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 by high throughput sequencing.
1b. Approach (from AD-416)
1. Aphid transmission 2. Reverse transcription (RT) polymerase chain reaction (PCR) 3. Real time PCR (qPCR) 4. Cloning, sequencing 5. Western blots, northern blots, hybridizations 6. Small RNA deep sequencing 7. Bioinformatics
3. Progress Report
This report documents research conducted under a Specific Cooperative Agreement in support of Objective 1B of the parent project, 5302-22000-009-00D. The goal is to identify genes that regulate disease expression of citrus tristeza virus (CTV). This research contributes to development of control measures for CTV and examines the putative role of gene silencing in cross protection of stem pitting and/or seedling yellows disease expression. Small interfering (si) RNAs and micro (mi) RNAs obtained through deep sequencing were analyzed. Eight miRNA families (miR156, miR157, miR166, miR167, miR168, miR172, miR482 and miR3951) showed differential expression and were selected for further examination. Using a custom analyses pipeline developed for this project, target genes (UniGene ESTs of sweet orange for NCBI) were identified for these miRNAs in the cross-protected and non-cross protected plants. Four hundred thirty three miRNA targets were common in CTV-infected plants; 873 unique miRNA targets were found in cross protected sour orange plants; and 753 unique targets were found in sour orange plants showing strong CTV symptoms (no cross protection). These results suggest a reprogramming of miRNA occurs in response to CTV infection, one leading to cross protection and the other to no cross protection. Research activities were monitored by the lead scientist through email and telephone communication with the cooperator.