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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #431779

Research Project: Design and Delivery of Therapeutic Proteins for HLB Protection

Location: Crop Improvement and Genetics Research

Project Number: 2030-21220-002-01-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Feb 1, 2016
End Date: Jan 31, 2019

Objective:
Peptides and proteins from the citrus host and the bacterial pathogen will be screened to identify targets suitable for disrupting the development of haunglongbing and protecting otherwise susceptible citrus. HLB-protective proteins that either clear CLas or block disease development will be designed and delivered. The project includes short-, medium- and long-term delivery components to protect existing trees and develop enduring resistance in improved cultivars for replanting.

Approach:
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 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 CLas genes and citrus genes in tissue from infected citrus trees to identify the list of possible candidate citrus-CLas proteins that may interact. HLB blocking therapeutic proteins will be designed based the knowledge of the Liberibacter-citrus protein-protein interactions critical to the onset and progression of HLB. 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. We will be involved in developing improved molecular tools, construction of identified priority transgenes, and development of recombinase stacking technology for huanglongbing resistance.