Production of Transgenic Citrus Resistant to Citrus Canker and Huanglongbing Diseases

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 cultivars identified with strong HLB resistance. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). As part of the USDA citrus breeding program we are trying to develop citrus resistant to both HLB and citrus canker through expression of antimicrobial peptides (AMPs). In grape, enhanced Xylella fastidiosa resistance was reported from expression of a chimeral AMP-peptide (comprised of an AMP and a peptide with high bacterial membrane affinity) vs. the AMP alone.  Our study is being conducted with the AMP D4E1, a plant thinion, and a chimeric AMP composed of D4E1 linked to thionin. We generated transgenic Carrizo and Hamlin with each of these constructs through Agrobacterium-mediated transformation. So far we have obtained verified transformants of 26 Carrizo and 10 Hamlin with chimeric AMP insertion, 28 Carrizo and 10 Hamlin with thionin, and 20 Carrizo and 20 Hamlin with D4E1 insertion.  Gene expression was confirmed by reverse transcriptase PCR (RT-PCR) in some transgenic lines. Transgenic Carrizo lines containing these constructs were infiltrated with X. citri strain 3213. Several transgenic lines expressing the chimeric AMP / thinion showed marked canker resistance. Through 30 days after infiltration, some transformants have shown no canker symptoms with low but detectable levels of X. citri. These promising transgenic plants have been replicated and will be tested for HLB resistance.