Involvement of BcElp4 in vegetative development, various environmental stress response and virulence of Botrytis cinerea

Summary The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1‐Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis and formation of modified wobble uridines in tRNA. In this study, we investigated the function of BcElp4 in Botrytis cinerea, which is homologous to S. cerevisiae Elp4. A bcelp4 deletion mutant was significantly impaired in vegetative growth, sclerotia formation and melanin biosynthesis. This mutant exhibited decreased sensitivity to osmotic and oxidative stresses as well as cell way‐damaging agent. Pathogenicity assays revealed that BcElp4 is involved in the virulence of B. cinerea. In addition, the deletion of bcelp4 led to increased aerial mycelia development. All these defects were restored by genetic complementation of the bcelp4 deletion mutant with the wild‐type bcelp4 gene. The results of this study indicated that BcElp4 is involved in regulation of vegetative development, various environmental stress response and virulence in B. cinerea.


Introduction
The transcriptional elongation complex was first identified based on its direct association with the RNA polymerase II holoenzyme in yeast . The protein complex is composed of two sub-complexes: one made of the Elp1, Elp2 and Elp3 subunits and an other one constituted by Elp4, Elp5 and Elp6 subunits forming the hexameric holo-Elongator (Petrakis et al., 2004). Elp3 forms a stable complex with the five other polypeptides called Elp1, Elp2, Elp4, Elp5 and Elp6 (Krogan and Greenblatt, 2001) and contains two domains. Previous studies showed that Elp3 contains an iron-sulphur cluster that can bind S-adenosylmethionine (Paraskevopoulou et al., 2006) and may be involved in DNA demethylation (Okada et al., 2010). Amyotrophic lateral sclerosis was shown recently to be linked to allelic variants of ELP3 (Simpson et al., 2009). Based on sequence comparisons, it was suggested that Elp4 and Elp6 could be inactive orthologues of ancestral ATPases involved in chromatin remodelling (Ponting, 2002). In Saccharomyces cerevisiae, deletions of the individual genes that encode the Elongator subcomplex Elp4-6 revealed that only the ELP5 gene is essential for growth (Krogan and Greenblatt, 2001). However, the mammalian ELP4 gene was recently implicated in rolandic epilepsy (Strug et al., 2009) and the eye anomaly aniridia (Crolla and van Heyningen, 2002;Kleinjan et al., 2002;Zhang et al., 2011). In yeast, the Elongator complex functions as a histone acetyltransferase (HAT) complex that was related to the hyper-phosphorylated elongating form of RNA polymerase II (Glatt et al., 2012). The known biological roles of Elongator are in RNAPII-mediated transcription through the acetylation of histone H3 and H4 in chromatin, the modification of certain tRNAs and the acetylation of alpha-tubulin Fellows et al., 2000;Jablonowski et al., 2001). Previous studies have shown that Elongator was involved in various cellular functions including tRNA modification, transcriptional silencing, sensitivity to DNA damaging agents and histone acetylation (Wittschieben et al., 1999;Huang et al., 2005;Esberg et al., 2006;Li et al., 2009;Okada et al., 2010). In addition, in yeast, Elongator mutants defective in vegetative growth exhibited sensitivity to rapamycin, high temperature, hydroxyurea, caffeine and various other stresses and were resistant to zymocin Li et al., 2009), a protein toxin secreted by the yeast Kluyveromyces lactis that kills other yeasts including S. cerevisiae (Jablonowski and Schaffrath, 2007). The Elongator was also involved in replication-coupled nucleosome assembly, transcriptional silencing and polarized secretion (Rahl et al., 2005;Li et al., 2009).
Botrytis cinerea is a necrotrophic plant pathogenic fungi causing pre-and post-harvest grey mould in multiple plant species, resulting in serious economic losses in fruit, vegetable and ornamental flower production (Elad et al., 2004;Willamson et al., 2007;An et al., 2016). All plant organs, including leaves, flowers, soil storage organs and shoots, are susceptible to B. cinerea attacks (Veloukas and Karaoglanidis, 2012). Under a wide range of environmental conditions, B. cinerea employs various modes to attack its different hosts (Qin et al., 2010). In addition, it can survive as conidia and mycelia or for extended periods of time as sclerotia in plant debris (Willamson et al., 2007), resulting in that the control of grey mould is full of challenges. Thus, research on the mechanism of development and pathogenesis of B. cinerea could provide strategies to reduce the damage caused by the grey mould. A whole-genome search uncovered that B. cinerea has an orthologue of Elp4 (hereafter named bcelp4). Based on the analysis of function of Elp4 in yeast, we hypothesized that in B.c., bcelp4 might have significant effects on development and virulence in B. cinerea. To clarify this hypothesis, the major target of this study was to explore the biological and biochemical function of bcelp4 using a target gene deletion strategy. Our study indicated that bcelp4 plays important roles in regulating the vegetative growth, asexual development, melanin synthesis, various stresses sensitivity and virulence in B. cinerea.

