Compared with the wild-type strain and the complemented strain, the sensitivity of ΔBcMtg2 to osmotic stress was significantly increased (Fig. 3A,B). Previous studies have indicated that glycerol content plays a significant role in the regulation of fungus response to osmotic stress 22,23 . Thus, we measured glycerol accumulation in the parental strain B05.10, the BcMtg2 deletion mutant and the complement strain ΔBcMtg2C. The result indicated that the parental strain B05.10, ΔBcMtg2 and ΔBcMtg2C generated little glycerol without osmotic stress. However, the glycerol content of the parental strain B05.10 and the complement strain ΔBcMtg2C were significantly increased compared with that of the BcMtg2 deletion mutant under osmotic stress conditions (Fig. 4A). Moreover, the previous study shown that the histidine kinase, BcBoS1 and the mitogen-activated protein kinase, BcSAK1 affect the sensitivity of B. cinerea responses to osmotic pressure 24 . The real-time PCR analysis showed that the transcription levels of the two genes were significantly decreased in ΔBcMtg2 compared with that in B05. 10 and ΔBcMtg2C under osmotic stress conditions (Fig. 4B). In addition, compared with B05.10 and ΔBcMtg2C, ΔBcMtg2 presented increased sensitivity to the cell-damaging agent (Congo red) and to oxidative pressure (Fig. 3A,B). Previous studies had shown that Mkk1 and Gls2 were core component genes of cell wall completion in S. cerevisiae 25 . To understand the function of BcMtg2 in cell wall completion in B. cinerea, the transcription levels of BcMkk1 and BcGls2 in each strain were analyzed. Compared with the parental strain B05.10 and the complement strain ΔBcMtg2C, the expression levels of the two genes were significantly decreased in ΔBcMtg2 in response to Congo red (Fig. 4B), indicating that BcMtg2 involved in various environmental stress response in B. cinerea.
BcMtg2 is involved in the sensitivity of B. cinerea response to cell wall lyase. The BcMtg2 deletion mutant exhibited increased sensitivity to a cell wall-damaging factor (Congo red). To further understand the role of BcMtg2 in cell wall integrity (CWI), we analyzed the sensitivity of ΔBcMtg2 to cell wall lyase. 0.1 g fresh mycelia of each strain was incubated with 15 mL 1.5% lyases buffer (0.6 M KCl, 50 mM CaCl 2 ) at 30 °C for 45 min. Then, the amount of protoplast was counted with a hemocytometer. The result showed that ΔBcMtg2 mycelia were well digested and released significantly more protoplasts than the parental strain B05.10 and the complement strain ΔBcMtg2C (Fig. 5A,B), indicating that the BcMtg2 plays an important role in regulating CWI of B. cinerea.

Effects of BcMtg2 deletion on sclerotia formation.
To investigate the function of BcMtg2 in sclerotia development in B. cinerea, strains were cultivated in the dark for 4 weeks. ΔBcMtg2 produced significantly fewer sclerotia than B05.10 and Δ BcMtg2C on CM medium. In addition, B05.10 and ΔBcMtg2C produced vast sclerotia, while the BcMtg2 deletion mutant did not produce any sclerotia on PDA medium (Fig. 5C,D). These results indicated that BcMtg2 had an important effect on sclerotial formation in B. cinerea. BcMtg2 is required for the virulence of B. cinerea. To investigate the involvement of BcMtg2 in virulence, the ability of the strain to infect various plant organisms was evaluated. As indicated in Fig. 6, ΔBcMtg2 caused significantly smaller disease spots than the parental strain B05.10 and the complement strain ΔBcMtg2C on wounded strawberry, grape, tomato and apple fruits, after 72 h of inoculation. Moreover, on wounded tomato leaves and strawberry leaves ΔBcMtg2 also caused obviously smaller disease spots than B05.10 and ΔBcMtg2C after 60 h of inoculation ( Fig. 7A-C). These results indicated that BcMtg2 owned significant effect on the virulence in B. cinerea.

Complementation of yeast Mtg2 deletion mutants with BcMtg2.
To further investigate the functions of BcMtg2, we determined whether BcMtg2 could complement the phenotype of the yeast Mtg2 mutant. Base on the BcMtg2 deletion mutant shown increased sensitivity to osmotic pressure (NaCl), cell wall inhibitor (Congo red) and oxidative stress (H 2 O 2 ), we used the same environment stresses in to analyze the strains growth phenotype in the yeast complement assay. The parental strain and yeast Mtg2 mutant grew well on YEPG medium. However, compared with the parental strain and the complement strain, the Mtg2 mutant was obviously inhibited when the culture medium was modified with 4 mM H 2 O 2 and 0.4 M NaCl. Additionally, the Mtg2 mutant and the complement strain were significantly hindered when the medium was modified with 130 mg ml −1   Congo red. These results indicated that the growth defects of Mtg2 were partially recovered by genetic complementation of the yeast Mtg2 mutant with BcMtg2 (Fig. 7D).

