Molecular and Biological Characterization of a New World Mono-/Bipartite Begomovirus/Deltasatellite Complex Infecting Corchorus siliquosus

Abstract

The genus Begomovirus (family Geminiviridae) is the largest genus in the entire virosphere, with more than 400 species recognized. Begomoviruses are single-stranded DNA plant viruses transmitted by whiteflies of the Bemisia tabaci complex and are considered one of the most important groups of emerging plant viruses in tropical and subtropical regions. Several types of DNA satellites have been described to be associated with begomoviruses: betasatellites, alphasatellites, and deltasatellites. Recently, a family of single-stranded DNA satellites associated with begomoviruses has been created, Tolecusatellitidae, including the genera Betasatellite and Deltasatellite. In this work, we analyzed the population of begomoviruses and associated DNA satellites present in Corchorus siliquosus, a malvaceous plant growing wild in Central America, southeastern North America and the Caribbean, collected in Cuba. The genomes of isolates of two New World begomoviruses [(Desmodium leaf distortion virus (DesLDV) and Corchorus yellow vein Cuba virus (CoYVCUV)] and two deltasatellites [tomato yellow leaf distortion deltasatellite 2 (TYLDD2) and Desmodium leaf distortion deltasatellite (DesLDD)] have been cloned and sequenced from plants showing yellow vein symptoms. Isolates of one of the begomoviruses, CoYVCUV, and one of the deltasatellites, DesLDD, represent novel species. Experiments with infectious clones showed the monopartite nature of CoYVCUV and that DesLDD utilizes the bipartite DesLDV as helper virus, but not the monopartite CoYVCUV. Also, CoYVCUV was shown to infect common bean in addition to Nicotiana benthamiana. This is the first time that (i) a monopartite New World begomovirus is found in a host other than tomato and (ii) deltasatellites have been found in C. siliquosus, thus extending the host and helper virus ranges of this recently recognized class of DNA satellites.

Keywords: Corchorus siliquosus; DNA satellites; Malvaceae; begomoviruses; deltasatellites; geminiviruses; phylogenetic analysis; virus taxonomy.

FIGURE 1

Corchorus siliquosus plants showing vein yellowing symptoms. (A) Sample 679. (B) Sample 705.

FIGURE 2

Iterons (arrows) of genome components of begomoviruses Desmodium leaf distortion virus (DesLDV) and Corchorus yellow vein Cuba virus (CoYVCUV). TATA motifs of the Rep promoter are highlighted with boxes.

FIGURE 3

Phylogenetic trees illustrating the relationships of isolates of Desmodium leaf distortion virus (DesLDV) DNA-A (A) and DNA-B (B) (bold in blue) and Corchorus yellow vein Cuba virus (CoYVCUV) DNA-A (bold in red) isolates with other New World begomoviruses. The trees were constructed by the maximum likelihood method (1000 replicates) with the MEGA 7 program using the best fit model, TN93 + G + I for DNA-A and HKY + G for DNA-B. AbMV, Abutilon mosaic virus; BGMV, bean golden mosaic virus; BlYSV, Blainvillea yellow spot virus; CabLCJV, cabbage leaf curl Jamaica virus; DaChMV, Dalechampia chlorotic mosaic virus; JMV, Jatropha mosaic virus; MClMV, melon chlorotic mosaic virus; PepGMV, pepper golden mosaic virus; PYMV, potato yellow mosaic virus; SiGMFlV, Sida golden mosaic Florida virus; SiGYVV, Sida golden yellow vein virus; TbYCV, tobacco yellow crinckle virus; ToYLDV, tomato yellow leaf distortion virus. The Old World begomovirus tomato leaf curl New Delhi virus (ToLCNDV) was used as an outgroup. The bars below the trees indicate the number of nucleotide substitutions per site. Nodes with bootstrap values higher than 50% are marked with filled circles.

FIGURE 4

Restriction fragment length polymorphism (RFLP) analysis of begomoviruses and deltasatellites infecting Corchorus siliquosus (samples 705 and 706). (A) RFLP performed by digestion of rolling circle amplification (RCA) products obtained from DNA extracts with the restriction enzyme HpaII revealed on a 1.5% agarose gel. (B) In silico RFLP analysis (HpaII) of the begomovirus and deltasatellite sequences obtained from each sample, performed with SnapGene (Insightful Science, available at snapgene.com). MW: molecular weight marker (HyperLadder 1 kb, Bioline).

FIGURE 5

Symptoms of leaf crinkle and mild yellowing developed in common bean plants agroinoculated with Corchorus yellow vein Cuba virus (CoYVCUV) DNA-A. Mock-inoculated controls are shown at the left of each panel.

FIGURE 6

Phylogenetic tree illustrating the relationships of Desmodium leaf distortion deltasatellite (DesLDD) (bold in blue) and tomato yellow leaf distortion deltasatellite 2 (ToYLDD2) (bold in red) isolates obtained in this work with representative isolates of other deltasatellite species. The tree was constructed by the maximum-likelihood method (1000 replicates) with the MEGA 7 program using the best fit model, T92 + G. CrYVD, Croton yellow vein deltasatellite; MaLCuD, Malvastrum leaf curl deltasatellite; SiGYVD1, Sida golden yellow vein deltasatellite 1; SiGYVD2, Sida golden yellow vein deltasatellite 2; SiGYVD3, Sida golden yellow vein deltasatellite 3; SPLCD1, sweet potato leaf curl deltasatellite 1; SPLCD2, sweet potato leaf curl deltasatellite 2; SPLCD3, sweet potato leaf curl deltasatellite 3; ToLCD, tomato leaf curl deltasatellite; ToYLDD1, tomato yellow leaf distortion deltasatellite 1; ToYLDD2, tomato yellow leaf distortion deltasatellite 2. Major deltasatellite clusters associated with bipartite New World begomoviruses (NW) and monopartite Old World begomoviruses and sweepoviruses (OW and SWEEPO) are indicated. The bar below the tree indicates the number of nucleotide substitutions per site. Nodes with bootstrap values higher than 50% are marked with filled circles.

FIGURE 7

Symptoms of leaf distortion and a significant reduction in plant growth developed in Nicotiana benthamiana plants agroinoculated with Desmodium leaf distortion virus (DesLDV) DNA-A and DNA-B, alone or in combination with Desmodium leaf distortion deltasatellite (DesLDD). Mock-inoculated control is shown at the left of the photograph.