DESIGN AND DELIVERY OF THERAPEUTIC PROTEINS FOR HUANGLONGBING (HLB) PROTECTION

We will design two types of protein therapeutics: (1) anti-Liberibacter protein chimeras that consist of Liberibacter recognition and lysis domains from the innate immune repertoire of citrus proteins and (2) protein inhibitors that block specific Liberibacter-citrus protein-protein interactions critical in the onset and progression. For the protein chimeras, first we need to identify the molecular entities on the Liberibacter membrane that can be targeted by citrus protein. We will perform structural and computational analysis to identify the Liberibacter recognition and lysis domains. For the protein inhibitors of huanglongbing (HLB), we will first identify the critical protein-protein interactions in Liberibacter-citrus interactions, for example the ones in the signaling pathways involving pathogen-associated molecular pattern triggered (PTI) and effector triggered signaling. Identification of Liberibacter-citrus protein-protein interactions will use three different methods: Computational prediction by supervised and unsupervised learning algorithms; Coordinated expression analysis of Liberibacter-citrus gene and protein pairs; and Validation using split-GFP technique. We will study expression of both Candidatus Liberibacter asiaticus (CLas) genes and citrus genes in tissue from infected citrus trees to identify the list of possible candidate citrus-CLas proteins that may interact. Huanglongbing blocking therapeutic proteins will be designed based the knowledge of the Liberibacter-citrus protein-protein interactions critical to the onset and progression of huanglongbing. We have developed a structure-based method to design a chimera starting from selected pathogen recognition and lysis domains. Briefly, first we will connect Liberibacter recognition and lysis domains by a flexible linker (e.g., a 7-amino acid segment GSTAPPA). Second, we will perform molecular dynamic and energy-minimization to identify stable structures that retain the individual functions of the recognition and lysis domains. Third, we will confirm that the chimera shows synergy in its in vitro antimicrobial activity, i.e., more active than the sum of the activities of individual domains. The Stover lab will be involved in developing improved transformation tools, transforming citrus with identified priority genes, screening for transgene expression, and assessing huanglongbing resistance.