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

Abstract

Huanglongbing {HLB [associated with Candidatus Liberibacter sp. (CLas)]} and asiatic citrus canker {ACC [causal organism Xanthomonas citri ssp. citri (XCC)]} are bacterial diseases that seriously threaten sustainability of the Florida citrus (Citrus sp.) industry. Sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) are highly susceptible to ACC and improvement through conventional breeding is a long-term process, making transgenic solutions attractive. No strong HLB resistance has been identified within cultivated citrus scion types: creation of transgenic citrus that would permit economic citrus production where HLB is endemic is a high priority. 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) for 44 AMPs was conducted using Sinorhizobium meliloti and Agrobacterium tumefaciens as surrogates for the unculturable CLas because they are closely related alpha proteobacteria (class Alphaproteobacteria). XCC is also a gram-negative bacterium and was included in these analyses in anticipation that HLB and ACC resistance can be achieved with the same AMP transgene if expressed using non-tissue-specific promoters. Twenty AMPs from diverse sources were initially tested. AMPs with the lowest MICs included tachyplesin I from horseshoe crab (Tachypleus tridentatus), SMAP-29 from sheep (Ovis aries), D4E1 and D2A21 (which are synthetic AMPs derived through evaluation of critical amino acid residues in AMPs, overall peptide structure, and AMP effectiveness), the human (Homo sapiens) LL-37, and the honeybee (Apis mellifera) venom AMP melittin. These AMPs inhibited growth of all three test bacterial species at 1 μM or less. An additional 20 synthetic AMPs were designed based on structures of the most effective AMPs 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 × AMP interactions were observed. Hemolytic activity was assessed by exposure of porcine erythrocytes (from Sus scrofa) to the AMPs. Hemolysis from most AMPs was not significantly different from water, whereas melittin was highly hemolytic.