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.
Results from this study are in support of objective 3A of the parent project. Viral cross protection is a natural plant defense system where a mild virus strain prevents subsequent infection of a second, more severe strain of the same virus. The mechanism of cross-protection is unknown, however, one possible mechanism may be RNA interference. During FY12, cross-protection of severe strains of Citrus Tristeza Virus was examined in citrus plants infected with a mixture of two strains (one mild and the other severe). Since RNA interference involves inactivation of the virus by cleaving viral RNA into short segments (21-28 nucleotides), a study was conducted to characterize small viral RNAs isolated from plants in cross-protection tests. Infected, but asymptomatic plants (protected) contained predominately small viral RNAs from the severe strain. In contrast, infected plants with severe symptoms (non-protected) had prominent small viral RNAs from the mild strain. The data supports the hypothesis that cross-protection occurs when the plant host RNA interference system targets the RNA from the severe strain viral for degradation.