The autophagy protein BcAtg2 regulates growth, development and pathogenicity in the gray mold fungus Botrytis cinerea

Autophagy is an intracellular degradation process that facilitates material recycling to maintain cellular homeostasis in eukaryotes. Atg2 is a phospholipid transfer protein involved in cellular autophagy in Saccharomyces cerevisiae . To date, the role of Atg2 in growth, development and pathogenicity of the gray mold fungus Botrytis cinerea remains unknown. In this study, we identified and characterized an Atg2 ortholog, designated as BcAtg2, in B. cinerea . Deletion of BcATG2 resulted in a block of the autophagic process in B. cinerea . The Δ BcAtg2 mutant failed to produce sclerotia, and showed significant reduction in mycelial growth rate, formation of aerial mycelium and conidiation. In addi-tion, the Δ BcAtg2 mutant lost the ability to form infection structures and cause symptom on host plants. All of these phenotypic changes in Δ BcAtg2 mutant were restored by targeted gene complementation. Moreover, BcAtg2 was demonstrated to physically interact with the phosphoinositide binding protein BcAtg18. Taken together, these results indicate that BcAtg2 plays an important role in vegetative growth, development and pathogenicity in B. cinerea .


Background
Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a filamentous fungal pathogen that causes gray mold on a wide range of hosts (more than 500 plant species), including fruits, vegetables and ornamental flowers (Fillinger and Elad 2016). Gray mold occurs in both pre-and post-harvest period, leading to considerable crop losses worldwide (Williamson et al. 2007). Currently, due to the lack of resistant varieties, chemical control is the most effective strategy for the control of gray mold (Dean et al. 2012), however, the control efficiency of some fungicides has been severely weakened with the development of fungicide-resistant populations in B. cinerea (Hu et al. 2016). Understanding the fundamental biology of this pathogen will provide the basis for establishment of more effective control strategies.
Autophagy is an evolutionarily conserved intracellular degradation process in eukaryotes, which plays an important role in the maintenance of cellular homeostasis (Yorimitsu and Klionsky 2005). The process of autophagy includes sequestration of bulky cytosolic contents such as long-lived proteins and damaged organelles into vesicular compartments, followed by delivery of these intracellular materials to lysosomes/vacuoles for degradation (Mizushima 2007). A central event in autophagy is the biogenesis of double-membrane vesicles termed autophagosomes, which sequester intracellular components for degradation in lysosomes/vacuoles (Xie and Klionsky 2007). Previous studies in yeast and mammals have shown that the autophagy-related protein Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 to initiate autophagosome biogenesis in the preautophagosomal structure  (Chowdhury et al. 2018;Kotani et al. 2018). In filamentous fungi, Atg2 is essential for autophagy, and mediates fungal development and pathogenicity in Magnaporthe oryzae and Fusarium graminearum, two important plant pathogens causing destructive diseases on gramineous crops (Khan et al. 2012;Lv et al. 2017).

Open Access
Up to now, the role of Atg2 in B. cinerea, a model organism for molecular studies of necrotrophic fungi, has not been reported. In the present study, we identified an ortholog of yeast Atg2 in B. cinerea and investigated its biological functions. Our data demonstrated that BcAtg2 plays important roles in mycelial growth, conidiation, sclerotial formation and pathogenicity in B. cinerea.

Identification of BcATG2 in B. cinerea
The BcAtg2 protein-coding gene BcATG2 (Gene ID: BCIN_14g01550) was identified through BLASTP search of the B. cinerea genome database (https:// fungi. ensem bl. org/ Botry tis_ ciner ea/ Info/ Index) using the Saccharomyces cerevisiae Atg2 protein as a query. BcATG2 is predicted to encode a 2160-aa protein, which shares 27% identity with Atg2 of S. cerevisiae. Protein domain architectures were characterized by using the Simple Modular Architecture Research Tool (SMART) (http:// smart. embl-heide lberg. de/), and the results showed that BcAtg2 contains typical functional domains of Atg2, including chorein-N, ATG-CAD and ATG-C (Fig. 1a). Phylogenetic analysis of BcAtg2 and its orthologs from different fungal species revealed that BcAtg2 is evolutionarily conserved in fungi (Fig. 1b).
To investigate the role of BcATG2 in B. cinerea, the ΔBcAtg2 mutant was generated by targeted deletion of BcATG2 using a homologous recombination strategy (Fig. 2a). The putative BcATG2 deletion mutants were preliminarily screened from the hygromycin-resistant transformants by PCR amplification (Fig. 2b). Southern blot analysis confirmed that the recombination event of BcATG2 mutant occurred at the right locus (Fig. 2c).