Characterization of the bcelp4 gene
The bcelp4 (BC1G_03169) was originally identified via a homology search of the B. cinerea genome database using BLAST with the elp4 gene from S. cerevisiae as a query (Glatt et al., 2012). To analyse the size and existence of introns, RNA was extracted from mycelia of the wild-type strain B05.10 with Tian-gen Reagent kit (Tiangen Biotech. Co., Beijing, China) and was used for reverse transcription with a cDNA synthesis kit (TaKaRa Biotech. Co., Dalian, China). The primer pair P1/P2 (Table S1) generated 1209-bp and 1297-bp fragments from cDNA and genomic DNA. Sequence results indicated that the coding region of bcelp4 has one intron of 88 bp, which was located after the first 451 nucleotides (Fig. S1).

Generation of bcelp4 deletion and complemented mutant
To research the biological function(s) of BcElp4 in B. cinerea, we constructed a mutant using a homologous recombination strategy (Fig. S2). The deletion mutant was identified from 189 hygromycin-resistant transformants by PCR and verified with different primer pairs. The P11/P12 primers amplified a 345-bp fragment from the wild-type strain B05.10 but did not amplify any fragment from the bcelp4 deletion mutant. The P13/P14, P15/P16 and P17/ P18 primers amplified 981-bp, 2688-bp and 2399-bp fragments from the bcelp4 deletion mutant, respectively, but did not amplify any fragment from the wild-type strain B05.10. (Fig. S3). In a southern hybridization assay, when probed with the 706-bp 3 0 -flanking region of bcelp4, the bcelp4 deletion mutant Dbcelp4 generated a 4243-bp band, but lacked a 3776-bp band which was present in the wild-type strain B05.10.
These results confirm that Dbcelp4 resulted from the anticipated homologous recombination events at the bcelp4 locus. The wild-type bcelp4 was ectopically integrated into the genome of the complemented strain (Dbcelp4C).

Involvement of bcelp4 in vegetative growth and sclerotial formation
To analyse the role of bcelp4 in vegetative colony growth, each strain was inoculated on potato dextrose agar (PDA), minimal medium (MM) and complete medium (CM) respectively. The growth rate of Dbcelp4 showed a significant reduction compared with that of the wild-type strain, but the growth rate did not differ between the complemented strain Dbcelp4C and the wild-type strain. (Fig. 1A and B). A previous study had shown that sclerotial generation in dying host tissues plays an important role in the survival of B. cinerea (Willamson et al., 2007). We explored the function of bcelp4 on sclerotial generation, the bcelp4 deletion mutant was unable to produce any sclerotia, and in contrast, the wild-type and the complemented strain Dbcelp4C produced vast sclerotia after incubation on PDA, MM and CM, respectively, in dark for 4 weeks ( Fig. 1C and D). These results shown that bcelp4 was required for vegetative growth and sclerotial formation in B. cinerea.

