Molecular Characterization and Pathogenicity of a Novel Soybean-Infecting Monopartite Geminivirus in China

Abstract

Soybean is a major legume crop that plays an important role in food production, industrial production, and animal husbandry. Here, we characterize a novel soybean-infecting monopartite geminivirus identified in China. Analysis of the contigs de novo assembled from sequenced small interfering RNAs, followed by PCR, cloning, and sequencing, the complete viral genome was determined to be 2782 nucleotides. The genome contains the conserved nonanucleotide sequence, TAATATTAC and other sequence features typical of the family Geminiviridae, and encodes two and four open reading frames in the virion-sense and the complementary-sense strands, respectively. Genome-wide pairwise identity analysis revealed that the novel virus shares less than 65.6% identity with previously characterized geminiviruses. Phylogenetic and recombination analysis indicated that this virus was placed in a unique taxon within the family Geminiviridae and potentially arose from recombination. An infectious clone of this virus was further constructed and its infectivity was tested in different species of plants. Successful infection and characteristic symptoms were observed in Glycine max, Nicotiana benthamiana, N. tabacum, N. glutinosa, and N. tabacum cv. Samsun plants. Taken together, this virus represents a member of an unclassified genus of the family Geminiviridae, for which the name soybean yellow leaf curl virus is proposed.

Keywords: geminivirus; recombination; soybean; ssDNA virus.

Figure 1

Genome organization of soybean yellow leaf curl virus (SbYLCV). (A) Schematic representation of SbYLCV genomic organization. Open reading frames encoded on the virion-sense (V) strand and complementary-sense (C) strand are denoted with different colors. (B) The stem-loop structure located within the intergenic region of SbYLCV. The conserved nonanucleotide sequence is shown in red color. The putative nick site within the nonanucleotide sequences is marked with an arrow.

Figure 2

Alignment of the N-terminal sequences of the predicted SbYLCV Rep protein with those of selected geminiviruses. The conserved motifs I, II, III, and GRS are shown, as indicated. ACMV, African cassava mosaic virus; ACSV, Axonopus compressus streak virus; BCTV, beet curly top virus; BGMV, bean golden mosaic virus; BYDV, bean yellow dwarf virus; CGMV, cowpea golden mosaic virus; CpYV, chickpea yellows virus; ECSV, Eragrostis curvula streak virus; HrCTV, horseradish curly top virus; HYVV, honeysuckle yellow vein virus; MSV, maize streak virus; PGA, prunus geminivirus A; SeCTV, sesame curly top virus; TPCTV, tomato pseudo-curly top virus; TYLCCNV, tomato yellow leaf curl China virus; TYLCV, tomato yellow leaf curl virus.

Figure 3

Phylogenetic relationships of SbYLCV and representative geminiviruses based on the nucleotide sequence of the full-length genome (A) and the amino acid sequence of the coat protein (CP) (B). The phylogenetic trees were constructed with MEGA 5.0 using the neighbor-joining method. The statistical significance of the branches was determined with a bootstrap of 1000 replicates.

Figure 4

Recombination analysis of SbYLCV. The recombinant event was detected using the Recombination Detection Program RDP4. The brown line indicates the pairwise identity between the minor parent (Stachytarpheta leaf curl virus, AJ810157.1) and SbYLCV. The grey region represents the location of predicted breakpoints. The displayed linearized genome organization of SbYLCV shows the position in the alignment.

Figure 5

Systemic infection of SbYLCV in Glycine max and Nicotiana benthamiana plants. (A) Characteristic symptoms induced by SbYLCV. Plants were agroinoculated with the pBinPLUS vector (mock) or the infectious clone of SbYLCV, as indicated. Photos were taken at 30 days post-inoculation (dpi). (B) Southern blot hybridization analysis of SbYLCV DNA with a specific DIG-labelled DNA probe. Total DNA was extracted from systemic leaves of SbYLCV-infected and mock-inoculated plants, as indicated. Ethidium bromide staining of total DNA was used as loading controls.

Figure 6

Systemic infection of SbYLCV in Nicotiana tabacum, N. glutinosa, N. tabacum cv. Samsun NN, and N. tabacum cv. Samsun nn plants. (A) Characteristic symptoms induced by SbYLCV. Plants were agroinoculated with the pBinPLUS vector (mock) or the infectious clone of SbYLCV, as indicated. Photos were taken at 30 days post-inoculation (dpi). (B) Southern blot hybridization analysis of SbYLCV DNA with a specific DIG-labelled DNA probe. Total DNA was extracted from systemic leaves of SbYLCV-infected and mock-inoculated plants, as indicated. Lanes 1, 3, 5, and 7 represent total DNA extracted from plants inoculated with the pBinPLUS vector; lanes 2, 4, 6, and 8 represent total DNA extracted from plants inoculated with the infectious clone of SbYLCV. Ethidium bromide staining of total DNA was used as a loading control.