BcAtg2 is essential for autophagy
GFP-BcAtg8 is a useful marker for monitoring autophagy in B. cinerea (Ren et al. 2018a). To determine the role of BcAtg2 in autophagy, the subcellular localization of GFP-BcAtg8 was analyzed in B. cinerea wild-type and BcATG2 deletion mutant strains. Under nitrogen-starvation conditions (nitrogen-deficient medium supplemented with phenylmethylsulfonyl fluoride, PMSF), the autophagic bodies were observed in the vacuole of the wild-type strain B05.10, whereas no autophagic bodies were observed in the vacuole of the BcATG2 deletion mutant ΔBcAtg2 (Fig. 3a). Additionally, the proteolysis of GFP-BcAtg8 was analyzed. Under nutrient-rich conditions, both the GFP-BcAtg8 fusion protein and free GFP protein were detected by anti-GFP western blotting in B05.10, and nitrogen starvation promoted the proteolysis of GFP-BcAtg8, however, the protein contents of GFP-BcAtg8 and GFP were not affected by nitrogen starvation in ΔBcAtg2 (Fig. 3b, c). These results indicate that BcAtg2 plays an important role in the regulation of autophagy in B. cinerea.

BcAtg2 is involved in growth, conidiation and sclerotial formation
To determine the role of BcAtg2 in growth and development of B. cinerea, we compared the characteristics of mycelial growth, conidiation and sclerotial production between the wild-type strain B05.10, BcATG2 deletion mutant ΔBcAtg2 and complemented strain BcAtg2-C. Compared with B05.10 and BcAtg2-C, ΔBcAtg2 showed significant reduction in aerial mycelium formation and mycelial growth rate after incubation on PDA, MM or CM medium at 25 °C for 3 days (Fig. 4a, b). After incubation on sterilized potato fragments at 20 °C under white light for 10 days, ΔBcAtg2 formed abnormal conidiophores (Fig. 5a), and its conidial morphology was also altered (approximately 25%) (Fig. 5b). In addition, the production of conidia in ΔBcAtg2 was drastically reduced compared with those in B05.10 and BcAtg2-C (Fig. 5c). As for sclerotial formation, B05.10 and BcAtg2-C produced abundant sclerotia, whereas ΔBcAtg2 failed to produce sclerotia after incubation on PDA at 10 °C in the dark for 3 weeks (Fig. 6). These results indicate that BcAtg2 is crucial for growth, conidiation and sclerotial formation in B. cinerea.

BcAtg2 is required for pathogenicity
To determine the role of BcAtg2 in the pathogenicity of B. cinerea, we conducted infection tests on cucumber leaves and apple fruits. At 3 days post-inoculation, the wild-type strain B05.10 and the complemented strain BcAtg2-C caused significant lesions in both cucumber leaves and apple fruits, whereas the ΔBcAtg2 mutant lost ability to infect both host tissues (Fig. 7a, b). Further investigation revealed that ΔBcAtg2 lost the ability to form infection structures at the initial stage of infection (Fig. 7c). These results indicate that BcAtg2 is essential for pathogenicity in B. cinerea.

BcAtg2 physically interacts with BcAtg18
In yeast, Atg2 forms a complex with Atg18, which is essential for autophagosome formation (Kobayashi et al. 2012). To test whether an interaction exists between their orthologs in B. cinerea, yeast two-hybrid (Y2H) assays were performed. The results demonstrated that an interaction exists between BcAtg2 and BcAtg18 (Fig. 8). These results suggest that the corresponding regulatory mechanism is evolutionarily conserved in B. cinerea.

