A plant virus evolved by acquiring multiple nonconserved genes to extend its host range

Abstract

Viruses have evolved as combinations of genes whose products interact with cellular components to produce progeny virus throughout the plants. Some viral genes, particularly those that are involved in replication and assembly, tend to be relatively conserved, whereas other genes that have evolved for interactions with the specific host for movement and to counter host-defense systems tend to be less conserved. Closteroviridae encode 1-5 nonconserved ORFs. Citrus tristeza virus (CTV), a Closterovirus, possesses nonconserved p33, p18, and p13 genes that are expendable for systemic infection of the two laboratory hosts, Citrus macrophylla and Mexican lime. In this study, we show that the extended host range of CTV requires these nonconserved genes. The p33 gene was required to systemically infect sour orange and lemon trees, whereas either the p33 or the p18 gene was sufficient for systemic infection of grapefruit trees and the p33 or the p13 gene was sufficient for systemic infection of calamondin plants. Thus, these three genes are required for systemic infection of the full host range of CTV, but different genes were specific for different hosts. Remarkably, either of two genes was sufficient for infection of some citrus hybrids. These findings suggest that CTV acquired multiple nonconserved genes (p33, p18, and p13) and, as a result, gained the ability to interact with multiple hosts, thus extending its host range during the course of evolution. These results greatly extend the complexity of known virus-plant interactions.

Fig. 1.

(A) Schematic diagram of the genomic organization of CTV (CTV9) with ORFs (open boxes) showing the putative papain-like proteases (PRO), large interdomain region (IDR), and the methyl transferase-like (MT), helicase-like (HEL), and RNA-dependent RNA polymerase-like (RdRp) domains. The ORFs with numbers and corresponding translation products are indicated. HSP 70h, homolog of heat shock protein 70; CPm, minor coat protein; CP, major coat protein. ORFs corresponding to replication gene block, quintuple gene module, and virion assembly are indicated. The enlarged view of the 3′ end ORFs showed below the genome organization. CTV deletion mutants are shown in B–H with deleted sequences shown as dotted lines, and nucleotide coordinates of deletions are indicated.

Fig. 2.

Detection of GFP fluorescence in Citrus macrophylla (A), sour orange (B), and grapefruit (C) plants inoculated with CTV9-GFPC3, CTV9Δp33-GFPC3, or CTV9Δp33Δp18Δp13-GFPC3. The internal side of the bark patches of citrus plants was observed at 12 wk after inoculation under Zeiss Stemi SV 11 UV-fluorescence dissecting microscope with an attached Olympus Q-color 5 camera.

Fig. 3.

Schematic diagram of duplex plant of CTV9Δp33-infected C. macrophylla and sour orange. (A) Sour orange budwood was grafted onto CTV9Δp33-infected C. macrophylla plant, and the branches of C. macrophylla were pruned to force the shoots of sour orange (SO) and C. macrophylla (CM-I) simultaneously. (B) Analysis of sour orange and C. macrophylla branches from duplex plants for virus infection by DAS-I-ELISA.

Fig. 4.

CTV9Δp33 failed to infect sour orange in bark patch experiment. (A) Schematic representation of bark patch from healthy sour orange onto C. macrophylla plants infected with GFP-tagged CTV deletion mutants (a). A small piece of healthy sour orange (SO) bark patch was graft inoculated onto C. macrophylla (CM) plants infected with CTV9-GFPC3 (b), CTV9Δp33-GFPC3 (c), and CTV9Δp33Δp18Δp13-GFPC3 (d). (B) Schematic diagram of C. macrophylla (CM) bark patch from GFP-tagged deletion mutants onto healthy sour orange (SO) plants (a). A small piece of C. macrophylla bark patch from CTV9-GFPC3– (b), CTV9Δp33-GFPC3– (c), CTV9Δp33Δp18Δp13-GFPC3–infected (d) plants were graft inoculated onto healthy sour orange plants. The substituted bark patches were allowed to establish vascular connections, and a small piece of bark patch junction of C. macrophylla and sour orange was excised and observed under Zeiss Stemi SV 11 UV-fluorescence dissecting microscope. Note that GFP fluorescence was not observed at detectable levels in sour orange bark patches in Ac, Ad, Bc, and Bd.

Fig. 5.

CTV9Δp33Δp18Δp13 failed to infect grapefruit in bark patch experiment. (A) Schematic representation of bark patch from healthy grapefruit (DG) onto C. macrophylla (CM) plants infected with GFP-tagged CTV deletion mutant (a). A small piece of healthy grapefruit bark patch was graft inoculated onto C. macrophylla plants infected with CTV9-GFPC3 (b), and CTV9Δp33Δp18Δp13-GFPC3 (c). (B) Schematic diagram of C. macrophylla (CM) bark patch from GFP-tagged deletion mutant onto healthy grapefruit (DG) plants (a). A small piece of C. macrophylla bark patch from CTV9-GFPC3– (b), and CTV9Δp33Δp18Δp13-GFPC3–infected (c) plants was graft inoculated onto healthy grapefruit plants. The substituted bark patches were allowed to establish vascular connections, and a small piece of bark patch junction of C. macrophylla and grapefruit was excised and observed for GFP fluorescence. GFP fluorescence was not observed at detectable levels in grapefruit bark patches in Ac and Bc.