Simone G. Ribeiro

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.

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

BackgroundHeterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity.ResultsTo validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions.ConclusionSince SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications.

Molecular and biological characterization of Tomato chlorotic mottle virus suggests that recombination underlies the evolution and diversity of Brazilian tomato begomoviruses

ABSTRACT Tomato chlorotic mottle virus (ToCMoV) is an emerging begomovirus species widely distributed throughout tomato-growing regions of Brazil. ToCMoV appears to have expanded its geographic range recently, invading tomato-growing areas that were free of begomovirus infection before 2004. We have determined the first complete genome sequence of an infectious ToCMoV genome (isolate BA-Se1), which is the first begomovirus species isolated in the northeast of Brazil. When introduced by particle bombardment into tomato, the cloned ToCMoV-[BA-Se1] DNA-A and DNA-B components caused typical chlorotic mottle symptoms. The cloned virus was whitefly-transmissible and, although it was infectious in hosts such as Nicotiana benthamiana, pepper, tobacco, and Nicandra physaloides, it was unable to infect Arabidopsis thaliana, bean, N. glutinosa, and Datura metel. Sequence and biological analyses indicate that ToCMoV-[BA-Se1] is a typical New World begomovirus sp. requiring both DNA-A and DNA-B components to establish systemic infections. Although evidence of multiple recombination events was detected within the ToCMoV-[BA-Se1] DNA-A, they apparently occurred relatively long ago, implying that recombination probably has not contributed to the recent emergence of this species.

Sources of resistance in Lycopersicon spp. to a bipartite whitefly-transmitted geminivirus from Brazil

Accessions of Lycopersicon chilense, L. peruvianum, L.hirsutum and sixteen L. esculentum genotypes were evaluated undergreenhouse conditions for resistance to a whitefly-transmitted geminivirusisolate from Brasilia-DF (DF1). Artificial cage inoculation oftomato plants at the two true-leaf stage, using 20 viruliferous whiteflies perplant in individual insect-proof cages, ensured 100% infection ofsusceptible tomato plants. Virus infection was confirmed by symptomdevelopment and dot-blot or squash-blot hybridization. In advanced testing,accessions of L. chilense (LA 1967), L. peruvianum (CNPH784) and L. hirsutum (PI-127827) and three selected inbred lines(TY 197, TY 198 and Tx 468-1) showed no symptoms and viral DNAwas barely detectable four weeks after inoculation, indicating good sourcesof resistance to the virus.Accessions of Lycopersicon chilense, L. peruvianum, L.hirsutum and sixteen L. esculentum genotypes were evaluated undergreenhouse conditions for resistance to a whitefly-transmitted geminivirusisolate from Brasília-DF (DF1). Artificial cage inoculation oftomato plants at the two true-leaf stage, using 20 viruliferous whiteflies perplant in individual insect-proof cages, ensured 100% infection ofsusceptible tomato plants. Virus infection was confirmed by symptomdevelopment and dot-blot or squash-blot hybridization. In advanced testing,accessions of L. chilense (LA 1967), L. peruvianum (CNPH784) and L. hirsutum (PI-127827) and three selected inbred lines(TY 197, TY 198 and Tx 468-1) showed no symptoms and viral DNAwas barely detectable four weeks after inoculation, indicating good sourcesof resistance to the virus.

A novel monopartite begomovirus infecting sweet potato in Brazil

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) present in a single sweet potato (Ipomoea batatas) plant collected in São Paulo, Brazil, are presented. Based on the current taxonomic criteria for the genus Begomovirus, one of the isolates was shown to represent a novel species, tentatively named Sweet potato leaf curl Sao Paulo virus (SPLCSPV). The other isolate represented a new strain of sweet potato leaf curl virus, named sweet potato leaf curl virus-Sao Paulo (SPLCV-SP). The full genome sequence of the SPLCSPV isolate shared the highest nucleotide identity (87.6%) with isolates of sweet potato leaf curl Spain virus (SPLCESV). Phylogenetic and recombination analyses were used to investigate the relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses.

A Study of Gene Expression in the Nematode Resistant Wild Peanut Relative, Arachis stenosperma, in Response to Challenge with Meloidogyne arenaria

Peanut (Arachis hypogaea) is amongst the most important legume crops in the world. One of its main yield constraints is the root-knot nematode Meloidogyne arenaria. A number of wild Arachis species, including A. stenosperma, are resistant to nematodes, and are a potential source of new resistance alleles for cultivated peanut. Using in silico subtraction of ESTs and macroarray analysis, we identified genes differentially expressed in A. stenosperma roots during its resistance response to M. arenaria. The three most differentially expressed genes [Auxin Repressed Protein (AsARP), Cytokinin Oxidase (AsCKX) and Metallothionein Type 2 (AsMET2)] were further analyzed using northern-blot and showed distinct expression profiles in the resistant A. stenosperma and susceptible A. hypogaea, both after, and sometimes even before, challenge with nematodes. Of the three most differentially expressed genes, AsARP and AsCKX are potentially involved in plant hormonal balance, and AsMET2 may be related to the reactive oxygen reaction triggered by the hypersensitive response (HR).

