While the stress-responsive alternative sigma factor B has been identified in different species of Bacillus, Listeria, and Staphylococcus, the B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify B -dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate B -dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted B -dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ⌬sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant B -dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-foldhigher expression in the wild type). Of the 55 genes exhibiting B -dependent expression, 54 were preceded by a sequence resembling the B promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the B -dependent nature of a subset of eight selected promoter regions. Notably, the B -dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, B also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that B contributes to L. monocytogenes gene expression during infection.

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems. However, a few studies have also reported Tyr phosphorylation in plants, but the relative contribution of tyrosine phosphorylation to plant signal transduction has remained an open question. We present an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We combined a (15)N stable isotope metabolic labeling strategy with an immuno-affinity purification using phospho-tyrosine (pY) specific antibodies. This single enrichment strategy was sufficient to reproducibly identify and quantify pY containing peptides from total plant cell extract in a single LC-MS/MS run. We succeeded in identifying 149 unique pY peptides originating from 135 proteins, including a large set of different protein kinases and several receptor-like kinases. We used flagellin perception by Arabidopsis cells, a model system for pathogen triggered immune (PTI) signaling, to test our approach. We reproducibly quantified 23 pY peptides in 2 inversely labeled biological replicates identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive MAP kinases and 4 novel MAP kinases. With this targeted approach, we elucidate a new level of complexity in flagellin-induced MAP kinase activation.

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