Arabidopsis SNARE SYP132 impacts on PIP2;1 trafficking and function in salinity stress.

In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary.

Engineering a K channel 'sensory antenna' enhances stomatal kinetics, water use efficiency and photosynthesis.

Stomata of plant leaves open to enable CO entry for photosynthesis and close to reduce water loss via transpiration. Compared with photosynthesis, stomata respond slowly to fluctuating light, reducing assimilation and water use efficiency. Efficiency gains are possible without a cost to photosynthesis if stomatal kinetics can be accelerated.

SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis.

The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane (PM) H+-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity.

K Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic.

Cell expansion requires that ion transport and secretory membrane traffic operate in concert. Evidence from Arabidopsis () indicates that such coordination is mediated by physical interactions between subsets of so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which drive the final stages of vesicle fusion, and K channels, which facilitate uptake of the cation to maintain cell turgor pressure as the cell expands. However, the sequence of SNARE binding with the K channels and its interweaving within the events of SNARE complex assembly for exocytosis remains unclear.

SNAREs SYP121 and SYP122 Mediate the Secretion of Distinct Cargo Subsets.

SNARE (soluble -ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion and contribute to homoeostasis, pathogen defense, cell expansion, and growth in plants. In Arabidopsis (), two homologous Qa-SNAREs, SYNTAXIN OF PLANTS121 (SYP121) and SYP122, facilitate the majority of secretory traffic to the plasma membrane, and the single mutants are indistinguishable from wild-type plants in the absence of stress, implying a redundancy in their functions. Nonetheless, several studies suggest differences among the secretory cargo of these SNAREs.

Gating control and K uptake by the KAT1 K channel leaveraged through membrane anchoring of the trafficking protein SYP121.

Vesicle traffic is tightly coordinated with ion transport for plant cell expansion through physical interactions between subsets of vesicle-trafficking (so-called SNARE) proteins and plasma membrane Kv channels, including the archetypal inward-rectifying K channel, KAT1 of Arabidopsis. Ion channels open and close rapidly over milliseconds, whereas vesicle fusion events require many seconds. Binding has been mapped to conserved motifs of both the Kv channels and the SNAREs, but knowledge of the temporal kinetics of their interactions, especially as it might relate to channel gating and its coordination with vesicle fusion remains unclear.

VAMP721 Conformations Unmask an Extended Motif for K+ Channel Binding and Gating Control.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a major role in membrane fusion and contribute to cell expansion, signaling, and polar growth in plants. The SNARE SYP121 of Arabidopsis thaliana that facilitates vesicle fusion at the plasma membrane also binds with, and regulates, K channels already present at the plasma membrane to affect K uptake and K-dependent growth. Here, we report that its cognate partner VAMP721, which assembles with SYP121 to drive membrane fusion, binds to the KAT1 K channel via two sites on the protein, only one of which contributes to channel-gating control.

Commandeering Channel Voltage Sensors for Secretion, Cell Turgor, and Volume Control.

Control of cell volume and osmolarity is central to cellular homeostasis in all eukaryotes. It lies at the heart of the century-old problem of how plants regulate turgor, mineral and water transport. Plants use strongly electrogenic H-ATPases, and the substantial membrane voltages they foster, to drive solute accumulation and generate turgor pressure for cell expansion.

Analysis of crRNA Using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC ESI MS).

Mass spectrometry is a powerful tool for characterizing RNA. Here we describe a method for the identification and characterisation of crRNA using liquid chromatography interfaced with electrospray ionization mass spectrometry (LC ESI MS). The direct purification of crRNA from the Cascade-crRNA complex was performed using denaturing ion pair reverse phase chromatography.

Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic.

A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake.

In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus.

Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present.

Characterization and quantification of RNA post-transcriptional modifications using stable isotope labeling of RNA in conjunction with mass spectrometry analysis.

Mass spectrometry has emerged as an increasingly powerful tool for the identification and characterization of nucleic acids, in particular RNA post-transcriptional modifications. High mass accuracy instrumentation is often required to discriminate between compositional isomers of oligonucleotides. We have used stable isotope labeling ((15)N) of E.

RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system.

Structural basis for CRISPR RNA-guided DNA recognition by Cascade.

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner.

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions.

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments.