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The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell. [ABSTRACT FROM AUTHOR]

NEP1 (necrosis- and ethylene-inducing peptide 1)-like proteins ( NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP-treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short-term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa ( Theobroma cacao L.). Approximately 100 protein spots were collected from 2- DE gels in each proteome, with one-third showing more than twofold differences in the expression values. Fifty-three such proteins were identified by mass spectrometry ( MS)/ MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide-binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry ( GC)/ MS and semi-quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo-inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction-oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP-mediated cell death signaling in plants. [ABSTRACT FROM AUTHOR]