The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

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).

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector.

Enhancement of soil DNA extraction by the use of a hand held mixer

A eficiência da extracão de DNA do solo utilizando um homogeneizador bead beater (Biospec Products - Alemanha) foi comparada àquela obtida utilizando um homogeneizador manual (Moulinex - Brasil). O homogeneizador manual tem custo 100 vezes menor e revelou-se mais eficiente que o bead beater extraindo sete vezes mais DNA.