Poliane Alfenas-Zerbini

Six novel begomoviruses infecting tomato and associated weeds in Southeastern Brazil

The incidence of tomato-infecting begomoviruses has sharply increased in Brazil following the introduction of the B biotype of the whitefly vector in the early 1990s. Five definitive species and six tentative species have been described since then. Here, we report the detection of members of an additional six novel species, three in tomato and three infecting weeds that are commonly associated with tomato fields: Blainvillea rhomboidea, Sida rhombifolia and Sida micrantha. Tomato and weed samples were collected in two major tomato-growing regions of southeastern Brazil in 2005 and 2007. Two of the novel viruses were present in tomato plants collected in Paty do Alferes, Rio de Janeiro state. Three novel viruses were present in weed samples collected in Coimbra, Minas Gerais state. One virus was present in tomato samples collected at both locations. Genome features indicate that all six species are typical New World, bipartite begomoviruses. However, the viruses belonging to two of the novel species did not cluster with the Brazilian viruses in a phylogenetic tree. These species could represent a distinct lineage of New World begomoviruses, found in Brazil for the first time.

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.

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.

Synergism and negative interference during co-infection of tomato and Nicotiana benthamiana with two bipartite begomoviruses

In Brazil, at least eight begomoviruses including Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) infect tomatoes. ToYSV symptoms in tomato and Nicotiana benthamiana appear earlier and are more severe compared to those of ToRMV. We investigated the role of several factors in this differential adaptation. To analyze infection kinetics, a single leaf was inoculated and subsequently detached after different periods of time. Viral DNA accumulation was quantified in plants, viral replication was analyzed in protoplasts, and tissue tropism was determined by in situ hybridization. Results indicate that ToYSV establishes a systemic infection and reaches a higher concentration earlier than ToRMV in both hosts. ToRMV negatively interferes with ToYSV during the initial stages of infection, but once systemic infection is established this interference ceases. In N. benthamiana, ToYSV invades the mesophyll, while ToRMV is phloem-restricted. During dual infection in this host, ToYSV releases ToRMV from the phloem.

A mosaic of beach bean (Canavalia rosea) caused by an isolate of Cowpea aphid-borne mosaic virus (CABMV) in Brazil

Beach bean (Canavalia rosea) plants showing mosaic symptoms were found at Massaguaçú beach, Caraguatatuba, Brazil. A potyvirus was found to be responsible for the symptoms, based on transmission assays and electron microscopy. A positive reaction in ELISA was obtained against cowpea aphid-borne mosaic (CABMV) antisera. Viral identity was confirmed by RT-PCR using specific primers to amplify part of the NIb and the entire CP coding region of the genome and the 3′NTR. Comparison of the amplified sequences with that of CABMV showed a nucleotide sequence identity of 97% for the CP coding region. Thus, the potyvirus from beach bean should be considered a CABMV isolate, referred to as CABMV-Cr.

Sustained NIK-mediated antiviral signalling confers broad-spectrum tolerance to begomoviruses in cultivated plants.

Begomovirus-associated epidemics currently threaten tomato production worldwide due to the emergence of highly pathogenic virus species and the proliferation of a whitefly B biotype vector that is adapted to tomato. To generate an efficient defence against begomovirus, we modulated the activity of the immune defence receptor nuclear shuttle protein (NSP)-interacting kinase (NIK) in tomato plants; NIK is a virulence target of the begomovirus NSP during infection. Mutation of T474 within the kinase activation loop promoted the constitutive activation of NIK-mediated defences, resulting in the down-regulation of translation-related genes and the suppression of global translation. Consistent with these findings, transgenic lines harbouring an activating mutation (T474D) were tolerant to the tomato-infecting begomoviruses ToYSV and ToSRV. This phenotype was associated with reduced loading of coat protein viral mRNA in actively translating polysomes, lower infection efficiency and reduced accumulation of viral DNA in systemic leaves. Our results also add some relevant insights into the mechanism underlying the NIK-mediated defence. We observed that the mock-inoculated T474D-overexpressing lines showed a constitutively infected wild-type transcriptome, indicating that the activation of the NIK-mediated signalling pathway triggers a typical response to begomovirus infection. In addition, the gain-of-function mutant T474D could sustain an activated NIK-mediated antiviral response in the absence of the virus, further confirming that phosphorylation of Thr-474 is the crucial event that leads to the activation of the kinase.

