Cactaceae comprise a diverse and iconic group of flowering plants which are almost exclusively indigenous to the New World. The wide variety of growth forms found amongst the cacti have led to the trafficking of many species throughout the world as ornamentals. Despite the evolution and physiological properties of these plants having been extensively studied, little research has focused on cactus-associated viral communities. While only single-stranded RNA viruses had ever been reported in cacti, here we report the discovery of cactus-infecting single-stranded DNA viruses. These viruses all apparently belong to a single divergent species of the family Geminiviridae and have been tentatively named Opuntia virus 1 (OpV1). A total of 79 apparently complete OpV1 genomes were recovered from 31 different cactus plants (belonging to 20 different cactus species from both the Cactoideae and Opuntioideae clades) and from nine cactus-feeding cochineal insects (Dactylopius sp.) sampled in the USA and Mexico. These 79 OpV1 genomes all share > 78.4% nucleotide identity with one another and < 64.9% identity with previously characterized geminiviruses. Collectively, the OpV1 genomes display evidence of frequent recombination, with some genomes displaying up to five recombinant regions. In one case, recombinant regions span ~40% of the genome. We demonstrate that an infectious clone of an OpV1 genome can replicate in Nicotiana benthamiana and Opuntia microdasys. In addition to expanding the inventory of viruses that are known to infect cacti, the OpV1 group is so distantly related to other known geminiviruses that it likely represents a new geminivirus genus. It remains to be determined whether, like its cactus hosts, its geographical distribution spans the globe.

The begomoviruses (BGVs) are plant pathogens that evolved in the Old World during the Cretaceous and arrived to the New World (NW) in the Cenozoic era. A subgroup of NW BGVs, the “Squash leaf curl virus (SLCV) lineage” (S-Lin), includes viruses with unique characteristics. To get clues on the evolutionary origin of this lineage, a search for divergent members was undertaken. Four novel BGVs were characterized, including one that is basal to the group. Comparative analyses led to discover a ~670 bp genome module that is nearly exclusive of this lineage, encompassing the replication origin, the AC4 gene, and 480 bp of the Rep gene. A similar DNA module was found in two curtoviruses, hence suggesting that the S-Lin ancestor acquired its distinctive genomic segment by recombination with a curtovirus. This hypothesis was definitely disproved by an in-depth sequence analysis. The search for homologs of S-Lin Rep uncover the common origin of Rep proteins encoded by diverse Geminiviridae genera and viral “fossils” integrated at plant genomes. In contrast, no homolog of S-Lin Rep was found in public databases. Consequently, it was concluded that the SLCV clade ancestor evolved by a recombination event between a primitive NW BGV and a virus from a hitherto unknown lineage.

Background: Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results: A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions: EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of their DNA-B component, short rep gene sequences located downstream to a CP-promoter-like domain. This assemblage of DNA-A-related sequences within the DNA-B IR is reminiscent of polyomavirus microRNAs and could be involved in the posttranscriptional regulation of the cognate viral rep gene, an intriguing possibility that should be experimentally explored

A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was characterized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP-responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed “TATA-associated composite element” or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP-responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues.

Geminiviruses are a group of plant-infecting viruses with single-stranded DNA genomes. Within this family, viruses in the genus Begomovirus are known to have a worldwide distribution causing a range of severe diseases in a multitude of dicotyledonous plant species. Begomoviruses are transmitted by the whitefly Bemisia tabaci, and their ssDNA genomes can be either monopartite or bipartite. As part of a viral survey, various plants including those in the families Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Malvaceae, Oleaceae and Solanaceae were sampled and screened for begomoviruses using both a high-throughput sequencing and a begomovirus-specific primer pair approach. Based on the sequences derived using these approaches, the full-length genome of various begomoviruses were amplified from plants using abutting primers. Squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified in Cactaceae (n = 25), Solanaceae (n = 7), Cucurbitaceae (n = 2) and Lamiaceae (n = 1) samples. WCSV is an Old World bipartite begomovirus that has only recently been discovered infecting watermelons in the Americas. Our discovery of WCSV in the USA is the first indication that it has reached this country and indicates that this virus might be widespread throughout North America. Phylogenetic analysis suggests WCSV was introduced to the New World twice. The detection of begomoviruses in cactus plants suggests possible spillover events from agricultural areas into native vegetation. Since WCSV and SLCV have previously been found in mixed infections, pseudo-recombination infection experiments were conducted. We demonstrate that WCSV DNA-B is successfully trans-replicated by SLCV DNA-A despite very low degree of similarity between the replication-associated iterative sequences present in their common region, an essential feature for binding of the replication associated protein. This study highlights the importance of viral surveys for the detection of spillover events into native vegetation, but also suggests the need for more surveillance of WCSV in the USA, as this virus is a serious threat to watermelon cultivation in the Middle East.