Reverse genetics systems and replication studies of a Crinivirus, Lettuce infectious yellows virus (LIYV)

Lettuce infectious yellows virus (LIYV) has a bipartite, positive-sense ssRNA genome (ca 15.3 Kb) and is the type member of the genus Crinivirus in the family Closteroviridae. LIYV is phloem-limited in plants and is specifically transmitted to plants by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. It has not been transmitted to plants by mechanical inoculation. LIYV infections achieve only a low virus titer in plants and protoplasts, impeding reverse genetics efforts to analyze LIYV gene/protein functions. I showed that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, transgenic plants and protoplasts expressing the TuMV P1/HC-Pro sequence enhanced the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8 fold in these plants and protoplasts relative to control plants. By increasing LIYV titers, it will be easier to transmit more LIYV genotypes to plants for future reverse genetics experiments. In addition, LIYV infections of N. benthamiana plants were achieved by using Agrobacterium tumefaciens to deliver and express complete genomic cDNAs, thereby obviating the need for whitefly-mediated transmission. This is the first example of agroinoculation of any crinivirus to plants and offers great opportunities for further research. Agroinoculation and targeted mutagenesis technologies should greatly enhance LIYV reverse genetics studies, such as investigations of viral replication, recombination, trafficking, symptom elicitation and virus-vector interactions in planta. A series of LIYV RNA 1 mutants was created to study cis and trans determinants of LIYV genomic RNA replication. These were studied using tobacco protoplasts. Mutants DeltaEcoRI, DeltaE-LINK, and Delta1B, having deletions in open reading frames (ORFs) 1A and 1B, did not replicate when individually inoculated to protoplasts, nor when co-inoculated with wild-type RNA 1 as a helper virus. A fragment of the green fluorescent protein (GFP) gene was inserted into the RNA 1 ORF 2 (P34) in order to provide a unique sequence tag that would allow identification of this mutant, P34-GFP TAG. The P34-GFP TAG mutant was capable of independent replication in protoplasts. However, mutants derived from P34-GFP TAG having frameshift mutations in the ORF 1A or 1B were unable to replicate in protoplasts alone, or in trans when co-inoculated with wild-type RNA 1 as a helper virus. Taken together, these data strongly suggest that LIYV RNA 1 replication is cis-preferential. Finally, I examined the role of the LIYV RNA 1 ORF 2-encoded P34, a trans enhancer for RNA 2 accumulation, in viral infection. I found that P34 is a sequence non-specific ssRNA-binding protein in vitro. Furthermore, P34 bound to ssRNA in a cooperative manner, and the RNA-binding domain was mapped to the C-terminal region of P34. In addition, fusions of P34 to GFP showed a perinuclear localization in transfected protoplasts, which also is the putative site of LIYV replication. Together, these data support the hypothesis that P34 enhancement of RNA 2 replication may rely on binding to LIYV RNA 2.