Genetically Modified Citrus Rootstocks Expressing a Synthetic Cationic Antimicrobial Peptide Slows Down Bacterial Infection in the Non-transgenic Scion

Synthetic cationic antimicrobial peptides with broad-spectrum antimicrobial activity have been developed that improve the molecular stability, antimicrobial activity and enhance disease resistance to several plant and animal pathogens. We expressed a gene encoding a synthetic chimera of the cecropin A and melittin antimicrobial peptides (AMP) into two citrus rootstocks. Twenty-two transgenic diploid Carrizo citrange and 14 complex tetraploid Orange#16 rootstock plants were regenerated expressing the cationic antimicrobial peptide. A conventional diploid sweet orange ‘Valencia’ scion was grafted onto these rootstocks and replicated trees were challenged for resistance to Huanglongbing (HLB), a fatal phloem-restricted disease of citrus caused by the Candidatus Liberibacter asiaticus (Clas) bacterium. Unbudded rootstock trees were also tested for their susceptibility to Clas. In greenhouse experiments, the rootstock trees (budded and unbudded) were exposed to free flying Clas infected Asian citrus psyllids. There were no significant differences in the infection rates between the unbudded transgenic trees and control trees, and all tested positive for HLB after a year of exposure. Grafted trees also tested positive for HLB, but the rate of infection was significantly slower than comparable non-transgenic rootstock-scion combinations under both greenhouse and field test conditions. A number of transgenic lines remained disease free for more than two years before testing positive for CLas using qPCR. These qPCR positive trees did not demonstrate any visual signs of disease even after three years in the field, and continued to grow and produce flowers and fruit. Differences among the various grafted transgenic trees and controls in the incidence of CLas infection did not correlate to gene expression patterns. Our results demonstrate the possibility of using a genetically modified rootstock to protect the aboveground scion against HLB infection.