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.
3. Progress Report
This agreement was established in support of Objective 2 of the in-house project “Characterization and Epidemiology of Citrus Tristeza Virus and Other Invasive and Emerging Graft-Transmissible Diseases of Citrus in California”. Aphid transmission and strain differentiation of Citrus Tristeza Virus (CTV) by real-time polymerase chain reaction (PCR) assays were used to examine CTV symptom expression. This is the second year of a 3-year grant to investigate cross protection of CTV using a mild strain derived from aphid transmission to control CTV disease expression. A severe strain of CTV was characterized and found to contain a mixture of strains. Infection of test plants by a mixture of strains which included the aphid-transmitted mild strain, severity of seedling yellows (SY) symptoms was reduced or eliminated. A library of small RNAs from sour orange plants expressing SY vs. “protected” symptomless treatments were obtained and sequenced using the Illumina Genome Analyzer. Forty percent of the sequences were associated with the Poncirus trifoliata CTV resistance gene locus in both cases. The largest host-derived small RNA in this locus was from the Gypsy-like retrotransposon C. This retrotransposon is now being investigated to determine if it has a role in CTV resistance.