Henrik U. Stotz

Oxalate Production by Sclerotinia sclerotiorum Deregulates Guard Cells during Infection

Oxalic acid is a virulence factor of several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, but the detailed mechanisms by which oxalic acid affects host cells and tissues are not understood. We tested the hypothesis that oxalate induces foliar wilting during fungal infection by manipulating guard cells. Unlike uninfected leaves, stomatal pores of Vicia faba leaves infected with S. sclerotiorum are open at night. This cellular response appears to be dependent on oxalic acid because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. In contrast to oxalate-deficient S. sclerotiorum, wild-type fungus causes an increase in stomatal conductance and transpiration as well as a decrease in plant biomass. Green fluorescent protein-tagged S. sclerotiorum emerges through open stomata from the uninfected abaxial leaf surface for secondary colonization. Exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening. Guard cells treated with oxalic acid accumulate potassium and break down starch, both of which are known to contribute to stomatal opening. Oxalate interferes with abscisic acid (ABA)-induced stomatal closure. The Arabidopsis (Arabidopsis thaliana) L. Heynh. mutants abi1, abi3, abi4, and aba2 are more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. We conclude that oxalate acts via (1) accumulation of osmotically active molecules to induce stomatal opening and (2) inhibition of ABA-induced stomatal closure.

Plant defensins: Defense, development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity, and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.

Effector-triggered defence against apoplastic fungal pathogens

Highlights • ETD is triggered by RLPs that engage the receptor-like kinase SOBIR1.• ETD triggers cell wall-related defence responses.• ETD does not eliminate apoplastic pathogens.

TGA transcription factors and jasmonate-independent COI1 signalling regulate specific plant responses to reactive oxylipins

Jasmonates and phytoprostanes are oxylipins that regulate stress responses and diverse physiological and developmental processes. 12-Oxo-phytodienoic acid (OPDA) and phytoprostanes are structurally related electrophilic cyclopentenones, which activate similar gene expression profiles that are for the most part different from the action of the cyclopentanone jasmonic acid (JA) and its biologically active amino acid conjugates. Whereas JA–isoleucine signals through binding to COI1, the bZIP transcription factors TGA2, TGA5, and TGA6 are involved in regulation of gene expression in response to phytoprostanes. Here root growth inhibition and target gene expression were compared after treatment with JA, OPDA, or phytoprostanes in mutants of the COI1/MYC2 pathway and in different TGA factor mutants. Inhibition of root growth by phytoprostanes was dependent on COI1 but independent of jasmonate biosynthesis. In contrast, phytoprostane-responsive gene expression was strongly dependent on TGA2, TGA5, and TGA6, but not dependent on COI1, MYC2, TGA1, and TGA4. Different mutant and overexpressing lines were used to determine individual contributions of TGA factors to cyclopentenone-responsive gene expression. Whereas OPDA-induced expression of the cytochrome P450 gene CYP81D11 was primarily regulated by TGA2 and TGA5, the glutathione S-transferase gene GST25 and the OPDA reductase gene OPR1 were regulated by TGA5 and TGA6, but less so by TGA2. These results support the model that phytoprostanes and OPDA regulate differently (i) growth responses, which are COI1 dependent but jasmonate independent; and (ii) lipid stress responses, which are strongly dependent on TGA2, TGA5, and TGA6. Identification of molecular components in cyclopentenone signalling provides an insight into novel oxylipin signal transduction pathways.

2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions

2-Cys peroxiredoxins and thylakoid ascorbate peroxidase act together to protect plants against high light damage by creating a water-water cycle and restricting light-induced redox signaling. Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.