Discussion
In the last decades, the study of autophagy has been extended from yeast to plants and mammals. Increasing evidence has demonstrated that autophagy plays a wide range of biological roles in eukaryotes by maintaining cellular homeostasis (Awan and Deng 2014; Parzych and Klionsky 2014). The role of autophagy in plant pathogenic fungi has been gradually revealed through investigation of gene functions (Pollack et al. 2009). In the present study, we identified and characterized the Atg2 ortholog in the gray mold fungus B. cinerea. The results indicated that BcAtg2 is essential for autophagy, and is involved in the regulation of growth, development and pathogenicity in B. cinerea.
Atg2 forms a complex with Atg18, which is essential for autophagosome formation in yeast (Yamamoto and Ohsumi 2014). In this study, the direct physical interaction between BcAtg2 and BcAtg18 was verified by Fig. 3 BcAtg2 is essential for autophagy in B. cinerea. a Subcellular localization of GFP-BcAtg8 in the wild-type strain B05.10 and the BcATG2 deletion mutant ΔBcAtg2 with or without nitrogen-starvation treatment. Vacuoles were stained with CMAC (7-amino-4-chloromethylcoumarin). b Analysis of proteolysis of GFP-BcAtg8 in the wild-type strain B05.10 and ΔBcAtg2 with or without nitrogen-starvation treatment using anti-GFP Western blot. GAPDH was used as an internal reference. c The percentage of free to total GFP. Error bars denote standard deviations from three independent experiments, and asterisks indicate statistically significant differences at P = 0.05, NS indicates no significant difference yeast two-hybrid assay. Additionally, the autophagic process was blocked in the BcATG2 deletion mutant. Functional domain analysis showed that BcAtg2 contains chorein-N, ATG-CAD and ATG-C, the typical conserved domains in yeast Atg2 that are involved in the regulation of autophagy (Osawa et al. 2019). Phylogenetic analysis suggested that Atg2-coding genes are conservatively evolved to maintain a similar function in fungi.
Endogenous nutrient recycling supplied by autophagy ensures normal growth and development of pathogenic fungi (Bartoszewska and Kiel 2010). Consistent with the crucial role of Atg2 in mycelial growth and conidiation in M. oryzae and F. graminearum (Khan et al. 2012;Lv et al. 2017), in this study, ΔBcAtg2 exhibited reduced mycelial growth rate and aerial mycelium formation, and poor conidiation. In addition, ΔBcAtg2 lost the ability to form sclerotia. These results suggest that BcAtg2-mediated autophagy is important for vegetative growth and differentiation in B. cinerea.
Autophagy is a cellular degradation process, through which a large amount of material and energy needs are met in pathogenic fungi during their infection of host plants (Deng et al. 2012). Previous studies have shown that autophagy is involved in pathogenicity of plant pathogens (Pollack et al. 2009). In this study, ΔBcAtg2 was shown to lose the ability to form infection structure and therefore failed to infect plant tissues, consistent with the ATG2 deletion mutants of M. oryzae and F. graminearum, which also lose pathogenicity (Khan et al. 2012;Lv et al. 2017). These results suggest that BcAtg2-mediated autophagy is required for pathogenicity of B. cinerea. cinerea. a Colony morphology of the wild-type strain B05.10, ΔBcAtg2 and complemented strain BcAtg2-C grown on PDA, MM and CM media at 25 °C for 3 days. b Column diagram showing mycelial growth rate of each strain. Error bars denote standard deviations from three independent experiments, and asterisks indicate statistically significant differences at P = 0.05

Fig. 5
BcAtg2 is crucial for conidiation in B. cinerea. a Conidia on conidiophores of the wild-type strain B05.10, BcATG2 deletion mutant ΔBcAtg2 and complemented strain BcAtg2-C incubated on sterilized potato fragments at 20 °C under white light for 10 days. b Conidia morphology of each strain. Red arrows indicate abnormal conidia. c Numbers of conidia produced by each strain. Error bars denote standard deviations from three independent experiments, and asterisks indicate statistically significant differences at P = 0.05

Conclusions
In this study, we identified and characterized the autophagy protein Atg2 in the gray mold fungus B. cinerea, and our results indicate that Atg2-mediated autophagy plays important roles in the regulation of growth, development and pathogenicity in B. cinerea. These results provide a better understanding of the biological role of autophagy in fungi.

