Transgenic Strategies for Huanglongbing-resistant Citrus at the USDA-ARS

Huanglongbing (HLB) seriously threatens the sustainability of the Florida citrus industry.  The predominate HLB pathogen is CandidatusLiberibacter asiaticus (Las), a phloem limited bacterium vectored by the Asian citrus psyllid. No HLB-resistance has been identified within cultivated citrus scions, making it a high priority to create transgenic citrus that would permit economic citrus production where HLB is endemic.  Several strategies are being explored to create practical transgenic citrus with HLB-resistance.  Single antimicrobial peptide (AMP) transgenics have had modest efficacy but transgenics expressing chimeral peptides, with a modified citrus thionin linked to an AMP are showing promise against citrus canker and are being challenged with Las.  Flagellin proteins from bacteria are often recognized by plant FLS2 receptors to elicit defense responses.  Las flagellin appears not to induce resistance responses in citrus but is recognized by other plants to trigger defense responses.  An FLS2 from a plant that recognizes Las flagellin is being expressed in citrus cultivars.  Las produces a LuxR protein required for quorum sensing, but not LuxI which provides the signal perceived by LuxR. We are expressing LuxI transgenically to see if early quorum sensing prevents systemic infection.  Efficacy of RNA interference has been demonstrated using a virus expression vector to produce dsRNA in citrus directed at psyllid genes encoding essential proteins. Transgenics producing these dsRNA are in early development. Small chain variable fragment antibodies have been designed against exterior epitopes of Las and numerous transgenics are now being analyzed for effects on Las survival and proliferation.  Recently, a family of Small Cyclic Amphipathic Peptides (SCAmpPs) has been demonstrated to be highly expressed in citrus phloem.  Even though the final predicted peptide sequences of diverse phloem SCAmpPs are quite variable, there is almost complete identity in the promoter, first exon and intron.  We are functionally dissecting  phloem SCAmpPs sequence components, analyzing expression of marker genes and ultimately resistance genes for control of phloem-limited Las.  A recombination/ exchange system is being implemented to control the integration and the excision of DNA allowing the precise integration of transgenes at specific sites, with the simultaneous removal of marker genes and/or any other unneeded sequences. This tool will permit future stacking of traits into transgenic cultivars and provides for efficient comparison of transgenic vector components to optimize construct development.