Jasmonate-Dependent and COI1-Independent Defense Responses Against Sclerotinia sclerotiorum in Arabidopsis thaliana: Auxin is Part of COI1-Independent Defense Signaling

The jasmonate receptor COI1 is known to facilitate plant defense responses against necrotrophic pathogens, including the ascomycete Sclerotinia sclerotiorum. However, it is not known to what extent jasmonates contribute to defense nor have COI1-independent defense pathways been sufficiently characterized. Here we show that the susceptibility to S. sclerotiorum of the aos mutant, deficient in biosynthesis of jasmonic acid (JA) and its precursor 12-oxophytadienoic acid, was elevated to a level reminiscent of that of hypersusceptible coi1 mutants. In contrast, susceptibility of the JA-deficient opr3 mutant was comparable with that of the wild type. A set of 99 genes responded similarly to infection with S. sclerotiorum in wild-type and coi1 mutant leaves. Expression of this COI1-independent gene set correlated with known differences in gene expression between wild-type plants and a mutant in the transcriptional repressor auxin response factor 2 (arf2). Susceptibility to S. sclerotiorum was reduced in two arf2 mutants early during infection, implicating ARF2 as a negative regulator of defense responses against this pathogen. Hypersusceptibility of an axr1 mutant to S. sclerotiorum confirmed the contribution of auxin action to defense responses against this fungal pathogen.

ABA signaling inhibits oxalate-induced production of reactive oxygen species and protects against Sclerotinia sclerotiorum in Arabidopsis thaliana

Oxalic acid is an essential virulence factor of Sclerotinia sclerotiorum that elicits wilting symptoms in infected host plants. Foliar wilting in response to oxalic acid is known to be dependent on an increase in stomatal conductance. To determine whether stomatal regulation controls susceptibility to S. sclerotiorum, abscisic acid-insensitive and open stomata mutants of Arabidopsis thaliana were analyzed. Whereas abscisic acid-insensitive mutants were hypersusceptible to S. sclerotiorum, open stomata mutants were as susceptible as wild type. It was concluded that stomatal regulation does not control susceptibility to S. sclerotiorum because open stomata mutants are known to only impair guard cells whereas abscisic acid-insensitive mutants also affect other cell types. Guard cell-independent processes also control sensitivity to oxalic acid because oxalic acid was more toxic to abscisic acid-insensitive mutants than to open stomata mutants. To explore a possible mechanism of toxicity, production of reactive oxygen species was measured in plant cells after exposure to oxalic acid. Oxalic acid was found to elicit reactive oxygen species production independently of abscisic acid. Nevertheless, cancellation of reactive oxygen species elicitation after co-stimulation of wild-type guard cells with oxalic acid and abscisic acid provided evidence for antagonistic interaction between both molecules.

Review of combinations of experimental and computational techniques to identify and understand genes involved in innate immunity and effector-triggered defence

The innate immune system includes a first layer of defence that recognises conserved pathogen-associated molecular patterns that are essential for microbial fitness. Resistance (R) gene-based recognition of pathogen effectors, which function in modulation or avoidance of host immunity, activates a second layer of plant defence. In this review, experimental and computational techniques are considered to improve understanding of the plant immune system. Biocomputation contributes to discovery of the molecular genetic basis of host resistance against pathogens. Sequenced genomes have been used to identify R genes in plants. Resistance gene enrichment sequencing based on conserved protein domains has increased the number of R genes with nucleotide-binding site and leucine-rich repeat domains. Network analysis will contribute to an improved understanding of the innate immune system and identify novel genes for partial disease resistance. Machine learning algorithms are expected to become important in defining aspects of the immune system that are less well characterised, including identification of R genes that lack conserved protein domains.

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.

TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2

Reactive electrophile species (RES), including prostaglandins, phytoprostanes and 12-oxo phytodienoic acid (OPDA), activate detoxification responses in plants and animals. However, the pathways leading to the activation of defense reactions related to abiotic or biotic stress as a function of RES formation, accumulation or treatment are poorly understood in plants. Here, the thiol-modification of proteins, including the RES-activated basic region/leucine zipper transcription factor TGA2, was studied. TGA2 contains a single cysteine residue (Cys186) that was covalently modified by reactive cyclopentenones but not required for induction of detoxification genes in response to OPDA or prostaglandin A1. Activation of the glutathione-S-transferase 6 (GST6) promoter was responsive to cyclopentenones but not to unreactive cyclopentanones, including jasmonic acid suggesting that thiol reactivity of RES is important to activate the TGA2-dependent signaling pathway resulting in GST6 activation We show that RES modify thiols in numerous proteins in vivo, however, thiol reactivity alone appears not to be sufficient for biological activity as demonstrated by the failure of several membrane permeable thiol reactive reagents to activate the GST6 promoter.