Sensitivity of the bcelp4 deletion mutant to various environmental stresses
In a previous study, it was shown that the elp4 plays an important role in the sensitivity of S. cerevisiae to various stresses (Krogan and Greenblatt, 2001). Therefore, we tested the sensitivity of the bcelp4 deletion mutant to various environmental stresses including cell wall-damaging agents (Caffeine and Congo red), osmotic and oxidative stresses. As shown in Fig. 2, the Dbcelp4 revealed significantly decreased sensitivity to osmotic stresses generated by 1.0 M NaCl or 1.2 M KCl. It has been reported that osmotic stress can induce glycerol accumulation in fungi primarily triggered by activation of the HOG pathway (Fillinger et al., 2001;de Vries et al., 2003). Then, we tested the glycerol content in the bcelp4 deletion mutant. As shown in Fig. 3A, in the absence of osmotic stress, very little glycerol was detected in the wild-type stain or the DBcElp4. However, NaCl stress induced an eightfold increase in the glycerol content of the B05.10 strain but not to the same extent in the bcelp4 deletion mutant. Additionally, as shown in Fig. 2, Dbcelp4 also exhibited significantly decreased sensitivity to oxidative stress generated by 24 mM H 2 O 2 or cell way-damaging agent generated by 5 mM caffeine or 0.25 mg ml À1 Congo red. In a previous study, it was shown that mkk1 and gls2 were cell wall integrity core genes in S. cerevisiae (Rodriguez et al., 1985). In addition, the Yap1 protein was a relevant regulation factor in oxidative stress response in S. cerevisiae (Kuge et al., 1997;Lu et al., 2003;He and Fassler, 2005). To further investigate the role of bcelp4 in the cell wall integrity and oxidative stress response in B. cinerea, we analysed the expression of bcmkk1, bcgls2 and bcyap1. Which were homologous to mkk1, gls2 and yap1 of S. cerevisiae, respectively. As shown in Fig. 3B, the expression levels of these three genes were down-regulated in Dbcelp4 relative to the wild-type strain (ABI 7500 SDS software; Applied Biosystems, Foster City, CA, USA). Meanwhile, as shown in Fig. 4, Dbcelp4 exhibited decreased sensitivity to low temperature (4°C and 15°C). The results indicated that the bcelp4 gene product affects on the sensitivity of B. cinerea to various stresses.
Effect of bcelp4 deletion on aerial mycelia development of B. cinerea Each strain was cultivated on PDA, MM and CM medium at 25°C in dark for 3 days. As shown in Fig. 5A, the bcelp4 deletion mutant generated more white aerial mycelia than the wild-type strain or the complemented strain. This result indicated that bcelp4 is involved in mycelia differentiation in B. cinerea.

Involvement of bcelp4 in the regulation of mycelial melanization
To investigate the bcelp4 involvement in the synthesis of melanin in mycelia, each of the strains were incubated on PDA at 25°C for 10 days (Thompson et al., 2000). As shown in Fig. 5B, compared to the wild-type strain B05.10 and the complemented strain Dbcelp4C, hyphal melanization of the bcelp4 deletion mutant decreased when incubated in PDA medium. This phenotype was further confirmed by measuring the level of expression of the melanin biosynthetic related genes bcpks13, BcCmr1 and bcscd1 (Eliahu et al., 2007;Liu et al., 2011), which were lower in Dbcelp4 than that in the wide-type strain B05.10 (Fig. 5C). These results support that bcelp4 is related to melanin synthesis in B. cinerea.
Requirement of bcelp4 in the virulence of B. cinerea The involvement of bcelp4 in virulence was analysed on various host plant tissues. Mycelial plugs of each strain were inoculated on host plant tissues. On wounded grapes, apples, tomato and leaves of strawberry, the bcelp4 deletion mutant caused significantly smaller disease lesions than the wild-type strain B05.10 and complemented strain Dbcelp4C (Fig. 6) after inoculated for 72 h with 16 h of daylight. The results indicated that the bcelp4 plays an important role in the virulence in B. cinerea.