Identification of host proteins modulated by the virulence factor AC2 of Tomato chlorotic mottle virus in Nicotiana benthamiana

Tomato, one of the most important crops cultivated worldwide, has been severely affected by begomoviruses such as the Tomato chlorotic mottle virus (ToCMoV). Virulence factor AC2 is considered crucial for a successful virus–plant interaction and is known to act as a transcriptional activator and in some begomoviruses to function as an RNA silencing suppressor factor. However, the exact functions of the AC2 protein of the begomovirus ToCMoV are not yet established. The aim of the present study was to identify differentially expressed proteins of the model plant Nicotiana benthamiana in response to the expression of the AC2 gene, isolated from ToCMoV. N. benthamiana plants were inoculated with Agrobacterium tumefaciens containing the viral vector Potato virus X (PVX) and with the PVX‐AC2 construction. 2DE was performed and proteins were identified by MS. The results showed that the expression of ToCMoV AC2 alters the levels of several host proteins, which are important for normal plant development, causing an imbalance in cellular homeostasis. This study highlights the effect of AC2 in the modulation of plant defense processes by increasing the expression of several oxidative stress‐related and pathogenesis‐related proteins, as well as its role in modulating the proteome of the photosynthesis and energy production systems.

Distribuição de geminivírus nas culturas do tomate e pimentão em doze municípios do Submédio do Vale São Francisco

In 1996 and 1997, whitefly-transmitted geminivirus symptoms were observed in tomato (Lycopersicon esculentum) and sweet pepper (Capsicum annuum) plants in the Lower basin of San Francisco Valley, located in the states of Pernambuco and Bahia, Northeastern Brazil. One thousand three hundred and sixty-eight leaf samples of tomato and 194 pepper leaf samples showing similar symptoms to those caused by geminivirus were randomly collected from October 1996 to December 1998 in 104 and 16 fields, respectively, from 12 counties of that region and two neighboring counties. The incidence of symptomatic plants was estimated from 5 to 100% in tomato and 10 to 20% in sweet pepper fields. For geminivirus detection, dot or squash-blots were hybridized with heterologous probes. For tomato, the probe consisted of full-length DNA-A components of Bean golden mosaic virus (BGMV) from Brazil and Bean golden yellow mosaic virus (BGYMV) from Guatemala, while for sweet pepper it consisted of a fragment of the DNA-A component of an isolate from tomato found in the Federal District. Out of 1,562 collected samples, 908 (58.1%) tested positive for geminivirus, 823 (60.2%) on tomato and 85 (43.8%) on sweet pepper. The presence of infected plants was detected in all 120 fields with an incidence varying from 20% to 100%, indicating a broad dissemination of geminivirus in these crops in the Lower Basin of San Francisco Valley.

Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes (‘Santa Clara’, susceptible, and its near-isogenic line ‘LAM 157’, resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

A novel geminivirus identified in tomato and cleome plants sampled in Brazil

Viruses in the family Geminiviridae have single-stranded DNA genomes encapsulated in geminate icosahedral particles. High throughput sequencing (HTS) for metagenomic approaches are being extensively used for the identification of known and novel viruses. Using a HTS approach, we identified a novel geminivirus in a tomato (Solanum lycopersicum) sample and a Cleome sp. sample collected in the midwest region of Brazil. The genomes from the two samples share 99.96% identity and ∼61-63% to genomes in the genus Capulavirus. The novel virus has been tentatively named tomato associated geminivirus 1 (TaGV1). No visual symptoms were observed in the field tomato plant or in the inoculated Nicotiana benthamiana where the virus established a systemic infection. The replication associated protein of TaGV1 is most similar to that encoded by capulaviruses (sharing 62-70% identity), whereas the CP is most similar to that of tomato pseudo curly top virus (sharing ∼31% identity). In the TaGV1 positive Cleome sp. sample, begomovirus DNA A and B components were also detected sharing 96% and 90% sequence identity to cleome leaf crumple virus DNA A and B components, respectively. Using a HTS approach, we identified TaGV1 in tomato and Cleome sp. samples and this is the first report of a geminivirus that is non-begomovirus in Brazil.