Genome Diversity and Intra- and Interspecies Recombination Events in <italic>Grapevine fanleaf virus</italic>

Mekuria, T. A., Gutha, L. R., Martin, R. R., and Naidu, R. A. 2009. Genome diversity and intraand interspecies recombination events in Grapevine fanleaf virus. Phytopathology 99:1394-1402. Grapevine fanleaf virus (GFLV) was documented in self-rooted vines of four grapevine (Vitis vinifera) cultivars in eastern Washington. GFLV was found as mixed infection in cvs. Pinot Noir, Chardonnay, and Cabernet Franc and as single infections in cv. Merlot. Fanleaf disease symptoms were only observed in the first two cultivars. The spatial distribution of GFLV-infected grapevines was random, suggesting primary spread through planting virus-infected cuttings rather than infield transmission. RNA1 sequences of Washington isolates showed 87 to 89% nucleotide sequence identity between them and with strain F13. RNA2 of Washington isolates was variable in size, showing 85 to 99% sequence identity between them and 81 to 92% with other isolates. As in other GFLV isolates, three conserved putative stem-loop structures were present in the 5′ noncoding regions of both RNAs of Washington isolates. Phylogenetic incongruence of GFLV isolates from Washington in 2AHPand 2BMP-based trees and identification of putative recombination events suggested that their genomic RNA2 originated from interand intraspecies recombination events between GFLV, Grapevine deformation virus, and Arabis mosaic virus. These results confirm interspecies recombination in RNA2 of grapevine-infecting nepoviruses as an important strategy for GFLV evolution. Grapevine fanleaf disease is a devastating virus disease of grapevine (Vitis spp.) in many grape-growing areas around the world (1). The virus causes a wide range of symptoms that include reduced vigor and general decline of vines; malformation of leaves, canes, and berries; and reduced yields due to poor berry set. The reduction in yield varies depending on the severity of symptoms and grape cultivar but >80% reduction has been recorded (7). Foliar symptoms produced by the disease are highly variable depending on cultivar and seasonal influences, and include fan-like distortions of leaves, ringspots, line patterns, vein banding, yellowish mottling, and mosaic in different cultivars. Infected grapevines exhibit foliar symptoms early in the season that tend to fade during the summer and fall. Plant-to-plant spread of the virus in the vineyard occurs only by the ectoparasitic dagger nematode Xiphinema index (17,27). The virus also is transmitted efficiently by grafting and via the distribution of infected vegetative propagation materials. Grapevine fanleaf virus (GFLV, genus Nepovirus, family Comoviridae) (21) is the causal agent of grapevine fanleaf disease. The genome of GFLV is composed of two single-stranded, positive-sense RNAs, termed RNA1 and RNA2 (Fig. 1). They are encapsidated separately in polyhedral virus particles of ≈28 nm in diameter (41). The size of RNA1 is 7,342 nucleotides (nt) but RNA2 is variable between 3,774 and 3,806 nt (44,47,57). Both RNAs are polyadenylated at their 3′ end and carry a small protein, VPg, covalently linked to their 5′ ends (36). It has been shown that RNA1 can replicate autonomously in protoplasts, whereas RNA2 replication occurs only when present together with RNA1 (55). However, both RNAs are required for systemic infection of plants. RNA1 and RNA2 are monocistronic and each encodes a single polyprotein that is processed proteolytically into functional proteins required to complete the virus life cycle. The RNA1encoded P1 polyprotein includes in its C-terminal region the domains for the putative nucleotide triphosphate-binding protein (NTB or 1B), the small viral protein linked to the genome (1C), the proteinase (1D), and the RNA-dependent RNA polymerase (1E) (Fig. 1). The functional role of the N-terminal region of P1 polyprotein is less characterized, although recent studies predicted two protein domains upstream of the NTB domain, designated as X1 and X2, separated by putative cleavage sites C/A and G/A between X1–X2 and X2-NTB domains, respectively (56). The RNA2-encoded P2 polyprotein contains (from the Nto C-terminus) the domains for the homing protein (2A), the movement protein (2B), and the coat protein (2C) (Fig. 1) (26). The 2A localizes in the replication site and has been implicated in RNA1-dependent replication of RNA2 (13). The 2B is a movement protein and is found in tubules observed in the plasmodesmata (43). The 2C is a multifunctional coat protein that is important in specific transmission by X. index, encapsidation of genomic RNAs, and systemic spread in plants (2,4,8,17). GFLV has been documented as a broad range of molecular variants in several countries in Europe, Africa, the Middle East, and North America (3,12,24,32,37,40,51). GFLV, but not other nepoviruses, was identified so far in Washington State (30) vineyards and poses a potential threat to the sustainability of the wine grape industry that has a $3 billion-plus impact to the state’s economy. GFLV could also become a major threat to the wine grape industry in the Pacific Northwest of the United States due Corresponding author: R.A. Naidu; E-mail address: [email protected] The GenBank accession numbers for the sequences reported in this article are GQ332364 to GQ332373. * The e-Xtra logo stands for “electronic extra” and indicates that the online version contains a figure showing the symptoms produced by Grapevine fanleaf virus in wine grape cultivars. doi:10.1094 / PHYTO-99-12-1394 © 2009 The American Phytopathological Society e-Xtra*