Discussion
A previous study had shown that Elongator is a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription . In yeast, the deletion of any of the six Elongator protein subunit (Elp1-Elp6) gene causes similar pleiotropic effects (Huang et al., 2005). In this study, we identified a S. cerevisiae Elp4 homologous gene bcelp4 in B. cinerea. The function analysis of bcelp4 indicated that bcelp4 is involved in various phenotypes in B. cinerea. A previous study showed that elp4 affected vegetative growth in S. cerevisiae (Krogan and Greenblatt, 2001). Consistent with this observation is that the bcelp4 B. cinerea mutant showed significant defect on mycelia growth on PDA, MM or CM medium compared with the wild-type strain B05.10 and the complemented strain Dbcelp4C. These results indicated that elp4 played a relevant role in development in different fungi. In a previous study, it was shown that sclerotia play an important role in sexual reproduction during the development of apothecia in B. cinerea (Willamson et al., 2007). In this study, we found that the wild-type strain and the complemented strain produced vast sclerotia, while the Dbcelp4 mutant lost the ability of sclerotia formation. This result indicated that the bcelp4 played an important role in sexual reproduction mechanism in B. cinerea.
In a previous study with S. . cerevisiae it was shown that the elp4 was involved in sensitivity to various environmental stresses (Krogan and Greenblatt, 2001). In our study, the results support that the bcelp4 deletion mutant showed significantly decreased sensitivity to cell wall-damaging compounds such as Congo red or caffeine, oxidative stress generated by H 2 O 2 , osmotic stresses generated by NaCl or KCl and low temperature stresses generated by 4 or 15°C. Gene expression of bcmkk1, bcgls2 and bcyap1 was significantly decreased in Dbcelp4 compared with that in B05.10 and Dbcelp4C. Additionally, when all of strains responded to osmotic stress, glycerol accumulation in the bcelp4 deletion mutant was lower than that in wild-type strain and the complemented strain. Based on these results, we deduced that the deletion of bcelp4 may interfere with regulated factors in various stress responses in B. cinerea leading in the deletion mutant to decreased sensitivity to various stresses. This hypothesis needs to be further explored. In addition, we found that the bcelp4 deletion mutant generated more aerial mycelia than that of wild-type strain and the complemented strain. This result indicated that the bcelp4 affects on the differentiation of mycelia vegetative growth in B. cinerea.
In the current study, we found that the bcelp4 deletion mutant generated lower levels of melanin than wild-type  strain and the complemented strain. In many fungal species, Paracoccidiodes brasiliensis, Cochliobolus heterostrophus and Cryptococcus gattii, melanin has been shown to play multiple roles in providing defence against various environmental stresses such as oxidizing factors, UV light and ionizing radiation and it contributed to the ability of fungi to survive in harsh environments (Eisenman and Casadevall, 2012). As the bcelp4 deletion mutant generated lower melanin than that of wild-type strain and the complemented strain, we explored whether decreased melanin generation in the bcelp4 deletion mutant conferred reduced sensitivity of B. cinerea to various environmental stresses, which was confirmed.
In current study, phenotypic characterization of B. cinerea bcelp4 mutant showed that it was also involved in virulence. The reduced virulence of the bcelp4 deletion mutant could be due to defects in multiple regulatory factors. First, the bcelp4 deletion mutant grew significantly slower than the wild-type strain or the complemented strain. We found also that the Dbcelp4 showed decreased sensitivity to various environmental stresses, which play important roles in the interaction of fungal with host plants (Rolke et al., 2004;Motoyama et al., 2005;Arbelet et al., 2010), what might be related to the low virulence of this mutant on some plant tissues (Esberg et al., 2006).
In conclusion, we have compared B. cinerea Dbcelp4 mutant with the wild-type strain and have shown that bcelp4 plays an important role in growth, melanin production, asexual development, responses to various stresses and virulence. Our results provided the basis for further exploration of function of Elongator complex in B. cinerea and may enhance the development of fungicides that target bcelp4 for the control of plant diseases caused by B. cinerea.
Mycelial growth assays under different condition were performed on PDA plates modified with the following: caffeine, Congo red, H 2 O 2 , NaCl and KCl at the concentrations indicated in the figure legends. Each plate was inoculated with a 5-mm-diameter mycelial plug taken from the edge of a 3-day-old colony grown on PDA. The percentage of inhibition of mycelial radial growth (PIMG) was calculated using the formula, PIMG = [(C -N)/(C -5)] 9 100, where C is the colony diameter of the untreated control and N is that of a treatment. Each experiment was repeated three times independently.

