Attacin A Gene from Tricloplusia ni Reduces Susceptibility to Xanthomonas axonopodis pv. citri in Transgenic Citrus sinensis `Hamlin'

Abstract

<jats:p>Citrus canker, caused by <jats:italic>Xanthomonas axonopodis</jats:italic> Starr and Garces pv. <jats:italic>citri</jats:italic> (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic <jats:italic>Citrus sinensis</jats:italic> (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived <jats:italic>attacin</jats:italic> A gene (<jats:italic>att</jats:italic>A) were obtained by <jats:italic>Agrobacterium tumefaciens</jats:italic> (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pC<jats:italic>att</jats:italic>A 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (<jats:italic>Allium cepa</jats:italic> L.) epidermal cells were transformed, via biobalistics, using plasmids containing the <jats:italic>att</jats:italic>A gene with or without SP, fused with the green fluorescent protein gene (p<jats:italic>att</jats:italic>A 1303 and pSP<jats:italic>att</jats:italic>A 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of <jats:italic>att</jats:italic>A gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 10<jats:sup>6</jats:sup> cfu/mL suspension of the pathogen <jats:italic>X. axonopodis</jats:italic> pv. <jats:italic>citri</jats:italic>. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.</jats:p>

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp. citri

“…The gene construct utilized for genetic transformation consisted of the pCattA 2300 plasmid binary vector containing the 35S-35S-attA-35ST expression cassette that codes for the attacin A antimicrobial peptide and a native signal peptide responsible for directing the insect protein to the extracellular space (Boscariol et al 2006). This expression cassette was cloned into pCambia 2300 binary vector (CAMBIA, Canberra, Australia), which carries the nptII selection gene, and introduced into Agrobacterium tumefaciens EHA 105 by the freeze-thaw method (Holsters et al 1978).…”

“…The protocols for A. tumefaciens cultivation, explant selection and preparation, and genetic transformation have been previously described (Mendes et al 2002;Almeida et al 2003). Adventitious buds regenerated were analyzed by polymerase chain reaction (PCR) to identify putative transgenic plants (Boscariol et al 2006) and in vitro grafted onto Carrizo citrange (C. sinensis×Poncirus trifoliata). The developed plants were acclimatized in a greenhouse (28± 2°C, 80±10% RU).…”

“…Specific research carried out in citrus revealed a reduction of 55-60% in ACC disease severity in attA Hamlin sweet orange transgenic plants (Citrus sinensis L. Osbeck), a cultivar highly susceptible to Xcc (Boscariol et al 2006). These promising results led us to hypothesize how transgenic plants of other sweet orange cultivars carrying attA gene would behave against ACC pathogen.…”

“…The gene that codes for this peptide has 1,601 bp with two introns and a signal peptide (Kang et al 1996). As it occurs in insect cells, the antimicrobial peptide is secreted to the apoplast, outside of the plant cell (Boscariol et al 2006).…”

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

“…Since the first report of citrus genetic transformation (Kobayashi and Uchimiya 1989), transgenes have been introduced in many citrus cultivars (Peña et al 2001, Boscariol et al 2006). Among the rootstock cultivars, transgenic plants can easily be obtained for Carrizo citrange (Peña et al 1995, Yu et al 2002, however, other rootstock cultivars, such as C. limonia, C. volkameriana, and C. aurantium, have shown to be recalcitrant (Gutiérrez.…”