Plant Pathol. J. 37(1) : 36-46 (2021) https://doi.org/10.5423/PPJ.OA.12.2020.0221 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 ©The Korean Society of Plant Pathology Research Article Open Access Fast Track Proteomic and Phenotypic Analyses of a Putative Glycerol-3-Phosphate Dehydrogenase Required for Virulence in Acidovorax citrulli Minyoung Kim1, Jongchan Lee1, Lynn Heo1, Sang Jun Lee 2*, and Sang-Wook Han 1* 1Department of Plant Science and Technology, Chung-Ang University, Anseong 17546, Korea 2Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea (Received on December 9, 2020; Revised on December 28, 2020; Accepted on December 29, 2020) Acidovorax citrulli (Ac) is the causal agent of bacterial presence of glycerol, indicating that GlpdAc is involved fruit blotch (BFB) in watermelon, a disease that poses in glycolysis. AcΔglpdAc(EV) also displayed higher cell- a serious threat to watermelon production. Because of to-cell aggregation than the wild-type bacteria, and tol- the lack of resistant cultivars against BFB, virulence erance to osmotic stress and ciprofloxacin was reduced factors or mechanisms need to be elucidated to control and enhanced in the mutant, respectively. These results the disease. Glycerol-3-phosphate dehydrogenase is the indicate that GlpdAc is involved in glycerol metabolism enzyme involved in glycerol production from glucose and other mechanisms, including virulence, demon- during glycolysis. In this study, we report the functions strating that the protein has pleiotropic effects. Our of a putative glycerol-3-phosphate dehydrogenase in study expands the understanding of the functions of Ac (GlpdAc) using comparative proteomic analysis and proteins associated with virulence in Ac. phenotypic observation. A glpdAc knockout mutant, AcΔglpdAc(EV), lost virulence against watermelon in Keywords : bacterial fruit blotch, glycerol-3-phosphate de- two pathogenicity tests. The putative 3D structure and hydrogenase, virulence, watermelon amino acid sequence of GlpdAc showed high similarity with glycerol-3-phosphate dehydrogenases from other Handling Editor : Chang-Jin Park bacteria. Comparative proteomic analysis revealed that many proteins related to various metabolic pathways, Acidovorax citrulli (Ac), previously known as Pseudomo- including carbohydrate metabolism, were affected by nas pseudoalcaligenes subsp. citrulli, causes bacterial fruit GlpdAc. Although AcΔglpdAc(EV) could not use glu- blotch (BFB) disease in cucurbit crops such as watermelon cose as a sole carbon source, it showed growth in the (Latin and Rane, 1990; Sowell and Schaad, 1979). The disease was first observed in 1989 in Florida, USA, and *Co-corresponding authors. S. J. Lee has since spread throughout the US and other countries, in- Phone) +82-31-670-3356, FAX) +82-2-675-3108 cluding Korea (Burdman and Walcott, 2012). Under favor- E-mail) [email protected] able conditions, the crop losses caused by BFB have been S.-W. Han greater than 90% on marketable crops. The disease gener- Phone) +82-31-670-3150, FAX) +82-2-670-8845 ally begins with contaminated seeds because Ac is known E-mail) [email protected] ORCID as a seed-borne pathogen (Latin and Hopkins, 1995). The Sang Jun Lee initial symptoms are water-soaked lesions on cotyledons https://orcid.org/0000-0002-2803-753X and wilt development in seedlings. In fruit, the infected Sang-Wook Han surfaces also show water-soaked lesions, which expand, https://orcid.org/0000-0002-0893-1438 leading to necrosis of the infected fruit (Latin and Hopkins, This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// 1995). Thus, BFB is a major threat to the production of creativecommons.org/licenses/by-nc/4.0) which permits unrestricted watermelon. Despite its negative impact on the industry, noncommercial use, distribution, and reproduction in any medium, resistant cultivars of watermelon have not been developed. provided the original work is properly cited. Therefore, virulence factors or mechanisms in Ac have to Articles can be freely viewed online at www.ppjonline.org. be elucidated to control the disease. Roles of GlpdAc in Acidovorax citrulli 37 There was a focus on controlling BFB spread in the coli strain DH5α was used, and for cloning Tn5-inserted 1990s, and various studies regarding virulence factors and DNA fragments, the E. coli strain EC100D was used (Epi- mechanisms in Ac were subsequently carried out. It was centre, Madison, WI, USA). E. coli strains were grown in elucidated that twitching motility and biofilm formation, LB (Luria-Bertani; 1% tryptone, 0.5% yeast extract, and which are mediated by type IV pili, are indispensable for 1% NaCl) with appropriate antibiotics. The final antibiotic Ac virulence (Bahar et al., 