Discussion
Previous studies showed that the nuclear gene Mtg2 played an important role in the assembly of mitochondrial ribosomes in S. cerevisiae 12,18 . The protein is member of the Obg GTPase family 19 . The assembly of cytoplasmic ribosomes requires the coordinated action of assembly proteins 26 . These assembly factors include nucleases, ATP-dependent RNA helicases, modifying enzymes, and a number of recently identified GTPases [26][27][28][29] . To investigate the function of BcMtg2 in B. cinerea, we analyzed the phenotype of the BcMtg2 deletion mutant. The previous study showed that a deletion mutant of the Rho4 GTPase gene was defective in vegetative growth and conidiation in Neurospora crassa 30 . In our study, the BcMtg2 deletion mutant exhibited a clear growth defect on MM medium (Fig. 1A). Moreover, compared with the parental strain and the complement strain, ΔBcMtg2 produced significantly fewer conidia and exhibited a lower conidia germination rate. The previous study indicated that the Ras small GTPase was involved in sclerotia development in Sclerotinia sclerotiorum 31 . The previous study showed that sclerotial development plays a significant role in the survival mechanism of B. cinerea in dying host organisms 21 . In our study, we found that ΔBcMtg2 produced significantly fewer sclerotia than the parental and complemented strains. In addition, in our study, we found that the BcMtg2 deletion mutant exhibited decreased virulence in various plant tissue. A similar phenotype was also observed in a Rho3 GTPase deletion mutant in B. cinerea 32 . These consequences indicated that GTPases may have a similar function in the regulation of asexual reproduction, vegetative growth, survival mechanism and virulence in plant pathogenic fungi.
In our study, the BcMtg2 deletion mutant exhibited significantly decreased virulence on host plant tissue. To date, the exact causes of the decreased pathogenicity of the BcMtg2 deletion mutant remain unclear, we inferred that the decreased pathogenicity of ΔBcMtg2 might be closely related to its reduced tolerance to various environmental stresses. First, the deletion of BcMtg2 leads to increased sensitivity of B. cinerea to cell wall-disrupting factors. The previous studies had shown that cell wall integrity is required for B. cinerea virulence 33,34 . Second, the BcMtg2 deletion mutant appeared to have increased sensitivity to oxidative stresses, which can be generated by a host plant during the host-fungus interaction 35,36 . However, B. cinerea possesses an effective ROS detoxification mechanism to address an oxidative burst 37 . The BcMtg2 deletion mutant may defect in ROS eliminated ability which resulted in the decreased pathogenicity. Third, a previous study showed that the fungal infection process might be related to osmotic stress 38,39 . The increased sensitivity of ΔBcMtg2 to osmotic stress may lead to defective virulence of B. cinerea on host plant tissue. Osmotic, oxidative stresses generated by host plant and cell wall integrity of B. cinerea significantly effect on the interaction of B. cinerea with host plant tissue [33][34][35][36] . To analyze the sensitivity of B. cinerea to osmotic, oxidative stresses, and to Congo red which can enhance our understanding the reasons for the defect of BcMtg2 deletion mutant on virulence.
In our study, ΔBcMtg2 exhibited increased sensitivity to various environmental stresses compared with the parental and complemented strains. In the cell wall tolerance and osmotic pressure assays, we found that ΔBcMtg2 showed increased sensitivity to Congo red but not caffeine and increased sensitivity to Na but not K, respectively. This result may be caused by the fact that ΔBcMtg2 is selective between Congo red and caffeine and has the ability to adapt to different osmotic pressures generated by Na or K. This hypothesis needs to be explored in the future. Additionally, in yeast complement assay, we found the BcMtg2 was able to partially complement the phenotype defects of yeast Mtg2 mutant under the various environment stresses (NaCl, Congo red and H 2 O 2 ). We deduced that the sequence difference between BcMtg2 and ScMtg2 leads to the biological function variation (Fig. S1). This assumption needs to be explore in the future research.
In conclusion, these results have indicated that the BcMtg2 protein possesses an important role in pathogenicity, vegetative growth, asexual reproduction, and tolerance to multiple environmental stresses. Additionally, the BcMtg2 deletion mutant may influence the function of mitochondrial ribosomes to cause the different phenotypes we observed in our study. This assumption is being investigated in our lab. Based on the conclusions in our study, the Mtg2 protein played an important role in growth and development in B. cinerea. This protein was hopeful as a new potential antifungal drug target for the development of novel fungicide against gray mold caused by B. cinerea.

