The closterovirus-derived gene expression and RNA interference vectors as tools for research and plant biotechnology

Abstract

Important progress in understanding replication, interactions with host plants, and evolution of closteroviruses enabled engineering of several vectors for gene expression and virus-induced gene silencing. Due to the broad host range of closteroviruses, these vectors expanded vector applicability to include important woody plants such as citrus and grapevine. Furthermore, large closterovirus genomes offer genetic capacity and stability unrivaled by other plant viral vectors. These features provided immense opportunities for using closterovirus vectors for the functional genomics studies and pathogen control in economically valuable crops. This review briefly summarizes advances in closterovirus research during the last decade, explores the relationships between virus biology and vector design, and outlines the most promising directions for future application of closterovirus vectors.

Keywords: Beet yellows virus; Citrus tristeza virus; Grapevine leafroll-associated virus-2; RNAi; closteroviruses; viral vector.

FIGURE 1

(A) Diagram of BYV genome with gene functions shown. L-Pro, papain-like leader protease; MET, methyltransferase (capping enzyme); S1H, superfamily I helicase; RdRp, RNA-dependent RNA polymerase; p6, 6-kDa protein; Hsp70h, Hsp70 homolog; p64, 64-kDa protein; CPm, minor capsid protein; CP, capsid protein; p20, 20-kDa protein; p21, 21-kDa protein. Homologous CP, CPm, and the C-terminal domain of p64 are shown in the same color. (B) Proteolytic gene expression cassette including β-glucuronidase ORF (GUS) fused in frame to papain-like, self-cleaving domain of potivirus HC-Pro. (C) Recombinant gene expression cassette including native BYV promoter, green fluorescent protein ORF (GFP) and heterologous BYSV promoter. (D) Dual expression cassette including heterologous BYSV and GLRaV-2 promoters, ER-targeted GFP and red fluorescent protein containing nuclear localization signal. (E) Confocal laser scanning microscopic image of the N. benthamiana leaf cell infected with BYV vector that expresses GFP^ER and RFP^NLS. Note the virus-induced, ER-derived, multivesicular body likely containing viral RNA replication complexes.

FIGURE 2

(A) Diagram of GLRaV-2-based gene expression vector vLR2-GFP with gene functions shown. L1 and L2, papain-like leader proteases 1 and 2; p19, 19-kDa protein; p24, 24-kDa protein; other designations as in Figure 1A. (B) vLR2-GFP-mediated GFP expression in grapevine (Vitis vinifera) berry. (C) Diagram of vLR2-based dual vector in which four variants of ChlI- or PDS-derived sequences in forward (F) or reverse (R) orientation were inserted downstream of ER-GFP ORF. (D) Spread of the dark, chlorophyll-less areas starts around the V. vinifera leaf cells infected with vLR2-ER-GFP-ChlI-F (green) as shown in the left panel, and later expands along the veins into the areas that contain no virus-infected cells (right panel). (E) Grapevine leaves showing chlorotic symptoms due to the RNAi targeting ChlI ~3 years post inoculation with vLR2-ER-GFP-ChlI-F.