Research Project: Transcriptome and Small Ribonucleic Acid (RNA) Profiling Studies in the Asian Citrus Psyllid

Location: Subtropical Insects and Horticulture Research

Project Number: 6034-22320-004-016-N
Project Type: Non-Funded Cooperative Agreement

Start Date: May 1, 2015
End Date: Apr 30, 2020

Objective:
Matched Proteome and Transcriptome (RNAseq) Analysis of Asian Citrus Psyllid (ACP) Digestive tract and Salivary Glands in Asian citrus psyllid (ACP) with and without the Huanglongbing-Causing Candidatus Liberibacter asiaticus bacterium. Goals: 1. Provide an understanding of genes important in digestive and salivation processes that could be interdiction points for psyllid control. 2. Differential gene expression as a result of the presence of Candidatus Liberibacter asiaticus (CLas) would provide information about molecular interactions and insect perception of the bacterium. 3. Could provide huanglongbing (HLB) resistance strategy in 3-4 years. 4. Implications for control strategies that are applicable across a broad range of insects similar to the psyllid that are major factors effecting global crop production. II. Small Ribonucleic acid (RNA) Micro RNA) Analysis of Asian Citrus Psyllids (ACP) with and without the Huanglongbing-Causing Candidatus Liberibacter asiaticus (CLas) Bacterium. Goals: 1. Provide an understanding of micro Ribonucleic acid (miRNA) profiles and relationship to important genes within the Asian citrus psyllid. 2. Showing involvement of micro Ribonucleic acid (miRNAs) in disease development/pathogen transmission would be a significant scientific advancement in our understanding of the huanglongbing disease complex. 3. Micro Ribonucleic acid therapy could be rapidly commercialized, if their nvolvement in citrus greening is proven.

Approach:
I. Matched Proteome and Transcriptome (Ribonucleic acid seq) Analysis of Asian Citrus Psyllid (ACP) Digestive tract and Salivary Glands in Asian citrus psyllid (ACP) with and without the Huanglongbing-Causing Candidatus Liberibacter asiaticus bacterium. 1. Develop 12 Ribonucleic acid (RNAseq) libraries from Polymerase chain reaction (PCR) positive and Polymerase chain reaction (PCR) negative Asian citrus psyllid (ACP) (six from each, three from each of digestive tract and salivary gland libraries). 2. Conduct Illumina Ribonucleic acid (RNAseq) analysis on these libraries (single run 50bp). 3. Provide Bioinformatic support to assemble reads onto a reference gene list developed from other psyllid Ribonucleic acid (RNAseq), Expressed sequence tag (EST) and genomic data. 4. Identify genes and proteins that are differentially regulated in response to the presence of Candidatus Liberibacer asiaticus within the Asian citrus psyllid. 5. Use this information to develop testable Ribonucleic acid interference (RNAi) based gene knockout experiments to study the effect of these knockouts on Candidatus Liberibacter asiaticus (CLas) acquisition by the Candidatus Liberibacter asiaticus. II. Small micro Ribonucleic acid (miRNA) Analysis of Asian Citrus Psyllids (ACP) with and without the Huanglongbing-Causing Candidatus Liberibacter asiaticus (CLas) Bacterium. 1. We have already conducted sequence analysis of two small Ribonucleic acid libraries. Vaccine and Gene Therapy Institute of Florida (VGTI) will provide bioinformatic analysis of these libraries to determine their quality, diversity and proof-of-concept to show that micro Ribonucleic acid (miRNAs) of plant origin can be identified from psyllids as a result of ingestion of these miRNAs. 2. If analysis of existing sequence data looks promising, Vaccine and Gene Therapy Institute of Florida will Conduct Illumina micro Ribonuclic acid sequencing on five other small Ribonucleic acid libraries. 3. Provide Bioinformatic support to assemble reads onto a reference gene list developed from other psyllid Ribonucleic acid seq, Expressed sequence tag (EST) and genomic data. 4. Identify differentially expressed micro Ribonuclic acid (miRNAs) in the psyllid as a result of the presence of Candidater Liberibacter asiaticus and associate these differences with information about the miRNA species origin (i.e. psyllid or citrus) and gene origin (to which gene and metabolic process does the miRNA belong). 5. Use the above information to develop testable hypotheses about the involvement of specific micro Ribonucleic acid (miRNAs) in disease development and psyllid transmission of Candidatus Liberibacter asiaticus (CLas). 6. Develop therapies targeting specific Micro Ribonucleic acid.