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Modes of Action of a Bi-Domain Plant Defensin Mtdef5 Against a Bacterial Pathogen Xanthomonas Campestris

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Modes of Action of a Bi-Domain Plant Defensin Mtdef5 Against a Bacterial Pathogen Xanthomonas Campestris fmicb-09-00934 May 14, 2018 Time: 15:47 # 1 ORIGINAL RESEARCH published: 16 May 2018 doi: 10.3389/fmicb.2018.00934 Modes of Action of a Bi-domain Plant Defensin MtDef5 Against a Bacterial Pathogen Xanthomonas campestris Siva L. S. Velivelli1, Kazi T. Islam1†, Eric Hobson1,2 and Dilip M. Shah1* 1 Donald Danforth Plant Science Center, St. Louis, MO, United States, 2 Department of Biology, Jackson State University, Jackson, MS, United States Defensins are small cysteine-rich endogenous host defense peptides expressed in all higher plants. They are thought to be important players in the defense arsenal of plants against fungal and oomycete pathogens. However, little is known regarding the antibacterial activity of these peptides. The genome of the model legume Medicago truncatula contains 63 genes each encoding a defensin with a tetradisulfide array. Edited by: A unique bi-domain defensin, designated MtDef5, was recently characterized for Santi M. Mandal, its potent broad-spectrum antifungal activity. This 107-amino acid defensin contains Indian Institute of Technology two domains, 50 amino acids each, linked by a short peptide APKKVEP. Here, we Kharagpur, India characterize antibacterial activity of this defensin and its two domains, MtDef5A and Reviewed by: Muhammad Saleem, MtDef5B, against two economically important plant bacterial pathogens, Gram-negative University of Kentucky, United States Xanthomonas campestris and Gram-positive Clavibacter michiganensis. MtDef5 inhibits Esther Orozco, Centro de Investigación y de Estudios the growth of X. campestris, but not C. michiganensis, at micromolar concentrations. Avanzados del IPN, Mexico MtDef5B, but not MtDef5A, exhibits more potent antibacterial activity than its parent *Correspondence: MtDef5. MtDef5 and each of its two domains induce distinct morphological changes Dilip M. Shah and cell death in X. campestris. They permeabilize the bacterial plasma membrane and [email protected] translocate across membranes to the cytoplasm. They bind to negatively charged DNA †Present address: Kazi T. Islam, indicating these peptides may kill bacterial cells by inhibiting DNA synthesis and/or Monsanto Company, Mason, MI, transcription. The cationic amino acids present in the two g-core motifs of MtDef5 United States that were previously shown to be important for its antifungal activity are also important Specialty section: for its antibacterial activity. MtDef5 and its more potent single domain MtDef5B have This article was submitted to the potential to be deployed as antibacterial agents for control of a Xanthomonas wilt Antimicrobials, Resistance and Chemotherapy, disease in transgenic crops. a section of the journal Keywords: antimicrobial peptides, antibacterial defensin, membrane permeabilization, Xanthomonas campestris, Frontiers in Microbiology Clavibacter michiganensis, Medicago truncatula Received: 13 December 2017 Accepted: 23 April 2018 Published: 16 May 2018 INTRODUCTION Citation: Velivelli SLS, Islam KT, Hobson E Plants possess a sophisticated innate immune system to counter pathogenic attack. They produce and Shah DM (2018) Modes several antimicrobial peptide (AMP) families via their canonical ribosomal gene expression of Action of a Bi-domain Plant machinery (Goyal and Mattoo, 2014; Breen et al., 2015; Tavormina et al., 2015). Defensins Defensin MtDef5 Against a Bacterial Pathogen Xanthomonas campestris. are small cationic cysteine-rich AMPs that are ubiquitous in higher plants and contribute Front. Microbiol. 9:934. to their innate immunity. Genes encoding antifungal defensins have been widely used for doi: 10.3389/fmicb.2018.00934 engineering broad-spectrum resistance to fungal pathogens in transgenic crops (Kaur et al., 2011; Frontiers in Microbiology| www.frontiersin.org 1 May 2018| Volume 9| Article 934 fmicb-09-00934 May 14, 2018 Time: 15:47 # 2 Velivelli et al. Antibacterial Activity of a Bi-domain Plant Defensin De Coninck et al., 2013). Based on their predicted subcellular X. campestris, but induce different morphological changes and localization, plant defensins are designated as class I or class bind to negatively charged DNA in vitro. Using site-directed II. Class I defensins are synthesized as precursor proteins mutagenesis, we demonstrate that cationic amino acid residues comprising the secretory signal peptide and the mature domain. present in each of the two g-core motifs of MtDef5 are critical for The precursor defensins enter the secretory pathway where they its antibacterial activity. are proteolytically processed into active mature peptides and released into apoplast. Class II defensins are synthesized as larger