Olga Valentová

Phosphatidylinositol 4-Kinase Activation Is an Early Response to Salicylic Acid in Arabidopsis Suspension Cells

Salicylic acid (SA) has a central role in defense against pathogen attack. In addition, its role in such diverse processes as germination, flowering, senescence, and thermotolerance acquisition has been documented. However, little is known about the early signaling events triggered by SA. Using Arabidopsis (Arabidopsis thaliana) suspension cells as a model, it was possible to show by in vivo metabolic phospholipid labeling with 33Pi that SA addition induced a rapid and early (in few minutes) decrease in a pool of phosphatidylinositol (PI). This decrease paralleled an increase in PI 4-phosphate and PI 4,5-bisphosphate. These changes could be inhibited by two different inhibitors of type III PI 4-kinases, phenylarsine oxide and 30 μm wortmannin; no inhibitory effect was seen with 1 μm wortmannin, a concentration inhibiting PI 3-kinases but not PI 4-kinases. We therefore undertook a study of the effects of wortmannin on SA-responsive transcriptomes. Using the Complete Arabidopsis Transcriptome MicroArray chip, we could identify 774 genes differentially expressed upon SA treatment. Strikingly, among these genes, the response to SA of 112 of them was inhibited by 30 μm wortmannin, but not by 1 μm wortmannin.

Mutual regulation of plant phospholipase D and the actin cytoskeleton

Membrane lipids and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane-cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD-actin interactions. Inhibition of PLD by n-butanol compromises pollen tube actin, and PA rescues the detrimental effect of n-butanol on F-actin, showing clearly the importance of the PLD-PA interaction for pollen tube F-actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDbeta1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP(2)-dependent PLD (PLDbeta) is specifically enhanced by F-actin and inhibited by G-actin. We then identified the NtPLDbeta1 domain responsible for actin interactions. Using sequence- and structure-based analysis, together with site-directed mutagenesis, we identified Asn323 and Thr382 of NtPLDbeta1 as the crucial amino acids in the actin-interacting fold. The effect of antisense-mediated suppression of NtPLDbeta1 or NtPLDdelta on pollen tube F-actin dynamics shows that NtPLDbeta1 is the active partner in PLD-actin interplay. The positive feedback loop created by activation of PLDbeta by F-actin and of F-actin by PA provides an important mechanism to locally increase membrane-F-actin dynamics in the cortex of plant cells.

Phospholipase D Activation Is an Early Component of the Salicylic Acid Signaling Pathway in Arabidopsis Cell Suspensions

Salicylic acid (SA) plays a central role in defense against pathogen attack, as well as in germination, flowering, senescence, and the acquisition of thermotolerance. In this report we investigate the involvement of phospholipase D (PLD) in the SA signaling pathway. In presence of exogenous primary alcohols, the production of phosphatidic acid by PLD is diverted toward the formation of phosphatidylalcohols through a reaction called transphosphatidylation. By in vivo metabolic phospholipid labeling with 33Pi, PLD activity was found to be induced 45 min after addition of SA. We show that incubation of Arabidopsis (Arabidopsis thaliana) cell suspensions with primary alcohols inhibited the induction of two SA-responsive genes, PATHOGENESIS-RELATED1 and WRKY38, in a dose-dependent manner. This inhibitory effect was more pronounced when the primary alcohols were more hydrophobic. Secondary or tertiary alcohols had no inhibitory effect. These results provide compelling arguments for PLD activity being upstream of the induction of these genes by SA. A subsequent study of n-butanol effects on the SA-responsive transcriptome identified 1,327 genes differentially expressed upon SA treatment. Strikingly, the SA response of 380 of these genes was inhibited by n-butanol but not by tert-butanol. A detailed analysis of the regulation of these genes showed that PLD could act both positively and negatively, either on gene induction or gene repression. The overlap with the previously described phosphatidylinositol-4-kinase pathway is discussed.

