Dissecting Novel Grapevine-Mealybug-Virus Interactions

The biological mechanisms underlying vector transmission of grapevine leafroll-associated virus 3 (GLRaV-3) remain poorly understood due to limitations of a technically challenging host- pathogen system in Vitis vinifera. GLRaV-3 was able to infect the model organism Nicotiana benthamiana by insect-vector mediated transmission using the vine mealybug, Planococcus ficus. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy (TEM) observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed identical sequences. High throughput sequencing was used to compare host response to GLRaV-3 infection between V. vinifera and N. benthamiana. General families of differentially expressed genes (DEGs) common in both hosts followed similar expression changes with six upregulated, seven downregulated, and two stably expressed genes in common. Overall, both hosts have many DEGs unique to each host as well as responses in common to GLRaV-3 infection. The vine mealybug, Planococcus ficus, fed through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana, and transmitted the virus to test plants.An immunofluorescence approach was used to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3 transmission by P. ficus can be disrupted. Our results indicate that our membrane feeding system and transmission blocking assays are a valid approach and can be used to screen other candidate blocking molecules. GLRaV-3 continues to impact grape-growing regions worldwide and the lack of knowledge surrounding virus-vector interactions remains limiting to the field. Elucidating the transmission biology of this important virus contributes to the eventual goal of blocking of transmission in insect vectors and the development of improved control strategies in vineyards