Formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes.

The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by cleavage of the nsp2/3 junction by the nsp2 protease, was nonessential for the formation of DMVs. However, the substitution of each of a cluster of four conserved cysteine residues, residing in a predicted luminal loop of nsp3, completely blocked DMV formation. Some of these mutant nsp3 proteins were also found to be highly cytotoxic, in particular, exerting a dramatic effect on the endoplasmic reticulum. The functionality of an engineered N glycosylation site in the cysteine-containing loop confirmed both its presence in the lumen and the transmembrane nature of nsp3. This mutant displayed an interesting intermediate phenotype in terms of DMV formation, with paired and curved membranes being formed, but DMV formation apparently being impaired. The effect of nsp3 mutations on replicase polyprotein processing was investigated, and several mutations were found to influence processing of the region downstream of nsp3 by the nsp4 main protease. When tested in an EAV reverse genetics system, none of the nsp3 mutations was tolerated, again underlining the crucial role of the protein in the arterivirus life cycle.

MYB72 is required in early signaling steps of rhizobacteria-induced systemic resistance in Arabidopsis.

Colonization of Arabidopsis thaliana roots by nonpathogenic Pseudomonas fluorescens WCS417r bacteria triggers a jasmonate/ethylene-dependent induced systemic resistance (ISR) that is effective against a broad range of pathogens. Microarray analysis revealed that the R2R3-MYB-like transcription factor gene MYB72 is specifically activated in the roots upon colonization by WCS417r. Here, we show that T-DNA knockout mutants myb72-1 and myb72-2 are incapable of mounting ISR against the pathogens Pseudomonas syringae pv tomato, Hyaloperonospora parasitica, Alternaria brassicicola, and Botrytis cinerea, indicating that MYB72 is essential to establish broad-spectrum ISR. Overexpression of MYB72 did not result in enhanced resistance against any of the pathogens tested, demonstrating that MYB72 is not sufficient for the expression of ISR. Yeast two-hybrid analysis revealed that MYB72 physically interacts in vitro with the ETHYLENE INSENSITIVE3 (EIN3)-LIKE3 transcription factor EIL3, linking MYB72 function to the ethylene response pathway. However, WCS417r activated MYB72 in ISR-deficient, ethylene-insensitive ein2-1 plants. Moreover, exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate induced wild-type levels of resistance in myb72-1, suggesting that MYB72 acts upstream of ethylene in the ISR pathway. Collectively, this study identified the transcriptional regulator MYB72 as a novel ISR signaling component that is required in the roots during early signaling steps of rhizobacteria-mediated ISR.

Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation.

This study aimed to evaluate the possibility of DNA sequences from genetically modified plants to persist in the gastrointestinal (GI) tract. PCR analysis and transformation assays were used to study DNA persistence and integrity in various ex vivo and in vivo systems using gnotobiotic rats. DNA studied was either plasmid DNA, naked plant DNA or plant DNA embedded in maize flour. Ex vivo experiments performed by incubating plant DNA in intestinal samples, showed that DNA is rapidly degraded in the upper part of the GI tract whereas degradation is less severe in the lower part. In contrast, plasmid DNA could be recovered throughout the GI tract when intestinal samples were taken up to 5 h after feeding rats with plasmid. Furthermore, DNA isolated from these intestinal samples was able to transform electro-competent Escherichia coli, showing that the plasmid was still biologically active. The results indicate that ingested DNA may persist in the GI tract and consequently may be present for uptake by intestinal bacteria.