Novel Plantibodies Show Promise to Protect Citrus from Greening Disease

Abstract

Candidatus Liberibacter asiaticus (CLas), the bacteria responsible for citrus greening disease [huanglongbing (HLB)], has become a worldwide threat to citrus (Citrus sp.) production. HLB has proven difficult to study and treat because of the complex interactions between CLas, the citrus host, and insect vectors. We have selected for single chain fragment variable (scFv) antibodies from a specialized bacteriophage library for binding activity against CLas proteins InvA and TolC. Portions of each protein were chosen as antigens based on predicted binding availability and theorized necessary functions in pathogenicity. Binding affinity for individual scFv-expressing clones was confirmed by phage enzyme-linked immunosorbent assay (ELISA). The scFv sequences were stably transformed under the control of a tandem Cauliflower mosaic virus 35S (CaMV 2x35S) promoter by Agrobacterium tumefacien–mediated transformation into ‘Carrizo’ citrange (Citrus sinensis × Poncirus trifoliate), a citrus rootstock cultivar. Replicated plants of single transformations were inoculated by infestation with CLas positive asian citrus psyllid (Diaphorina citri), a CLas vector. Inoculation and disease progression was monitored through quantitative real-time polymerase chain reaction. Inoculated transgenic plants showed significantly reduced CLas titer compared with wild types. A subpopulation of transgenic plants displayed no measurable surviving bacteria after 12 months. Interestingly, individual replicated plants from the same transgenic events strongly segregated into two populations by resistance phenotype: a minority that were indistinguishable from wild-type plants and a majority that were highly resistant. Our results are the first step in developing a novel protection strategy for HLB.