Methods
Strains, media and growth conditions. Botrytis cinerea strain B05.10 was isolated from a grape in California, USA used as the wild-type progenitor for the construction of the gene deletion mutant in this study 32 . The wild-type strain B05.10 and mutant strains were grown on PDA, MM or CM 37 for the mycelial growth assays. For stress tests, strains were cultured on PDA modified with various supplements. Each plate was inoculated with a 5-mm diameter mycelial plug taken from the margin of a 3-day-old colony grown on PDA. In the yeast complement assay, all strains were grown on YEPG (1% yeast extract, 2% peptone, 1% galactose, 1% raffinose and 2% agar) 37 . The experiments were repeated three times independently. BcMtg2 in B. cinerea. Based on the B. cinerea genome sequence database (http://www. broadinstitute.org/), BcMtg2 (BC1G_14222) was initially identified using the BLASTP algorithm and the Mtg2 protein from S. cerevisiae as the query. To determine the size and existence of introns, RNA was extracted from the hypha of the parental strain B05.10 using the RNAsimple Total RNA Kit (Tiangen Biotech. Co., Beijing, China) and used for first-strand cDNA synthesis with the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). PCR amplification of the cDNA was performed with the P1/P2 primer pair (Table S1). The resultant PCR product was purified, cloned and sequenced.

Identification of
Generation of the BcMtg2 deletion mutant. The BcMtg2 deletion vector was constructed as described previously 40 . First, fragments 1.4 kb upstream and 1.3 kb downstream of BcMtg2 were amplified from the genomic DNA of the parental strain B05.10 using the primer pairs P3/P4 and P5/P6 (Table S1, Fig. S2A), respectively. The primer pair P7/P8 was used to amplify a 1.7-kb HPH cassette containing the hygromycin resistance gene and the Aspergillus nidulans trpC promoter 41 . Then, the three amplicons (upstream, HPH cassette and downstream) were fused using the primer pair P9/P10 42 (Table S1, Fig. S2A). The resultant PCR product was gel purified with the AxyPreTP DNA Gel Extraction Kit (Axygen, USA) and used to transform protoplasts of the parental strain B05.10. Protoplasts were prepared, and transformation was performed as described previously 43 .

Complementation of the BcMtg2 deletion mutant.
To confirm that the defects of the BcMtg2 deletion mutant were caused by the gene deletion, the BcMtg2 deletion mutant was complemented with the entire BcMtg2 gene. The complemented BcMtg2 gene was amplified from the genome of the parental strain B05.10 using the P20/P21 primer pair, which included the 325-bp promoter sequence and 543-bp terminator region (Table S1). The resultant PCR product was cloned into the SmaI-XbaI site of pNEO to generate the complement vector, pNEO-BcMtg2-C. BcMtg2 in this vector was sequenced to verify that no errors were present in this sequence before the complement was transformed into the BcMtg2 deletion mutant. The transformation of ΔBcMtg2 with the complement vector was performed as previously described 40 . Southern blot assay. Genomic DNA was extracted from B. cinerea using the previously described method 44 and digested with XhoI. Southern blot analysis was performed with the 241-bp downstream fragment of BcMtg2 as a probe. The fragment was amplified from the B05.10 genome using the primer pair P23/P24 (Table S1, Fig. S2A). The probe was labeled with digoxigenin (DIG) using the High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics).