Deletion of the bcelp4 gene and complemented mutant
To investigate the functions of bcelp4 in B. cinerea, we constructed a bcelp4 deletion mutant. The gene replacement cassette was generated as described previously  (Laleve et al., 2014;Zheng et al., 2014). First, 1409-bp upstream and 1362-bp downstream flanking fragment of bcElp4 were amplified from the wild-type strain B05.10 genomic DNA with the primer pairs P3/P4 and P5/P6 respectively (Fig. S2, Table S1). Subsequently, the 1764-bp HPH cassette (resistance to hygromycin B) containing a trpC promoter was amplified form the pKHT plasmid with the primer pair P7/P8 according to previous study (Duan et al., 2013). Then, the two bcelp4 flanking fragments were mixed with the HPH cassette in a molar proportion of 1:1:3 and used as a template for the fusion PCR (Fig. S2) (Yu et al., 2004). The 4676-bp DNA fragment amplified from the fusion PCR production using the primer pair P9/P10 and used for transforming protoplasts of the wild-type strain B05.10.
Protoplast generation and transformation of B. cinerea were performed referring to previous study with some modification (Schulze et al., 2001;Wang et al., 2015). Mycelial plugs cut from the edge of 3-day-old colony on PDA were placed in 250-ml flask containing 100 ml of liquid YEPD (10 mg/ml peptone, 3 mg/ml yeast extract, 20 mg/ml glucose). After the flasks have been shaken at 175 rpm and 25°C for 18 h, fresh mycelia were collected using a sterile filter and washed twice with distilled water. Then, 0.15 g of fresh mycelia was incubated with 15 ml 2% lysing enzymes buffer (0.6 M KCl, 50 mM CaCl 2 ; Sigma, St Louis, MO, USA). After 2 h at 30°C and 85 rpm, the enzyme solution was filtered to eliminate mycelial residues. The protoplasts in the filtrate were washed twice with STC buffer (0.8 M sorbitol, 0.05 M Tris, pH 8.0, 50 mM CaCl 2 ) and resuspended in (STC with 40%, w/v, PEG6000) buffer (STC: SPTC = 4:1). The transformation of protoplasts was performed as described previously (Zheng et al., 2014). For transformation, 10 7 in 500 ll of SPTC buffer and 35 lg replacement vector in 20 ll of spermidine were mixed and incubated on ice for 90 min; 1 ml SPTC was added into the suspension and incubated at 25°C for 30 min. Protoplasts were mixed into 200 ml RM medium (0.5 g/l yeast extract, 0.5 g/l casein hydrolysate, 0.7 M sucrose and 16 g/l agar powder) at 42°C, plated on petri plate (15 ml per plate) and incubated at 25°C for 16 h, and then, RM plates were overlaid with 10 ml of SRM medium (0.5 g/l yeast extract, 0.5 g/l casein hydrolysate, 1 M sucrose and 12 g/l agarose) containing 100 lg/ml hygromycin B. After incubation at 25°C for 4 days, the transformants were transferred to fresh PDA containing 100 lg/ml hygromycin B for PCR detection assay. Transformants indicating the homologous replacement of the vector were detected by PCR amplification of genomic DNA using different special primer pairs, P11/P12, P13/P14, P15/P16 and P17/P18 (Table S1, Fig. 2A) and confirmed by southern analysis of genomic DNA digested with EcoRV and hybridized with a labelled probe generated by PCR amplification using primer pair P21/P22 (Table 1, Fig. 1A).
To confirm that the phenotype of the bcelp4 deletion mutant resulted from deletion of the gene, the bcElp4 deletion mutant was complemented with the full-length bcElp4 gene refer to a previous study (Duan et al., 2013). The bcelp4 complement plasmid pNEO-bcelp4-Com was constructed using the backbone of pCAMBIA 1300 (CAMBIA, Canberra, Australia). First, a BstXI-XhoI NEO cassette containing a trpC promoter (resistance to neomycin) was amplified form plasmid PII99-Pro (DOHH) GFP with primers P19/P20 (Table S1) and cloned into the BstXI-XhoI site of pCAMBIA 1300 to create plasmid pNEO. Then, the full-length bcelp4 gene including the 415-bp upstream and 319-bp terminator regions was amplified from genomic DNA of the wildtype strain with primers P19/P20 (Table S1) and subsequently cloned into the Sma I -Hind III site of pNEO to generate the complement plasmid pNEO-bcelp4-Com. Before the plasmid, pNEO-bcelp4-Com was transformed into strain Dbcelp4, and bcelp4 in this plasmid was sequenced to ensure sequence correctness. Transformation of Dbcelp4 with plasmid pNEO-bcelp4-Com was conducted as described above, except that neomycin (100 lg/ml) was used as a selection agent.

