Abstract
Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the US citrus industry. There are no proven strategies to eliminate HLB disease and no cultivar has been identified with strong HLB resistance. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. Since no HLB-resistant citrus cultivars have been identified, we attempted to develop citrus resistant to both HLB and citrus canker through overexpression of a modified plant thionin. To improve effectiveness for disease resistance, we modified and synthesized the sequence encoding a plant thionin and cloned into the binary vector pBinPlus/ARS. The construct was then introduced into Agrobacterium strain EHA105 for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were generated by Agrobacterium-mediated transformation. Successful transformation and transgene gene expression was confirmed by molecular analysis. Transgenic Carrizo plants expressing the modified thionin gene were challenged with X. citri 3213 at a range of concentrations, and a significant reduction in canker symptoms and a decrease in bacterial growth were demonstrated compared to nontransgenic plants. Furthermore, the transgenic citrus plants were challenged with HLB via graft inoculation. Our results showed significant Las titer reduction in roots of transgenic Carrizo compared with control plants and reduced scion Las titer 12 months after graft inoculation. These data provide promise for engineering citrus disease resistance against HLB and canker.
Highlights
Citrus greening (Huanglongbing, HLB) is considered to be the most devastating citrus disease worldwide (Bové, 2006)
In the C. sinensis genome, we identified one thionin homolog designed as CsthiF (KDO61571)
We showed that transgenic citrus overexpressing the Mthionin had enhanced disease resistance to citrus canker and greatly reduced X. citri growth
Summary
Citrus greening (Huanglongbing, HLB) is considered to be the most devastating citrus disease worldwide (Bové, 2006). The disease is mainly associated with Candidatus Liberibacter asiaticus (Las) transmitted by the Asian citrus psyllid (ACP), Diaphorina citri. Citrus production in Florida continues to decline, fruit. Thionin Confers Citrus Disease Resistance quality is diminished, and production costs have greatly increased. Las is the species associated with HLB-affected trees in North America and its genome sequence has been determined (Duan et al, 2009). No effective strategies to eliminate HLB disease and no HLBresistant citrus cultivars have been identified, though thermaltherapy, chemotherapy, and psyllid management may prolong production in infected trees (Hoffman et al, 2013; Stansly et al, 2014; Zhang et al, 2014). Citrus canker disease incited by Xanthomas citri imposes additional pressure on the Florida industry threatened by HLB (Gottwald, 2007)
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