Francisco Murilo Zerbini

Distribution and genetic diversity of tomato-infecting begomoviruses in Brazil*

Summary. Tomato-infecting begomoviruses have been reported throughout Brazil since the introduction of the B biotype of Bemisia tabaci. Here, we report a large scale survey on the distribution and genetic diversity of tomato-infecting begomoviruses. Tomato samples with typical begomovirus symptoms were collected in seven different states, comprising the major tomato growing areas of the country. Viruses were detected by polymerase chain reaction (PCR) using universal primers for the genus Begomovirus. PCR-amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, at least seven previously undescribed species of begomoviruses were found. Four of the new viruses were found exclusively in the Southeastern states, two exclusively in the Northeastern states, and one was found in both regions. Sequence comparisons reveal strong evidence of recombination among the Brazilian begomoviruses. Together, the results indicate the existence of a high degree of pre-existing genetic diversity among tomato-infecting begomoviruses in Brazil and suggest that these viruses have emerged after being transferred from natural hosts to tomatoes, due to the introduction into Brazil of a novel polyfagous biotype of the whitefly vector.

Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017)

This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2017.

Characterization of a New World Monopartite Begomovirus Causing Leaf Curl Disease of Tomato in Ecuador and Peru Reveals a New Direction in Geminivirus Evolution

ABSTRACT All characterized whitefly-transmitted geminiviruses (begomoviruses) with origins in the New World (NW) have bipartite genomes composed of a DNA-A and DNA-B component. Recently, an NW begomovirus lacking a DNA-B component was associated with tomato leaf curl disease (ToLCD) in Peru, and it was named Tomato leaf deformation virus (ToLDeV). Here, we show that isolates of ToLDeV associated with ToLCD in Ecuador and Peru have a single, genetically diverse genomic DNA that is most closely related to DNA-A components of NW bipartite begomoviruses. Agroinoculation of multimeric clones of the genomic DNA of three ToLDeV genotypes (two variants and a strain) resulted in the development of tomato leaf curl symptoms indistinguishable from those of ToLCD in Ecuador and Peru. Biological properties of these ToLDeV genotypes were similar to those of Old World (OW) monopartite tomato-infecting begomoviruses, including lack of sap transmissibility, phloem limitation, a resistance phenotype in tomato germplasm with the Ty-1 gene, and functional properties of the V1 (capsid protein) and C4 genes. Differences in symptom phenotypes induced by the ToLDeV genotypes in tomato and Nicotiana benthamiana plants were associated with a highly divergent left intergenic region and C4 gene. Together, these results establish that ToLDeV is an emergent NW monopartite begomovirus that is causing ToLCD in Ecuador and Peru. This is the first report of an indigenous NW monopartite begomovirus, and evidence is presented that it emerged from the DNA-A component of a NW bipartite progenitor via convergent evolution and recombination.

Brazilian Begomovirus Populations Are Highly Recombinant, Rapidly Evolving, and Segregated Based on Geographical Location

ABSTRACT The incidence of begomovirus infections in crop plants sharply increased in Brazil during the 1990s following the introduction of the invasive B biotype of the whitefly vector, Bemisia tabaci. It is believed that this biotype transmitted begomoviruses from noncultivated plants to crop species with greater efficiency than indigenous B. tabaci biotypes. Either through rapid host adaptation or selection pressure in genetically diverse populations of noncultivated hosts, over the past 20 years various previously unknown begomovirus species have became progressively more prevalent in cultivated species such as tomato. Here we assess the genetic structure of begomovirus populations infecting tomatoes and noncultivated hosts in southeastern Brazil. Between 2005 and 2010, we sampled and sequenced 126 DNA-A and 58 DNA-B full-length begomovirus components. We detected nine begomovirus species in tomatoes and eight in the noncultivated host samples, with four species common to both tomatoes and noncultivated hosts. Like many begomoviruses, most species are obvious interspecies recombinants. Furthermore, species identified in tomato have probable parental viruses from noncultivated hosts. While the population structures of five well-sampled viral species all displayed geographical subdivision, a noncultivated host-infecting virus was more genetically variable than the four predominantly tomato-infecting viruses.

