Role of Defective RNAs in Citrus Tristeza Virus Diseases

Abstract

This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.