2009). It was also reported that concentrations in the media were as follows: kanamycin, quorum sensing and ferric uptake systems are involved 50 µg/ml; rifampicin, 50 µg/ml; gentamicin, 10 µg/ml; and in the regulation of its virulence (Johnson and Walcott, ampicillin, 100 µg/ml. 2013; Liu et al., 2019). Additionally, research focusing on the type III secretion system is actively ongoing (Jiménez- Generation of AcΔglpdAc and its complementary strain. Guerrero et al., 2019). Recently, Kim et al. (2020) reported To generate the knockout of glpdAc, the EZ-Tn5 <R6Kγori/ that CmpAc, which is a bifunctional chorismate mutase and KAN-2> Insertion Kit (Lucigen, Middleton, WI, USA) prephenate dehydratase, is involved in virulence and other was used. Following the kit procedure, Tn5 was randomly mechanisms in Ac. However, other genes and mechanisms inserted on one site of the Ac genome, and this insertion that are related to virulence in Ac have not been definitively was verified using Tn5-specific primers 5′-GGATCGC- identified. GATGGATATGTTCT-3′ and 5′-AACAGGAATC- Glycerol-3-phosphate (G3P) dehydrogenase is a cytosol- GAATGCAACC-3′. Genomic DNA was extracted, and ic enzyme involved in carbohydrate and lipid metabolism, the Tn5-disrupted DNA fragment was cloned following and it controls the conversion process between dihydroxy- the manufacturer's protocol (Lucigen). After cloning the acetone phosphate and G3P (Guindalini et al., 2010). This DNA fragment, its DNA sequence was verified by Sanger G3P dehydrogenase has been shown to be the key enzyme sequencing with the following primers: forward (KAN-2F), that produces glycerol from glucose during glycolysis 5′-ACCTACAACAAAGCTCTCATCAACC-3′ and re- (Hao et al., 2015). Further, G3P dehydrogenase plays an verse (R6KAN-2R), 5′-CTACCCTGTTGGAACACCTA- important role in the virulence of Pseudomonas aeruginosa CATCT-3′ (Lucigen). The confirmed strain was named (Daniels et al., 2014). In Magnaporthe oryzae, the dele- AcΔglpdAc. To create the construct for the complemented tion of G3P dehydrogenase had negative effects on fungal strain, the open reading frame of glpdAc was amplified development and virulence (Shi et al., 2018). The function using the following gene-specific primers: 5′-CTCGAGA- of G3P dehydrogenase in Colletotrichum gloeosporioides TGAAGATCATCGT-3′ and 5′-AAGCTTCAGTGGTG- is also related to conidiation (Wei et al., 2004). Thus, G3P GTGGTGGTGGTGGCAGGAGCGCAA-3′. The ampli- and glycerol assimilation and carbon utilization through con was cloned into the pGem-T Easy vector (Promega, G3P dehydrogenase play important roles in pathogenicity. Madison, WI, USA), generating pGem-GlpdAc, and the In this study, we report the functions of a putative G3P cloned DNA was confirmed by Sanger sequencing. The dehydrogenase in Ac (GlpdAc) in virulence and other bio- construct was digested with restriction enzymes XhoI and logical mechanisms using a G3P dehydrogenase knockout HindIII, and the digested DNA fragment was subcloned mutant (AcΔglpdAc). To predict and characterize the func- into the pBBR1-MCS5 vector, which is a broad host range tions of GlpdAc, the label-free shotgun proteomics follow- vector containing the lacZ promoter (Kovach et al., 1995), ing clusters of orthologous groups (COGs) was carried out. generating pBBR1-GlpdAc. The generated plasmid was In addition to comparative proteomic analysis, diverse phe- introduced into AcΔglpdAc and selected on tryptic soy agar notypic assays were also conducted to elucidate the roles of (TSA) with appropriate antibiotics. The selected trans- GlpaAc in Ac. formant was confirmed using PCR with GlpdAc-specific primers and called AcΔglpdAc(GlpdAc). To control for Materials and Methods vector side-effects, an empty pBBR1-MCS5 vector was also introduced into Ac and AcΔglpdAc, generating Ac(EV) Bacterial strains and growth conditions. For this study, and AcΔglpdAc(EV) strains, respectively. The transfor- the Ac group II strain, KACC17005, whose genome infor- mants were selected on TSA with appropriate antibiotics mation has been fully sequenced (Park et al., 2017), was and then confirmed by PCR with the following pBBR1- used as the wild-type strain. Ac strains were grown in TSB MCS5-specific primers: 5′-CAGGGTTTTCCCAGTCAC- (tryptic soy broth, 30 g/l) with appropriate antibiotics at GA-3′ and 5′-ATGCTTCCGGCTCGTATGTT-3′. The 28°C. For generating the plasmid constructs, Escherichia generated plasmids and strains are described in Supple- 38 Kim et al. mentary Table 1. two punches were ground in 200 μl sterilized water using a tissue grinder. The leaf extracts were serially diluted (10- Prediction of 3D structure. To predict the putative 3D fold). The diluted suspensions were spotted onto TSA with structure