RESUMEN
An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar 'Florex Gold') is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l(-1) kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.
Asunto(s)
Ferredoxinas/genética , Enfermedades de las Plantas/genética , Zantedeschia/genética , Agrobacterium tumefaciens/genética , Southern Blotting , Western Blotting , ADN de Plantas/análisis , ADN de Plantas/genética , Ferredoxinas/metabolismo , Pectobacterium carotovorum/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Transformación Genética , Zantedeschia/metabolismo , Zantedeschia/microbiologíaRESUMEN
Agrobacterium tumefaciens causes crown gall disease in a wide range of plants by transforming plants through the transfer and integration of its transferred DNA (T-DNA) into the host genome. In the present study, we used two-dimensional gel electrophoresis to examine the protein expression profiles of A. tumefaciens in response to the phenolic compound acetosyringone (AS), a known plant-released virulence (vir) gene inducer. Using mass spectrometry, we identified 11 proteins consisting of 9 known AS-induced Vir proteins and 2 newly discovered AS-induced proteins, an unknown protein Y4mC (Atu6162) and a small heat shock protein HspL (Atu3887). Further expression analysis revealed that the AS-induced expression of Y4mC and HspL is regulated by the VirA/VirG two-component system. This report presents the first proteomics study successfully identifying both known and new AS-induced proteins that are implicated in Agrobacterium virulence.
