Attacin A gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis 'Hamlin'

Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (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 Citrus sinensis (L.) Osb. 'Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 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 (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 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 attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 10(6) cfu/mL suspension of the pathogen X. axonopodis pv. citri. 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.