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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Research Project #435718

Research Project: A Screen of Nodule-Specific Cysteine-Rich Peptides for Control of the HLB Bacterium in Citrus

Location: Emerging Pests and Pathogens Research

Project Number: 8062-22410-006-47-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Oct 1, 2018
End Date: Sep 30, 2019

Objective:
The citrus industry in the US has been crippled by the spread of Huanglongbing (HLB), a bacterial disease widely considered to be the worst disease of citrus. There is no effective control strategy and due to the extended asymptomatic period, HLB can spread throughout a grove long before it is recognized. The causal agent, ‘Candidatus Liberibacter asiaticus’ (CLas), is genetically related to the soil bacterium Sinorhizobium meliloti, an important nitrogen-fixing plant symbiont. S. meliloti infects the roots of leguminous crop plants, triggering the formation of specialized plant nodules that are colonized by the bacteria. Within these organs, the plant expresses hundreds of nodule-specific cysteine-rich peptides (NCRs) that halt S. meliloti multiplication and transforms the cells into the symbiotic form called the bacteroid. The objective of this research is to test whether NCRs have antimicrobial activity against CLas.

Approach:
Objective 1 – Bioinformatics analysis and NCR peptide library synthesis (2 months). We propose to work with a commercial specialist in high-throughput peptide synthesis to synthesize NCRs from M. truncatula. There are 604 M. truncatula proteins annotated as NCRs in the UniProt database. Sequence analysis indicates that these peptides range from 16 to 924 residues in length, with the majority being 90 residues or smaller. We will use bioinformatics to analyze these NCR sequences and create a prioritized list for peptide synthesis and screening. Secretion signals, which are not required for antimicrobial activity, will be removed prior to synthesis. Objective 2 – Screen candidate antimicrobial peptides for bactericidal activity against CLas (16 months). We propose to implement a peptide screening assay designed to evaluate therapeutic agents for their efficacy in the control of CLas. We will first validate two assays which are under development by our USDA ARS collaborators, and we will proceed to screen our NCRs with the preferred bioassay based on data from both screens’ effectiveness and efficiency with positive and negative control samples.