Stable integration and expression of a plant defensin in tomato confers resistance to fusarium wilt

Abstract

Plant defensins are small cysteine-rich peptides which belong to a group of pathogenasis related defense mechanism proteins. The proteins inhibit the growth of a broad range of microbes and are highly stable under extreme environmental stresses. Tomato cultivation is affected by fungal disease such as Fusarium wilt. In order to overcome fungal damages, transgenic tomato plants expressing the Medicago sativa defensin gene MsDef1 under the control of the CaMV 35S promoter were developed. The Fusarium-susceptible tomato (Lycobersicum esculentum Mill) cultivar CastleRock was used for transformation to acquire fungal resistance. Hypocotyl with a part of cotyledon (hypocotyledonary) for young tomato seedlings were used as an explant material and transformation was performed using the biolistic delivery system. Bombarded shoots were selected on regeneration medium supplemented with hygromycin and suitable concentrations of BA, zeatin ripozide and AgNO(3). Putative transgenic plantlets of T(0) were confirmed by PCR analysis using primers specific for the transgene and the transformation frequency obtained was 52.3%. Transformation and transcription of transgenes were confirmed in T(1) by PCR, Southern hybridizations, and reverse-transcription PCR (RT-PCR). The copy numbers of integrated transgene into tomato genome ranged between 1-3 copies. Greenhouse bioassay was performed on the transgenic T(1) and T(2) young seedlings and non-transgenic controls by challenging with a vigorous isolate of the fungal pathogen Fusarium oxysporum f. sp. Lycopersici. The level of fungal infectivity was determined using RT-PCR with tomatinase specific primers. Transgenic lines were more resistant to infection by fusarium than the control plants. These results indicated that overexpressing defensins in transgenic plants confer resistance to fungal pathogens.

Insect antimicrobial peptides and their applications

“…Plant defensins have been expressed in transgenic rice (Choi et al, 2009; Jha and Chattoo, 2010), wheat (Li et al, 2011), banana (Ghag et al, 2012), tomato (Abdallah et al, 2010; Portieles et al, 2010), “Egusi” melon (Ntui et al, 2010), peanut (Swathi Anuradha et al, 2008), tobacco (Portieles et al, 2010; Swathi Anuradha et al, 2008), and Arabidopsis (Kaur et al, 2012). Transgenic expression of an insect defensin ( G. mellonella gallerimycin) and cecropin (sarcotoxin-IA) in tobacco also confers resistance to pathogenic fungi (Mitsuhara et al, 2000; Ohshima et al, 1999).…”

Management of tomato diseases caused by Fusarium oxysporum

“…Be that as it may, progress has been made in engineering tomato for resistance to Fol especially through transformation with genes derived from other plant species. Transgenic tomato plants expressing pathogenesis-related defense protein (defensin) genes from Medicago sativa, and Wasabia japonica were more resistant to Fol than non-transformants (Abdallah et al, 2010;Khan et al, 2011). Transgenic tomato lines expressing a high level of another pathogen related protein, chitinase, from wheat were found to be highly resistant to Fol and the trait was inheritable by the T 1 and T 2 generations (Girhepuje and Shinde, 2011).…”

Overexpression of a Defensin Enhances Resistance to a Fruit-Specific Anthracnose Fungus in Pepper

“…Thus, it is necessary to build up novel genetic resources for the development of anthracnose disease-resistant peppers, and genetic engineering is a feasible approach to generate anthracnose-resistant pepper plants [32]. Although constitutive expression of plant defensins has shown enhanced resistance in tomato, potato, and canola against various pathogens [33]–[37], anthracnose disease-resistant plants have not yet been developed.…”