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):
Aphid transmission, reverse transcription (RT) polymerase chain reaction (PCR), real time PCR (qPCR), cloning, sequencing, western and northern blots, hybridizations, deep sequencing for small RNAs, bioinformatics. Documents SCA with INSTITUTO DI VIROLOGIA VEGETALE.
Results from this study are in support of Objective 1.B of the parent project. To study the mechanism of cross-protection in which a mild strain of Citrus tristeza virus (CTV) protects against virulent effects of a severe strain of the same virus, a natural plant defense system called RNA interference was studied. Initial stages of this defense involves inactivation of the virus by cleaving viral RNA into short sequences. During FY12, sequences of small viral RNAs in plants with mixtures of tristeza strains were mapped. Results showed specific sites on the virus genome which may be active if RNA interference was occurring. Profiles of small viral RNAs were analyzed and microRNAs differentially produced in asymptomatic versus symptomatic plants were identified. Since plant microRNAs modulate gene expression, association of specific microRNAs with disease symptoms can lead to; therapies to mitigate disease, use as biomarkers for early detection of CTV infection, and development of screening methods to evaluate CTV cross-protection. Cross-protection is a well-utilized virus control strategy but its mechanism is poorly understood. The present study is designed to provide new knowledge that may lead to more stable and predictable use of mild CTV strains to control losses due to severe tristeza strains.