Tomato Apical Leaf Curl Virus: A Novel, Monopartite Geminivirus Detected in Tomatoes in Argentina

Abstract

Plant viruses that are members of the Geminiviridae family have circular single-stranded DNA (ssDNA) genome and are responsible for major crop diseases worldwide. We have identified and characterized a novel monopartite geminivirus infecting tomato in Argentina. The full-length genome was cloned and sequenced. The genome-wide pairwise identity calculation that resulted in a maximum of 63% identity with all of other known geminiviruses indicated that it is a new geminivirus species. Biolistic infected plants presented interveinal yellowing, apical leaf curling and extreme root hypotrophy. Thus, the name proposed for this species is tomato apical leaf curl virus (ToALCV). The phylogenetic inferences suggested different evolutionary relationships for the replication-associated protein (Rep) and the coat protein (CP). Besides, the sequence similarity network (SSN) protein analyses showed that the complementary-sense gene products (RepA, Rep and C3) are similar to capulavirus while the viron-sense gene products (CP, MP and V3) are similar to topocuvirus, curtovirus and becurtovirus. Based on the data presented, ToALCV genome appears to have "modular organization" supported by its recombination origin. Analyses of the specificity-determining positions (SDPs) of the CP of geminiviruses defined nine subgroups that include geminiviruses that share the same type of insect vector. Our sequences were clustered with the sequences of topocuvirus, whose vector is the treehopper, Micrutalis malleifera. Also, a set of the highest scored amino acid residues was predicted for the CP, which could determine differences in virus transmission specificity. We predict that a treehopper could be the vector of ToALCV, but transmission assays need to be performed to confirm this. Given everything we demonstrate in this paper, ToALCV can be considered a type member of a new putative genus of the Geminiviridae family.

Keywords: Argentina; ToALCV; coat protein; recombination; sequence similarity network; specificity-determining positions; tomato; treehopper.

FIGURE 1

Genomic organization and ORFs sizes of the putative species tomato apical leaf curl virus (ToALCV). The table characterize of the open reading frames of ToALCV. Length (nt/aa), pairwise identity by the BLAST algorithm, protein motif identification by the Conserved Domain Tool (NCBI) and the SMART algorithm.

FIGURE 2

Sequence similarity networks (SSNs) including 2910 geminiviral complementary-sense coded protein sequences (codified by C1, C1:C2, C2, C3, C4, and C5 genes). Sequences are represented as nodes and the pairwise relationships, as edges (lines) between nodes. Nodes are colored according to the virus genera to which they belong, while the query sequences are colored in red. (A) SSNs using a permissive threshold (E-value = 1E^-26). When sequences break out into clusters where the annotation is coincident, the name of the protein/function is indicated. One of the query sequences (putative C3) (red circle) clusters with the C3 sequences of capulavirus. Dotted red boxes enclose a group of sequences that was chosen for further analysis using more stringent thresholds (nodes are associated with more significant relationships). (B) Subnetwork 1, composed by 905 sequences, is shown at E-value threshold = 1E^-93. The two query sequences cluster with capulavirus (enclosed in red dotted line). The subnetwork is further analyzed in (C) Subnetwork 2, which includes 91 sequences, and is shown at a more stringent threshold (E-value = 1E^-150). Here the shape of the node indicates the protein annotation/function. The query sequences are closely related to the Rep associated protein of capulaviruses.

FIGURE 3

Sequence similarity networks (SSNs) including 1541 sense coded protein geminiviral sequences (codified by V1, V2, V3, V4, BC1-MP and BV1-NSP genes). Sequences are represented as different colored nodes and the pairwise relationships, as edges (lines) between nodes. The nodes are colored according to the virus genera to which the sequence belongs, while the query sequences are colored in red. (A) SSNs using a permissive threshold (E-value = 3). When sequences break out into clusters where the annotation is coincident, the name of the coded protein is indicated. (B) The network is shown at E-value threshold = 0.03. (C) The subnetwork that comprises 898 sequences at an E-value threshold = E^-35.

FIGURE 4

(A) Maximum likelihood phylogenetic tree of 66 full length genomes or DNA-A (bipartite begomovirus). (B) Maximum likelihood phylogenetic tree obtained with amino acid sequence alignments of the CP of the same 66 geminiviruses. Filled circles indicate 70 or more bootstrap percentages.

FIGURE 5

Schematic representation of the recombination event detected by RDP4.67 in the ToALCV genome and table with corresponding the p-values.

FIGURE 6

Biolistic infected plants symptoms. (A,B) Leaf interveinal yellowing and curling symptoms exhibited by ToALCV of inoculated plants with [AR: Yuto: Tom424:2008] RCA product by the biolistic method. (C) Root hypotrophy in plants infected with [AR: Yuto: Tom424:2008].