A PERK-like receptor kinase interacts with the geminivirus nuclear shuttle protein and potentiates viral infection

ABSTRACT The nuclear shuttle protein (NSP) from bipartite geminiviruses facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts in concert with the movement protein (MP) to promote the cell-to-cell spread of the viral DNA. A proline-rich extensin-like receptor protein kinase (PERK) was found to interact specifically with NSP of Cabbage leaf curl virus (CaLCuV) and of tomato-infecting geminiviruses through a yeast two-hybrid screening. The PERK-like protein, which we designated NsAK (for NSP-associated kinase), is structurally organized into a proline-rich N-terminal domain, followed by a transmembrane segment and a C-terminal serine/threonine kinase domain. The viral protein interacted stably with defective versions of the NsAK kinase domain, but not with the potentially active enzyme, in an in vitro binding assay. In vitro-translated NsAK enhanced the phosphorylation level of NSP, indicating that NSP functions as a substrate for NsAK. These results demonstrate that NsAK is an authentic serine/threonine kinase and suggest a functional link for NSP-NsAK complex formation. This interpretation was corroborated by in vivo infectivity assays showing that loss of NsAK function reduces the efficiency of CaLCuV infection and attenuates symptom development. Our data implicate NsAK as a positive contributor to geminivirus infection and suggest it may regulate NSP function.

Synonymous site variation due to recombination explains higher genetic variability in begomovirus populations infecting non-cultivated hosts.

Begomoviruses are ssDNA plant viruses that cause serious epidemics in economically important crops worldwide. Non-cultivated plants also harbour many begomoviruses, and it is believed that these hosts may act as reservoirs and as mixing vessels where recombination may occur. Begomoviruses are notoriously recombination-prone, and also display nucleotide substitution rates equivalent to those of RNA viruses. In Brazil, several indigenous begomoviruses have been described infecting tomatoes following the introduction of a novel biotype of the whitefly vector in the mid-1990s. More recently, a number of viruses from non-cultivated hosts have also been described. Previous work has suggested that viruses infecting non-cultivated hosts have a higher degree of genetic variability compared with crop-infecting viruses. We intensively sampled cultivated and non-cultivated plants in similarly sized geographical areas known to harbour either the weed-infecting Macroptilium yellow spot virus (MaYSV) or the crop-infecting Tomato severe rugose virus (ToSRV), and compared the molecular evolution and population genetics of these two distantly related begomoviruses. The results reinforce the assertion that infection of non-cultivated plant species leads to higher levels of standing genetic variability, and indicate that recombination, not adaptive selection, explains the higher begomovirus variability in non-cultivated hosts.

Genome-wide analysis of differentially expressed genes during the early stages of tomato infection by a potyvirus.

Plant responses against pathogens cause up- and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e.g., pathogenesis-related protein 5), regulation of the cell cycle (e.g., cytokinin-repressed proteins), signal transduction (e.g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e.g., WRKY and SCARECROW transcription factors), stress response proteins (e.g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e.g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein

SUMMARY In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear-exported complexes and their subsequent trans-cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP-interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP-binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP-NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co-factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.

Three distinct begomoviruses associated with soybean in central Brazil.

We report the complete nucleotide sequences of geminiviruses of the genus Begomovirus infecting soybean (Glycine max) in central Brazil. Samples obtained from soybean plants collected at Santo Antonio de Goiás, Goiás State, showing typical symptoms of viral infection, were analyzed. Infection was confirmed by PCR-based amplification of a DNA-A fragment with universal begomovirus primers. Total DNA from infected plants was then subjected to rolling-circle amplification (RCA), and 2.6-kb molecules were cloned into plasmid vectors. Sequencing of the three DNA-A and two DNA-B clones thus obtained confirmed infection by three distinct begomoviruses: bean golden mosaic virus, Sida micrantha mosaic virus and okra mottle virus, the last of which was reported recently to be a novel virus infecting okra plants in Brazil. Begomovirus infection of soybean plants has been reported sporadically in Brazil and has generally not been considered to be of economic relevance.

ICTV Virus Taxonomy Profile: Potyviridae

The Potyviridae is the largest family of RNA plant viruses, members of which have single-stranded, positive-sense RNA genomes and flexuous filamentous particles 680–900 nm long and 11–20 nm wide. There are eight genera, distinguished by the host range, genomic features and phylogeny of the member viruses. Genomes range from 8.2 to 11.3 kb, with an average size of 9.7 kb. Most genomes are monopartite but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Potyviridae, which is available at www.ictv.global/report/potyviridae.