Transgenic citrus strategies for control of the citrus root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae)

Diaprepes abbreviatus (L.) (DRW) is a major economic threat to the U.S. citrus industry since its arrival in the early 1960s and represents a worldwide threat as it spreads. Long-lived larvae cause the most damage that is exacerbated by pathogenic organisms entering plants through feeding sites. We propose a transgenic citrus rootstock approach of DRW control: manipulating citrus rootstocks to synthesize a set of molecules selectively toxic to DRW. A Bacillus thuringiensis toxin active against DRW- Cyt2Ca1, a cysteine proteinase inhibitor (CPI) and TMOF (a decapeptide trypsin proteinase inhibitor) are being evaluated as combinatorial control agents.  The Cyt2Ca1 protein was shown to provide resistance against larval feeding in alfalfa,a model plant system. Meanwhile, q-RT-PCR and western blot analysis of Cyt2Ca1-citrus cohorts, show high-expression of the recombinant gene and studies are on-going to investigate resistance to DRW. We show that TMOF alone can reduce DRW fitness but mortality is limited, presumably due to compensatory cathepsin activity.  Analyses of DRW cathepsin activity lead to the discovery of a CPI that reversibly binds DRW cathepsin L, to modulate activity. As a strategy to block DRW cathepsin activity, we have developed a mutant inhibitor hypothesized to bind irreversibly. Results of combined protease inhibitor and Cyt2Ca1 effect on DRW larvae survival will be presented.