Conidiation and sclerotial development analysis.
To evaluate conidiation, mycelial plugs (5 mm in diameter) obtained from the margin of a 3-day-old colony were cultivated on PDA medium and grown at 25 °C for 8 days. Spores from each plate were acquired using sterile H 2 O. The suspension was filtered using three layers of gauze, and the number of spores was calculated by a hemocytometer. To assay sclerotial production, 5-mm diameter mycelial plugs taken from the edge of a 3-day-old colony of each strain were inoculated onto PDA or CM and cultivated at 25 °C for 4 weeks in the dark. Then, the number of sclerotia was measured using a previously described method 45 . The experiment was repeated three times.
Assays for glycerol accumulation. To determine intracellular glycerol content, each strain was cultivated in YEPD medium (w/v, 1% peptone, 0.3% yeast extract, 2% glucose) for 2 days at 175 rpm at 25 °C in a shaker. After supplementing with 1.0 M NaCl and further incubation for 2 h, the mycelia of each strain were collected and ground in liquid nitrogen. Then, the mycelial powder (150 mg) was transferred to a 2-ml microcentrifuge tube containing 1 ml of glycerol extraction buffer (Applygen Technologies Inc., Beijing, China). After vortexing sufficiently for 1 min, the tubes were centrifuged at 6000× g for 10 min. Then, 10 μ L of the resulting supernatant of each sample was transferred to a new tube, and a 5-μ L aliquot of each supernatant was mixed with 190 μ L of glycerol determination buffer (Applygen Technologies Inc., Beijing, China). The coloration process was cultivated at 37 °C for 15 min. Then, the glycerol content was measured with a microplate reader (Molecular Devices Inc.) at 550 nm 37 . The experiment was independently repeated three times.

Quantitative real-time PCR.
To extract total RNA, the mycelial plugs of the wild-type strain B05.10, the BcMtg2 deletion mutant or the complemented strain BcMtg2C were transferred to YEPD medium and cultured for 2 days at 25 °C in dark. Before the mycelia were harvested for RNA extraction, the culture was treated for 2 h with NaCl or Congo red at the concentrations indicated in the legend of Fig. 4. Total RNA was extracted from the mycelia of each strain using the RNeasy Kit (Tiangen Biotech. Co). Then, total RNA of each strain was purified Scientific RepoRts | 6:28673 | DOI: 10.1038/srep28673 using DNase to remove the contaminated DNA with Reagent Kit (TaKaRa), and 10 μ g of each RNA sample was used for First Strand cDNA synthesis with the PrimeScript ® RT Reagent Kit (TaKaRa). Real-time PCR was conducted in an ABI 7500 real-time system (Applied Biosystems) using SYBR Green I fluorescent dye detection.
Amplification was conducted in a 20 μ L volume containing 10 μ L of iTaq ™ Universal SYBR ® Green Supermix (Bio-Rad Laboratories), 2 μ L of the reverse transcription product and 1 μ L each of the forward and reverse primers (Table S1). There were three replicates for each sample. The expression of the measured genes in each sample was normalized to the expression level of reference gene actin, and relative changes in gene expression levels were analysed with the ABI 7500 SDS software (Applied Biosystems), which automatically sets the baseline. Data from three biological replicates were used to calculate the mean and standard deviation 46 . The experiment was repeated three times.
Determination of the sensitivity of ΔBcMtg2 to environmental stress. Mycelial growth assays were performed on PDA plates modified with the following elements: Congo red, caffeine, KCl, NaCl, H 2 O 2, and paraquat at the concentrations shown in the figures. The percentage of inhibition of mycelial radial growth (PIMG) was calculated using the formula, PIMG = [(C − N)/(C − 5)] × 100, where C is the colony diameter of the untreated control and N is that of stress treatment 47 . The experiments were repeated twice.
Yeast complementation assays. The yeast wild type strain BY4741 and the MTG2 deletion mutant BY4741Δ MTG2 was provided by EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/). The full-length BcMtg2 cDNA was amplified from total RNA of B05.10 using the primer pair P35/P36 (Table S1). The PCR product was digested with BamHI and XbaI and cloned into the pYES2 vector (Invitrogen), and transformed into the yeast mutant BY4741Δ MTG2. Yeast transformants were selected on synthetic medium lacking uracil (Clontech). Additionally, the wild-type strain BY4741 and BY4741Δ MTG2 mutant transformed with empty pYES2 vector were used as controls. In the complementation assays, the yeast transformants were grown at 30 °C for 4 days on YPRG medium (1% yeast extract, 2% peptone, 1% galactose, 1% raffinose, 2% agar) modified with various stress agents including Congo red, NaCl, and H 2 O 2 at concentrations shown in figure legends. The experiments were repeated three times independently.
Plant tissue infection assays. Five-millimeter diameter mycelial plugs of 3-day-old colony grown on PDA were inoculated onto fully expanded leaves of tomato and strawberry plants, and grape, tomato, strawberry and apple fruits. Inoculated plant tissues were cultivated at 25 °C with 16 h of daylight 45 . The disease lesions were recorded after the indicated times in the figure legends. The experiment was repeated four times.