Screening Antimicrobial Peptides in-Vitro for Use in Developing Huanglongbing and Citrus Canker Resistant Transgenic Citrus

Huanglongbing (HLB, associated with Candidatus Liberibacter sp.) and Asiatic citrus canker (ACC, causal organism Xanthomonas citri subsp. citri (Xcc)) seriously threaten the sustainability of the Florida citrus industry.  No HLB-resistance has been identified within cultivated citrus, making it a high priority to create transgenic citrus that would permit economic citrus production where HLB is endemic.  Little is known about the HLB pathosystem and thus broad spectrum antimicrobial peptides (AMPs) have been the focus for current work, and identification of safe and effective transgenes is essential to our efforts. In vitro assessment of minimum inhibitory concentration (MIC) was conducted using Sinorhizobium meliloti and Agrobacterium tumefaciens as surrogates for the uncultivable Liberibacter as they are closely related alpha proteobacteria.  Xcc is also a gram negative bacterium and was included in these analyses, in the hope that HLB and ACC resistance can be achieved with the same AMP transgene, if expressed using non-tissue-specific promoters. Nineteen AMPs were initially tested from diverse sources.  The  most active AMPs included Tachyplesin 1 from horseshoe crab, SMAP-29 from sheep and D4E1 (which is a synthetic AMP derived through evaluation of critical amino acid residues in AMPs, overall peptide structure, and AMP effectiveness).  These AMPs inhibited growth of all three test bacterial species at 1 µM or less. A further 20 synthetic AMPs were designed based on initial results and seven of these showed effectiveness at 1 µM or less across all three test bacteria.  Most AMPs were comparable in effectiveness across the three bacterial species, but some species x AMP interactions were observed. Hemolytic activity was assessed by exposure of porcine erythrocytes to a range of AMP concentrations during incubation at 38.5 °C for 30 min.  Contrasting hemolysis of AMPs at 10 µM revealed that most were not significantly different from water, while melittin (from bee venom) was highly hemolytic as expected.   AMPs which suppress bacterial growth at 1 µM or less and show negligible hemolysis are the focus for further work, including production of transgenic citrus to express resistance to HLB and ACC.