Extracellular ATP targets Arabidopsis RIBONUCLEASE 1 to suppress mycotoxin stress-induced cell death.

Extracellular ATP is a purinergic signal with important functions in regulating plant growth and stress-adaptive responses, including programmed cell death. While signalling events proximate to receptor activation at the plasma membrane have been characterised, downstream protein targets and the mechanism of cell death activation/regulation are unknown. We designed a proteomic screen to identify ATP-responsive proteins in Arabidopsis cell cultures exposed to mycotoxin stress via fumonisin B1 (FB1) application. Arabidopsis RIBONUCLEASE 1 (RNS1) was identified by the screen, and transgenic plants overexpressing native RNS1 showed greater susceptibility to FB1, while a gene knockout rns1 mutant and antisense RNS1 transgenic plants were resistant to FB1-induced cell death. Native RNS1 complemented rns1 mutants and restored the cell death response to FB1, while a catalytically inactive version of the ribonuclease could not. The FB1 resistance of salicylic acid (SA)-depleted nahG-expressing plants was abolished by transformation with native RNS1, but not the catalytically dead version. The mechanism of FB1-induced cell death is activation of RNS1-dependent RNA cleavage, which is blocked by ATP via RNS1 suppression, or enhanced by SA through induction of RNS1 expression. Our study reveals RNS1 as a previously unknown convergence point of ATP and SA signalling in the regulation of stress-induced cell death.

Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors.

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.

The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination.

Gibberellic acid (GA)-mediated cell expansion initiates the seed-to-seedling transition in plants and is repressed by DELLA proteins. Using digital single-cell analysis, we identified a cellular subdomain within the midhypocotyl, whose expansion drives the final step of this developmental transition under optimal conditions. Using network inference, the transcription factor ATHB5 was identified as a genetic factor whose localized expression promotes GA-mediated expansion specifically within these cells. Both this protein and its putative growth-promoting target EXPANSIN3 are repressed by DELLA, and coregulated at single-cell resolution during seed germination. The cellular domains of hormone sensitivity were explored within the Arabidopsis (Arabidopsis thaliana) embryo by putting seeds under GA-limiting conditions and quantifying cellular growth responses. The middle and upper hypocotyl have a greater requirement for GA to promote cell expansion than the lower embryo axis. Under these conditions, germination was still completed following enhanced growth within the radicle and lower axis. Under GA-limiting conditions, the athb5 mutant did not show a phenotype at the level of seed germination, but it did at a cellular level with reduced cell expansion in the hypocotyl relative to the wild type. These data reveal that the spatiotemporal cell expansion events driving this transition are not determinate, and the conditional use of GA-ATHB5-mediated hypocotyl growth under optimal conditions may be used to optionally support rapid seedling growth. This study demonstrates that multiple genetic and spatiotemporal cell expansion mechanisms underlie the seed to seedling transition in Arabidopsis.

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection.

The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.

UDP-glucose pyrophosphorylase is a novel plant cell death regulator.

Programmed cell death (PCD) is an essential process that functions in plant organ sculpture, tissue differentiation, nutrient recycling, and defense against pathogen attack. A full understanding of the mechanism of PCD in plants is hindered by the limited identification of protein components of the complex signaling circuitry that underpins this important physiological process. Here we have used Arabidopsis thaliana and fumonisin B1 (FB1) to identify proteins that constitute part of the PCD signaling network. We made an inadvertent, but important observation that exogenous sucrose modulates FB1-induced cell death and identified sucrose-induced genes from publicly available transcriptomic data sets for reverse genetic analyses. Using transfer-DNA gene knockout plants, UDP-glucose pyrophosphorylase 1 (UGP1), a sucrose-induced gene, was demonstrated to be a critical factor that regulates FB1-induced PCD. We employed 2D-DiGE to identify proteomic changes preceding PCD after exposure of Arabidopsis to FB1 and used UGP1 knockout plants to refine the analysis and isolate downstream candidate proteins with a putative PCD regulatory function. Our results reveal chloroplasts as the predominantly essential organelles in FB1-induced PCD. Overall, this study reveals a novel function of UGP1 as a cell death regulator and provides candidate proteins likely recruited downstream in the activation of plant PCD.

Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase beta-subunit as a novel plant cell death regulator.

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase ß-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase ß-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.

A proteomics study of the induction of somatic embryogenesis in Medicago truncatula using 2DE and MALDI-TOF/TOF.

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.

The Top 10 oomycete pathogens in molecular plant pathology.

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.

A growth quantification assay for Hyaloperonospora arabidopsidis isolates in Arabidopsis thaliana.

There is a considerable interest in determining the role of individual oomycete effectors in promoting disease. Widely used strategies are based on manipulating effector-expression levels in the pathogen and by over-expressing particular effectors in the host by genetic transformation. In the case of the oomycete, Hyaloperonospora arabidopsidis (Hpa) genetic manipulation is not yet possible, so over-expression of predicted effectors in stably transformed Arabidopsis lines is used to investigate their capability for promoting virulence. Here, we describe a technique for quantifying pathogen growth based on the counting of asexual reproductive structures called sporangiophores in the compatible interaction between the Hpa isolate Noks1 and the Col-0 Arabidopsis accession.

A simple and fast protocol for the protein complex immunoprecipitation (Co-IP) of effector: host protein complexes.

Plant pathogens are responsible for enormous damage in natural and cultured ecosystems. One strategy most pathogenic organisms follow is the secretion of effector proteins that manipulate the host immune system to suppress defense responses. There is considerable interest in finding host targets of pathogen effectors as this helps to shape our understanding of how those proteins work in planta. The presented protocol describes a protein complex immunoprecipitation method aimed at verifying protein-protein interactions derived from protein complementation assays like Yeast-two-Hybrid.

Extracellular ATP: a modulator of cell death and pathogen defense in plants.

Living organisms acquire or synthesize high energy molecules, which they frugally conserve and use to meet their cellular metabolic demands. Therefore, it is surprising that ATP, the most accessible and commonly utilized chemical energy carrier, is actively secreted to the extracellular matrix of cells. It is now becoming clear that in plants this extracellular ATP (eATP) is not wasted, but harnessed at the cell surface to signal across the plasma membrane of the secreting cell and neighboring cells to control gene expression and influence plant development. Identification of the gene/protein networks regulated by eATP-mediated signaling should provide insight into the physiological roles of eATP in plants. By disrupting eATP-mediated signaling, we have identified pathogen defense genes as part of the eATP-regulated gene circuitry, leading us to the discovery that eATP is a negative regulator of pathogen defense in plants.(1) Previously, we reported that eATP is a key signal molecule that modulates programmed cell death in plants.(2) A complex picture is now emerging, in which eATP-mediated signaling cross-talks with signaling mediated by the major plant defense hormone, salicylic acid, in the regulation of pathogen defense and cell death.