precursors containing an additional carboxy-terminal propeptide MATERIALS AND METHODS sequence of 27–33 amino acids and are targeted to the vacuole (Lay et al., 2014). The three-dimensional protein structure of Bacterial Strains and Growth Conditions plant defensins is highly conserved and comprises one a-helix The Gram-positive pathogenic bacterial strain, Clavibacter and three antiparallel b-strands that are internally stabilized by michiganensis subsp. Nebraskensis strain CIC 395 (hereafter four disulfide cross-links. Despite their structural conservation, referred to as C. michiganensis) and the model Gram- plant defensins are diverse in their amino acid sequences. The negative pathogenic bacterial strain, Xanthomonas campestris sequence diversity contributes to a variety of biological functions pv. campestris 8004 (hereafter referred to as X. campestris), attributed to these peptides (Carvalho Ade and Gomes, 2009; were kindly provided by Dr. Dean Malvick of the University of Sagaram et al., 2013; Vriens et al., 2014). Minnesota, MN and Dr. Rebecca Bart of the Donald Danforth Past studies on plant defensins have primarily focused on Plant Science Center, St. Louis, MO, United States, respectively. their antifungal activity, with relatively few studies addressing These two bacterial pathogens are economically important their antibacterial activity. These peptides have been extensively pathogens of plants (Mansfield et al., 2012; Saleem et al., 2017). characterized for their ability to inhibit the growth of fungal C. michiganensis and X. campestris were routinely grown at ◦ and oomycete pathogens in vitro and in plants (Kaur et al., 28 ± 2 C for 2–3 days on nutrient broth-yeast extract agar 2011; De Coninck et al., 2013; Cools et al., 2017; Parisi (NBY, 8 g/l nutrient broth, 2 g/l yeast extract, 2.5 g/l glucose et al., 2018). However, the modes of action (MOA) of only and 2 g/l K2HPO4, 0.5 g/l KH2PO4, 1 mM MgSO4, 15 g/l − a few antifungal defensins have been studied in detail (Cools agar) containing cycloheximide (75 mg ml 1) and nutrient- et al., 2017; Parisi et al., 2018). Very few defensins with yeast extract glycerol agar (NYGA, 5 g/l bacto peptone, 3 g/l antibacterial activity have been reported (van der Weerden and yeast extract, 20 ml glycerol, 10 g/l agar) containing rifampicin − Anderson, 2013). For example, Cp-thionin from cowpea (Franco (100 mg ml 1), respectively. et al., 2006; Kraszewska et al., 2016), DmAMP1 from Dahlia merckii, CtAMP1 from Clitoria ternatea, AhAMP1 from Aesculus Expression and Purification of Defensins hippocastanum (Osborn et al., 1995), ZmESR-6 from maize The chemically synthesized MtDef5A with four disulfide (Balandin et al., 2005), fabatin-2 from broad bean (Zhang and bonds was obtained from JPT Peptide Technologies (Berlin, Lewis, 1997; Kraszewska et al., 2016), and SOD1-7 from spinach Germany). MtDef5, MtDef5B, and MtDef5_V1 (MtDef5 H36A, (Segura et al., 1998) have been reported to exhibit antibacterial R37A/H93A, R94A) were generated by recombinant expression activity against a range of Gram-positive and Gram-negative in Pichia pastoris and purified using the CM-Sephadex C-25 bacterial pathogens. Among antibacterial defensins, only SOD2 cation-exchange chromatography as described previously (Islam from spinach has been demonstrated to confer resistance to et al., 2017). Asiatic citrus canker (ACC) and Huanglongbing (HLB) caused by Xanthomonas citri ssp. citri and Candidatus Liberibacter sp., Antibacterial Assay respectively, in transgenic citrus (Stover et al., 2013). The MOA The antibacterial activity of MtDef5, MtDef5_V1, MtDef5A, of antibacterial plant defensins have yet to be deciphered in detail. and MtDef5B against X. campestris and C. michiganensis was Medicago truncatula genome contains 63 genes each encoding determined as described previously with minor modifications a defensin with four disulfide bonds (Maróti et al., 2015). One of (Balandin et al., 2005; Farkas et al., 2017). A single colony of these genes encodes a bi-domain defensin MtDef5 containing two each bacterial strain was inoculated into NBY and NYG broth, defensin domains, 50 amino acids each, connected by a 7-amino respectively, and grown overnight at 28 ± 2◦C on a rotary acid linker sequence APKKVEP. This defensin is predicted to be shaker at 225 rpm. Bacterial cells were diluted to 2 × 104 cfu/ml targeted to the apoplast. Recently, we have reported the potent in 2X NBY and NYG broth, respectively. The antibacterial antifungal activity and MOA of MtDef5 (Islam et al., 2017). Here, assay was performed in a 0.5 ml eppendorf tube, in which we report the antibacterial activity and MOA of this defensin and 50 ml of 2 × 104 cfu/ml bacterial inoculum corresponding to its two domains, MtDef5A and MtDef5B. We show that MtDef5 a final test concentration of 1 × 104 cfu/ml was incubated inhibits the growth of the Gram-negative bacterial pathogen
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