Comparative analysis of the genomes of two isolates of cowpea aphid-borne mosaic virus (CABMV) obtained from different hosts

The complete genomic sequences of two cowpea aphid-borne mosaic virus (CABMV) isolates from Brazil, MG-Avr from passion fruit (which also infects cowpea), and BR1 from peanut (which also infects cowpea, but not passion fruit), were determined. Their nucleotide sequences are 89% identical and display 85% identity to that of CABMV-Z. Both isolates have the typical potyvirus genome features. P3 and VPg are the most conserved proteins, with 99% amino acid sequence identity between the two isolates, and P1 is the most variable, with 50% identity. A significant variation exists at the 5’-end of the genome between the Brazilian isolates and CABMV-Z. However, this variation does not correlate with the biological properties of these three isolates.

Genetic variability of papaya lethal yellowing virus isolates from Ceará and Rio Grande do Norte States, Brazil

The papaya (Carica papaya) is a fruit crop of great economic importance throughout the Brazilian northeast, which is responsible for 60% of the national output. Papayas in the states of Ceara and Rio Grande do Norte are affected by lethal yellowing disease, caused by papaya lethal yellowing virus (PLYV). Previous work suggested that PLYV is a putative sobemovirus. To assess the genetic variability of PLYV, foliar samples were collected in October 2008 and October 2009 in commercial fields from Ceara and Rio Grande do Norte states, and total RNA was extracted. Specific primers based on the sequence of a previously characterized PLYV isolate were used for the RT-PCR-based amplification of a 900 bp fragment corresponding to the central region of the viral genome. Fragments from 21 viral isolates were cloned and sequenced. Sequence analyses indicated >97% nucleotide sequence identity among the isolates, 94-100% identity with the previously sequenced PLYV isolate, and a lower but significant identity with sobemoviruses (43-48.5%). These results suggest a low genetic variability among PLYV isolates, and are in agreement with the provisional placement of PLYV in the genus Sobemovirus. Definitive taxonomic conclusions, however, can only be drawn after the determination of the full-length genomic sequence.

Analysis of the full-length genome sequence of papaya lethal yellowing virus (PLYV), determined by deep sequencing, confirms its classification in the genus Sobemovirus

Papaya lethal yellowing virus (PLYV) causes an economically important disease in papayas in northeastern Brazil. Based on biological and molecular properties, PLYV has been tentatively assigned to the genus Sobemovirus. We report the sequence of the full-length genome of a PLYV isolate from Brazil, determined by deep sequencing. The PLYV genome is 4,145 nt long and contains four ORFs, with an arrangement identical to that of sobemoviruses. The polyprotein and CP display significant sequence identity with the corresponding proteins of other sobemoviruses. Pairwise comparisons and phylogenetic analysis based on complete nucleotide sequences confirm the classification of PLYV in the genus Sobemovirus.

Translationally controlled tumour protein (TCTP) from tomato and Nicotiana benthamiana is necessary for successful infection by a potyvirus

Translationally controlled tumour protein (TCTP) is a ubiquitously distributed protein in eukaryotes, involved in the regulation of several processes, including cell cycle progression, cell growth, stress protection, apoptosis and maintenance of genomic integrity. Its expression is induced during the early stages of tomato (Solanum lycopersicum) infection by the potyvirus Pepper yellow mosaic virus (PepYMV, a close relative of Potato virus Y). Tomato TCTP is a protein of 168 amino acids, which contains all the conserved domains of the TCTP family. To study the effects of TCTP silencing in PepYMV infection, Nicotiana benthamiana plants were silenced by virus-induced gene silencing (VIGS) and transgenic tomato plants silenced for TCTP were obtained. In the early stages of infection, both tomato and N. benthamiana silenced plants accumulated less virus than control plants. Transgenic tomato plants showed a drastic reduction in symptoms and no viral accumulation at 14 days post-inoculation. Subcellular localization of TCTP was determined in healthy and systemically infected N. benthamiana leaves. TCTP was observed in both the nuclei and cytoplasm of non-infected cells, but only in the cytoplasm of infected cells. Our results indicate that TCTP is a growth regulator necessary for successful PepYMV infection and that its localization is altered by the virus, probably to favour the establishment of virus infection. A network with putative interactions that may occur between TCTP and Arabidopsis thaliana proteins was built. This network brings together experimental data of interactions that occur in other eukaryotes and helps us to discuss the possibilities of TCTP involvement in viral infection.