1a.Objectives (from AD-416)
1) Develop qPCR probes for the different Dekopon sub-isolates and quantify each in single and mixed infections to assess interference in accumulation/replication of each sub-isolate.
2) Examine P20, P23, and CP (suppressors of gene silencing) for each sub-isolate for genetic variation and associate with symptom expression.
3) Characterize siRNA profiles in mixed and single infected plants.
1b.Approach (from AD-416)
Aphid transmission, reverse transcription (RT) polymerase chain reaction (PCR), real time PCR (qPCR), cloning, sequencing, western blots, northern blots, hybridizations. Documents Reimbursable with Citrus Research Board. Log 37119.
This Trust Agreement supports Objective 2 of the parent project. Management of tristeza by cross-protection is achieved by preinfecting citrus with a mild CTV strain to prevent infection or reduce damage by a superinfecting severe citrus tristeza virus (CTV) strain. Cross-protection can fail after 10-20 years (sometimes much sooner) and citrus cultivars vary in response to cross-protection. The mechanism of cross-protection is unknown and identification of the mode of action could define characters that may be used as tools to select durable, cross-protective strains. Co-infection of citrus with mild and severe CTV strains was used to characterize the role of regulatory small interfering RNAs (siRNAs) in cross-protection using high throughput sequencing technology. Most viral-derived siRNAs were found associated with the conserved portion of the CTV genome (3’ region). Using a search algorithm to align short nucleotide sequences, “hot spots” of homologous sequences were found in the 282 Kb CTV resistance locus of Poncirus trifoliata and suggested this region is involved in the silencing (antiviral) pathways. Fifty micro (mi) RNAs families had significant occurrence and 8 miRNA families showed differential expression after CTV infection. Further analysis of these miRNAs showed putative messenger (m) RNA (chemical blueprint for protein products) targets in the citrus Expressed Sequence Tags (EST) database which were differentiated into groups:. 1)common to both cross-protected and non-protected sour orange plants;. 2)present only in cross-protected sour orange plants (no or mild symptoms);. 3)present only in sour orange with failed cross-protection (severe symptoms). These results suggested different CTV strains can induce different miRNA-mRNA interactions but whether this is in response to a specific virus strain or simply a host response to CTV is being further investigated. This research can lead to development of a biomarker to identify strains effective in CTV cross-protection for mitigation of losses due to CTV infection.