The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.

Publisher Correction: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guard cells are of an oscillatory nature.

Changes in cytosolic free calcium ([Ca(2+)]cyt) are an early and essential element of signalling networks activated by the perception of pathogen-associated molecular patterns (PAMPs), such as flg22. The flg22-induced calcium signal has been described on whole-plant, but not on single-cell scale so far. Also, the Ca(2+) sources and channels contributing to its generation are still obscure. Ratiometric fluorescence imaging employing the calcium reporter Yellow Cameleon 3.6 was performed to analyse the flg22-induced calcium signature in single guard cells of Arabidopsis thaliana. Calcium stores and channel types involved in its generation were determined by a pharmacological approach. In contrast to the calcium signal determined on whole-plant level, the signature on single-cell level is not characterized by one sustained response, but by oscillations in [Ca(2+)]cyt. These oscillations were abolished by EGTA and lanthanum, as well as by U73122, neomycin and TMB-8, but only partially or not at all affected by inhibitors of glutamate receptor-like channels and cyclic nucleotide-gated channels. Our analyses suggest that the response observed on whole-plant level is the summary of oscillations occurring in single cells. Parallel to external calcium, influx via channels located at internal stores contributes to the signal.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

AtPTR4 and AtPTR6 are differentially expressed, tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family.

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.

The Small Ras Superfamily GTPase Rho4 of the Maize Anthracnose Fungus Colletotrichum graminicola Is Required for ß-1,3-glucan Synthesis, Cell Wall Integrity, and Full Virulence.

Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the ß-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343-350). Here, we show that Rho4 is required for ß-1,3-glucan synthesis. Accordingly, Δrho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Δrho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δrho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δrho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling.

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of "non-self") or damaged plant cells (indicative of "infected-self"), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.

Calcium-Nutrient and Messenger.

Calcium is an essential element needed for growth and development of plants under both non-stressed and stress conditions. It thereby fulfills a dual function, being not only an important factor for cell wall and membrane stability, but also serving as a second messenger in many developmental and physiological processes, including the response of plants to biotic stress. The perception of non-self hereby induces an influx of calcium ions (Ca2+) into the cytosol, which is decoded into downstream responses ultimately leading to defense. Maintaining intracellular Ca2+ homeostasis is crucial for the ability to generate this signal. This review will describe the current knowledge of the mechanisms involved in uptake and transport of calcium as well as cellular homeostasis and signal generation, describing known genes involved and discussing possible implications the plant's nutritional status with regard to calcium might have on immunity.

No Silver Bullet - Canonical Poly(ADP-Ribose) Polymerases (PARPs) Are No Universal Factors of Abiotic and Biotic Stress Resistance of Arabidopsis thaliana.

Abiotic and biotic stress can have a detrimental impact on plant growth and productivity. Hence, there is a substantial demand for key factors of stress responses to improve yield stability of crops. Members of the poly(ADP-ribose)polymerase (PARP) protein family, which post-translationally modify (PARylate) nuclear proteins, have been suggested as such universal determinants of plant stress responses. A role under abiotic stress has been inferred from studies in which a genetic or, more commonly, pharmacological inhibition of PARP activity improved the performance of stressed plants. To further elucidate the role of PARP proteins under stress, T-DNA knockout mutants for the three Arabidopsis thaliana PARP genes were subjected to drought, osmotic, salt, and oxidative stress. To exclude a functional redundancy, which was indicated by a transcriptional upregulation of the remaining parp genes, a parp triple mutant was generated. Surprisingly, parp mutant plants did not differ from wild type plants in any of these stress experiments, independent from the number of PARP genes mutated. The parp triple mutant was also analyzed for callose formation in response to the pathogenassociated molecular pattern flg22. Unexpectedly, callose formation was unaltered in the mutant, albeit pharmacological PARP inhibition robustly blocked this immune response, confirming previous reports. Evidently, pharmacological inhibition appears to be more robust than the abolition of all PARP genes, indicating the presence of so-far undescribed proteins with PARP activity. This was supported by the finding that protein PARylation was not absent, but even increased in the parp triple mutant. Candidates for novel PARP-inhibitor targets may be found in the SRO protein family. These proteins harbor a catalytic PARP-like domain and are centrally involved in stress responses. Molecular modeling analyses, employing animal PARPs as templates, indeed indicated a capability of the SRO proteins RCD1 and SRO1 to bind nicotinamide-derived inhibitors. Collectively, the results of our study suggest that the stress-related phenotypes of parp mutants are highly conditional, and they call for a reconsideration of PARP inhibitor studies. In the context of this study, we also propose a unifying nomenclature of PARP genes and parp mutants, which is currently highly inconsistent and redundant.

AtPTR1 and AtPTR5 transport dipeptides in planta.

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.

Salicylic acid (SA)-dependent gene activation can be uncoupled from cell death-mediated gene activation: the SA-inducible NIMIN-1 and NIMIN-2 promoters, unlike the PR-1a promoter, do not respond to cell death signals in tobacco.

SUMMARY Tobacco pathogenesis-related (PR) genes of group 1 are induced during pathogen defence (hypersensitive response, HR, and systemic acquired resistance, SAR), after exogenous application of salicylic acid (SA), and by developmental cues. Likewise, SA enhances transcripts for Arabidopsis NIMIN-1 and NIMIN-2, which interact with NPR1/NIM1, a key regulator of SAR. To further illuminate gene activation during pathogen defence, reporter gene expression from the NIMIN-1 and NIMIN-2 promoters was analysed in transgenic tobacco plants in direct comparison to PR-1 gene expression. NIMIN[GUS] chimeric genes were highly sensitive to SA, whereas NIMIN[GUS], unlike PR1a[GUS], expression was only weak in necrotic tissue exhibiting HR. Furthermore, PR-1a, but not NIMIN, promoter constructs were activated systemically in response to local cell death elicited by expression of the proapoptotic Bax gene. Conversely, NIMIN-1[GUS] expression was completely suppressed during pathogen defence in plants depleted from SA, whereas PR-1 proteins still accumulated in necrotic tissue. These findings demonstrate that SA-dependent gene activation can be uncoupled from cell death-induced gene activation. Whereas PR-1a induction during the HR and SAR responses is mediated by HR-associated signals and SA, activation of the NIMIN-1 and NIMIN-2 promoters in infected tobacco relies on SA, but not on cell death signals.

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis.

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.