Strains and culture conditions
The B. cinerea wild-type strain B05.10 and derivative transformants were routinely cultured on potato dextrose agar (PDA), minimal medium (MM) and complete medium (CM) at 25 °C as described previously (Ren et al. 2018a). Mycelia were cultured in liquid yeast   The combination of pGADT7/pGBKT7-53 was used as a positive control, and pGADT7/pGBKT7-Lam as a negative control extract peptone dextrose (YEPD) medium with shaking (180 rpm) at 25 °C for DNA/RNA extraction. Sterilized potato fragments were used for conidia production. PDA was used for sclerotial formation. MM-N [MM without (NH 4 ) 2 SO 4 ], a nitrogen-deficient medium, was used for induction of autophagy.

Targeted gene deletion and complementation
To replace BcATG2 in the wild-type strain B05.10, 1296-bp upstream and 1323-bp downstream flanking sequences of the gene were amplifies by PCR using the genomic DNA of B05.10 as template. The resulting amplicons were fused with hygromycin phosphotransferase gene (HPH) through double-joint PCR (Yu et al. 2004). Protoplast preparation and transformation were performed as described previously (Gronover et al. 2001). The resulting hygromycin-resistant transformants were preliminarily screened by PCR with primers shown in Additional file 1: Table S1, and further confirmed by Southern blot analysis. For complementation assays, BcAtg2-GFP cassette was constructed according to the method described previously (Ren et al. 2018b). Briefly, the entire open reading frame (ORF) of BcATG2 (without stop codon) was amplified and cloned into pNAN-OGG vector using the One Step Cloning Kit (Vazyme, Nanjing, China). The resulting constructs were confirmed by sequencing and transformed into the BcATG2 deletion mutant.

Yeast two-hybrid assay
To construct vectors for yeast two-hybrid tests, the fulllength cDNA of BcATG2 and BcATG18 were amplified from the genomic cDNA of B05.10, and then cloned into the yeast GAL4 activation domain vector pGADT7 and GAL4 binding domain vector pGBKT7, respectively, using a One Step Cloning Kit (Vazyme, Nanjing, China). The resulting vectors were confirmed by sequencing and co-transformed into the yeast reporter strain AH109 following the standard protocol (Schiestl and Gietz 1989). The yeast transformants growing on synthetic medium (SD) lacking Leu and Trp were collected and assayed for growth on SD (5 mM 3-aminotriazole) lacking Leu, Trp and His. The interactions between pGADT7-T and pGBKT7-53, and between pGADT7-T and pGBKT7-Lam were served as the positive and negative control, respectively. The experiments were repeated three times.

Western blot assay
Total proteins were extracted as described previously (Gu et al. 2015). Equal amounts of proteins were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membrane with a Tanon electroblotting apparatus. The anti-GFP antibody (Abcam, Cambridge, Cat#Ab32146) and anti-GAPDH antibody (Hangzhou HuaAn Biotechnology Co. Ltd, Hangzhou, China, Cat#EM1101) were used for immunoblot analyses. The experiments were repeated three times.

Pathogenicity assay
Infection tests were performed on cucumber leaves and apple fruits. Briefly, the plant samples were point-inoculated with mycelial plugs (5 mm in diameter) from 3-dayold cultures of the B. cinerea wild-type strain B05.10 and derivative strains. Before inoculation, plant cuticles were punctured with a sterilized needle tip to facilitate penetration. Water agar plugs without fungal mycelia were used as negative controls (mock). The inoculated samples were incubated under conditions of high relative humidity (about 95%) at 25 °C with 16 h of daylight. The experiments were repeated three times and each time with at least ten samples.