Chloroplast Proteome of Nicotiana benthamiana Infected by Tomato Blistering Mosaic Virus.

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase ß subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein-protein interaction studies to confirm their roles in virus replication and disease development.

Complete genome sequence of a proposed new tymovirus, tomato blistering mosaic virus.

In a previous work, a distinct tymovirus infecting tomato plants in Brazil was reported and tentatively named tomato blistering mosaic virus (ToBMV). In this study, the complete genome sequence of ToBMV was determined and shown to have a size of 6277 nucleotides and three ORFs: ORF 1 encodes the replication-complex polyprotein, ORF 2 the movement protein, and ORF 3 the coat protein. The cleavage sites of the replication-complex polyprotein (GS/LP and VAG/QSP) of ToBMV were predicted by alignment analysis of amino acid sequences of other tymoviruses. In the phylogenetic tree, ToBMV clustered with the tymoviruses that infect solanaceous hosts.

Unique RNA 2 sequences of two Brazilian isolates of Pepper ringspot virus, a tobravirus.

Pepper ringspot virus (PepRSV) is a tobravirus reported only in Brazil. Here, the sequences of the complete RNA 2 segments and the 3' end of the RNA 1 genomic regions of two new isolates from tomato plants were analyzed. The main ORF encodes the CP gene as other tobraviruses and termed ORF 1 of RNA 2. The second ORF was found only in one of the new isolates, although this gene was absent in the type isolate, CAM (collected in the 1960's). Interestingly, this ORF 2 gene did not show any nucleotide and amino acid sequence similarities with known 2b genes of tobraviruses, an essential gene of tobraviruses for nematodes-transmission. The 5'UTR sequence of RNA 2 segment of CAM isolate was previously reported showing two impaired direct repeats; however, the direct-repeats were absent in these new isolates. An additional ORF was predicted upstream of the CP gene. This putative protein possessed a transmembrane domain similar to the ORFN1 of RNA 2 of Tobacco rattle virus SYM isolate, although there was no sequence similarity. This is the first report on the diversity of the RNA 2 sequences of PepRSV.

A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.

Characterization of a novel tymovirus on tomato plants in Brazil.

A tymovirus was isolated in Brazil from tomato plants with severe symptoms of leaf mosaic and blistering. The virus was mechanically transmissible to solanaceous indicator host species. The infected plants contained icosahedral particles and chloroplasts with membrane deformations which are typical cytopathic effects caused by tymoviruses. Its coat protein amino acid sequence shares the maximum of 64 % identity with the tymovirus Chiltepin yellow mosaic virus, which suggested that it can be considered as a distinct member of the genus Tymovirus. In a phylogenetic tree, this tymovirus was clustered with other solanaceous-infecting tymoviruses. It was tentatively named as Tomato blistering mosaic virus (ToBMV).

The complete genome sequence of a Brazilian isolate of yam mild mosaic virus.

In this study, the complete genome of an isolate of yam mild mosaic virus (YMMV) from Brazil was sequenced, and the predicted amino acid sequence was analyzed. The YMMV RNA genome consists of 9538 nt without the poly(A) tail, encoding a putative typical potyvirus polyprotein of 3084 amino acids. Furthermore, the small overlapping ORF (PIPO) in the P3 gene was also deduced, and the cleavage sites of the polyprotein were predicted. Multiple alignment with other potyviruses showed a maximum nucleotide sequence identity of 64 % to wild tomato mosaic virus. A phylogenetic tree showed that YMMV clustered with Asian potyviruses that mainly infect solanaceous plants.

Presença dos gêneros Trichoderma e Fusarium em solo rizosférico e não rizosférico cultivado com tomateiro e pepineiro, em horta e estufa / Presence of the genus Trichoderma and Fusarium in rhizosphere and bulk soil cultivated with tomato and cucumber, in vegetable garden and greenhouse

Para a elaboração adequada de programas de biocontrole de patógenos de solo, é necessário conhecer a distribuição de patógenos e antagonistas nesse ambiente. O objetivo deste trabalho foi estudar a presença dos gêneros Trichoderma e Fusarium em solo rizosférico e não-rizosférico, cultivado com tomateiro e pepineiro, em horta e estufa, e identificar os isolados de Fusarium spp. patogênicos às culturas. Para isso, em horta e estufa, foram realizadas 40 amostragens de solo rizosférico (tomateiro e pepineiro) e 20 de solo não-rizosférico. As suspensões dos solos foram diluídas, incubadas em meio BDA e os fungos identificados. Posteriormente, foi realizado o teste de patogenicidade para o tomateiro e o pepineiro, com os isolados de Fusarium spp. obtidos dos solos coletados. Em estufa, para o tomateiro e para o pepineiro, o número de pontos de amostragem com a presença de Trichoderma spp. em solo rizosférico foi significativamente maior (95 e 45 por cento, respectivamente) do que em solo não-rizosférico (10 por cento). Neste ambiente, ocorreu diferença na presença de Trichoderma spp. e Fusarium spp., sendo encontrados, respectivamente, em 10 e 55 por cento dos pontos de amostragem. No teste de patogenicidade, cinco isolados de Fusarium oxysporum do tomateiro e seis do pepineiro foram patogênicos às respectivas culturas. Em estufa, Trichoderma spp. ocorre com maior freqüência na rizosfera, enquanto Fusarium spp. está distribuído no solo, e a maioria dos isolados de Fusarium spp. não é patogênica ao tomateiro nem ao pepineiro.

For the adequate elaboration of biocontrol programs of soil borne pathogens, it is necessary to understand the distribution of pathogens and antagonists in this environment. This research was aimed at studing the presence of the fungi Trichoderma and Fusarium in rhizosphere and bulk soil, cultivated with tomato and cucumber, in vegetable garden and greenhouse, and to quantify the isolates of Fusarium spp. pathogenic to the crops. For that, in a vegetable garden and a greenhouse, 40 samples of rhizosphere (cucumber and tomato) and 20 of bulk soil were taken,. The soil suspensions were diluted and plated in PDA media and the fungi identified. After that, the test of pathogenicity of the isolates for tomato and cucumber found in the samples, was performed. For tomato and cucumber, in greenhouse, the number of sampling points with the presence of Trichoderma spp. in the rhizosphere (95 e 45 percent, respectively) was significantly greater than in the bulk soil (10 percent). In the bulk soil, the presence of Trichoderma spp. and Fusarium spp. differed significantly, being found, respectively, in 10 and 55 percent of the sampling points. In the pathogenicity test, 5 isolates of Fusarium oxysporum of tomato and 6 of cucumber were pathogenic to the respective crops. In greenhouse, Trichoderma spp. occurs more frequently in the rhizosphere, whereas Fusarium spp. is distributed in the soil and the majority of the Fusarium spp. isolates are neither pathogenic to tomato nor to cucumber.