Nucleic acid manipulations
Fungal genomic DNA was extracted according to the previous described method ( McDonald and Martinez, 1990). Southern blot hybridization analysis of bcelp4 gene in the transformants of B. cinerea was performed using a 706-bp fragment downstream of bcelp4 as a probe. The probe was labelled with digoxigenin using the High Prime DNA labelling and detection starter kit II referring to the protocol of the manufacturer (Roche Diagnostics, Mannheim, Germany). DNA isolated from B. cinerea was digested with EcoR V and used for southern hybridization analysis.

Quantitative RT-PCR assay
To isolate total RNA, the mycelial plugs of each strain were inoculated into liquid YEPD (10 mg/ml peptone, 3 mg/ml yeast extract, 20 mg/ml glucose) and cultured at 25°C for 2 days in a shaker (175 rpm). RNA was isolated from mycelia with the RNeasy kit (Tiangen Biotech. Co., Beijing, China). First-strand cDNA was synthesized with the PrimeScript â RT reagent kit (TaKaRa). The real-time PCR amplifications were conducted in an ABI 7500 detection system (Applied Biosystems) (Zheng et al., 2014). The primers used for Quantitative RT-PCR assay are showed in Table S1. The expression level of the measured gene in all samples was normalized to actin gene expression, and relative changes in gene expression levels were analysed by ABI 7500 SDS software (Applied Biosystems), which automatically generates the baseline. Data from three biological replicates were applied to calculate the mean standard deviation.

Assay of intracellular glycerol accumulation
To determinate glycerol accumulation, each strain was cultivated in YEPD (10 mg/ml peptone, 3 mg/ml yeast extract, 20 mg/ml glucose) for 2 days at 25°C in a shaker. After treatment with 1.2 M NaCl, mycelia of each strain were harvested and ground in liquid nitrogen. The mycelial powder (0.15 g) was transferred to a 2-ml microcentrifuge tube containing 0.5 ml of glycerol isolation buffer (Applygen, Beijing, China). After vortexing three times for 35 s each, the tubes were centrifuged at 6000 g for 25 min. The supernatant was transferred to a 1.5-ml tube, and then, 10 ll of each supernatant in tube was mixed with 190 ll detection buffer from glycerol determination kit (Applygen). After the mixture was incubated for 15 min at 37°C, the glycerol concentration was determined by a microplate reader (SpectraMax M5) at 550 nm (Yang et al., 2012). The experiment was independently repeated twice.

Virulence assays
To investigate the role of bcelp4 in the virulence of B. cinerea, 3-week-old strawberry leaves, and grape, tomato and apple fruits were inoculated with 5-mm-diameter mycelial plugs cut from 3-day-old colony margin, respectively. Before inoculation, the leaves and fruits were wounded with a sterile needle tip to facilitate the penetration of the fungus into host plant tissue. Inoculated tissues were cultivated at 25°C with 16 h of daylight (Yang et al., 2012). Diameter of disease lesions was recoded after the indicated times in figure legends. The experiment was repeated three times. Fig. S1. The structure of bcelp4 and amino acid alignments of Botrytis cinerea elp4 (bcelp4) with those of Neurospora crassa elp4 (ncelp4), Magnaporthe oryzae elp4 (moelp4), Fusarium graminearum elp4 (fgelp4), Saccharomyces cerevisiae elp4 (scelp4) and Sclerotinia sclerotiorum elp4 (sselp4). Boxshade program was used to highlight identical (black shading) and similar (grey shading) amino acids. Fig. S2. Generation and identification of the bcelp4 deletion mutant of Botrytis cinerea. Fig. S3. Identification of the bcelp4 deletion mutant of Botrytis cinerea with PCR method. Table S1. Primers used in this study.