Bio-based resistance inducers for sustainable plant protection against pathogens

An increasing demand for environmentally acceptable alternative for traditional pesticides provides an impetus to conceive new bio-based strategies in crop protection. Employing induced resistance is one such strategy, consisting of boosting the natural plant immunity. Upon infections, plants defend themselves by activating their immune mechanisms. These are initiated after the recognition of an invading pathogen via the microbe-associated molecular patterns (MAMPs) or other microbe-derived molecules. Triggered responses inhibit pathogen spread from the infected site. Systemic signal transport even enables to prepare, i.e. prime, distal uninfected tissues for more rapid and enhanced response upon the consequent pathogen attack. Similar defense mechanisms can be triggered by purified MAMPs, pathogen-derived molecules, signal molecules involved in plant resistance to pathogens, such as salicylic and jasmonic acid, or a wide range of other chemical compounds. Induced resistance can be also conferred by plant-associated microorganisms, including beneficial bacteria or fungi. Treatment with resistance inducers or beneficial microorganisms provides long-lasting resistance for plants to a wide range of pathogens. This study surveys current knowledge on resistance and its mechanisms provided by microbe-, algae- and plant-derived elicitors in different crops. The main scope deals with bacterial substances and fungus-derived molecules chitin and chitosan and algae elicitors, including naturally sulphated polysaccharides such as ulvans, fucans or carageenans. Recent advances in the utilization of this strategy in practical crop protection are also discussed.

The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress

Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3 h to 6 h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of PNPC4:GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30 min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.

Phosphoglycerolipids are master players in plant hormone signal transduction.

Phosphoglycerolipids are essential structural constituents of membranes and some also have important cell signalling roles. In this review, we focus on phosphoglycerolipids that are mediators in hormone signal transduction in plants. We first describe the structures of the main signalling phosphoglycerolipids and the metabolic pathways that generate them, namely the phospholipase and lipid kinase pathways. In silico analysis of Arabidopsis transcriptome data provides evidence that the genes encoding the enzymes of these pathways are transcriptionally regulated in responses to hormones, suggesting some link with hormone signal transduction. The involvement of phosphoglycerolipid signalling in the early responses to abscisic acid, salicylic acid and auxins is then detailed. One of the most important signalling lipids in plants is phosphatidic acid. It can activate or inactivate protein kinases and/or protein phosphatases involved in hormone signalling. It can also activate NADPH oxidase leading to the production of reactive oxygen species. We will interrogate the mechanisms that allow the activation/deactivation of the lipid pathways, in particular the roles of G proteins and calcium. Mediating lipids thus appear as master players of cell signalling, modulating, if not controlling, major transducing steps of hormone signals.

Recognition of Avirulence Gene AvrLm1 from Hemibiotrophic Ascomycete Leptosphaeria maculans Triggers Salicylic Acid and Ethylene Signaling in Brassica napus

Interaction of a plant with a fungal pathogen is an encounter with hundreds of molecules. In contrast to this, a single molecule often decides between the disease and resistance. In the present article, we describe the defense responses triggered by AvrLm1, an avirulence gene from a hemibiotrophic ascomycete, Leptosphaeria maculans, responsible for an incompatible interaction with Brassica napus. Using multiple hormone quantification and expression analysis of defense-related genes, we investigated signaling events in Rlm1 plants infected with two sister isolates of L. maculans differentiated by the presence or absence of AvrLm1. Infection with the isolate carrying AvrLm1 increased the biosynthesis of salicylic acid (SA) and induced expression of the SA-associated genes ICS1, WRKY70, and PR-1, a feature characteristic of responses to biotrophic pathogens and resistance gene-mediated resistance. In addition to SA-signaling elements, we also observed the induction of ASC2a, HEL, and CHI genes associated with ethylene (ET) signaling. Pharmacological experiments confirmed the positive roles of SA and ET in mediating resistance to L. maculans. The unusual cooperation of SA and ET signaling might be a response to the hemibiotrophic nature of L. maculans. Our results also demonstrate the profound difference between the natural host B. napus and the model plant Arabidopsis in their response to L. maculans infection.

Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine-hydrolysing phospholipase C in tobacco cells.

• Aluminium ions (Al) have been recognized as a major toxic factor for crop production in acidic soils. This study aimed to assess the impact of Al on the activity of phosphatidylcholine-hydrolysing phospholipase C (PC-PLC), a new member of the plant phospholipase family. • We labelled the tobacco cell line BY-2 and pollen tubes with a fluorescent derivative of phosphatidylcholine and assayed for patterns of fluorescently labelled products. Growth of pollen tubes was analysed. • We observed a significant decrease of labelled diacylglycerol (DAG) in cells treated with AlCl(3). Investigation of possible metabolic pathways that control DAG generation and consumption during the response to Al showed that DAG originated from the reaction catalysed by PC-PLC. The growth of pollen tubes was retarded in the presence of Al and this effect was accompanied by the decrease of labelled DAG similar to the case of the BY-2 cell line. The growth of pollen tubes arrested by Al was rescued by externally added DAG. • Our observation strongly supports the role of DAG generated by PC-PLC in the response of tobacco cells to Al.

β-aminobutyric acid protects Brassica napus plants from infection by Leptosphaeria maculans. Resistance induction or a direct antifungal effect?

Resistance to infection in plants can be induced by treatment with various chemicals. One such compound is β-aminobutyric acid (BABA). Its positive effect on disease resistance has been noted in several pathosystems. Here we demonstrate that treatment with BABA protects Brassica napus plants from infection by the fungal pathogen Leptosphaeria maculans. Surprisingly, BABA also displayes in vitro antifungal activity against L. maculans with EC50 similar to the fungicide tebuconazole. Both spore germination and hyphal growth were affected. The toxic effect can be reverted by addition of trypton to the culture medium. We hypothesised that BABA might inhibit inorganic nitrogen assimilation. Suppression of disease progression in plants and antifungal activity in vitro was weaker for α-aminobutyric acid and negligible for γ-aminobutyric acid. In contrast to a resistance inducer benzothiadiazole, the effect of BABA on disease development was nearly independent of the timing of treatment, indicating possible antifungal activity in planta. On the other hand, quantification of multiple hormones and an expression analysis have shown that treatment with BABA induces a synthesis of salicylic acid (SA) and expression of SA marker gene PR-1, but no evidence was observed for priming of SA responses to L. maculans. While we have not conclusively demonstrated how BABA suppresses the disease progression, our results do indicate that antifungal activity is another mechanism by which BABA can protect plants from infection.

Plant hormones in defense response of Brassica napus to Sclerotinia sclerotiorum – Reassessing the role of salicylic acid in the interaction with a necrotroph

According to general model, jasmonic acid (JA) and ethylene (ET) signaling pathways are induced in Arabidopsis after an attack of necrotroph, Sclerotinia sclerotiorum (Lib.) de Bary. However, abscisic acid (ABA) and salicylic acid (SA) also seem to play a role. While signaling events in Arabidopsis have been intensively studied recently, information for the natural host Brassica napus is limited. In this study, multiple plant hormone quantification and expression analysis of marker genes of the signaling pathways was used to gain a complete view of the interaction of B. napus with S. sclerotiorum. Strong response of ET biosynthetic gene ACS2 was observed, accompanied by increases of SA and JA levels that correspond to the elevated expression of marker genes PR1 and LOX3. Interestingly, the level of ABA and the expression of its marker gene RD26 were also elevated. Furthermore, induction of the SA-dependent defense decreased disease symptoms. In addition, SA signaling is suggested as a possible target for manipulation by S. sclerotiorum. A gene for putative chorismate mutase SS1G_14320 was identified that is highly expressed during infection but not in vitro. Our results bring the evidence of SA involvement in the interaction of plant with the necrotroph that conflict with the current model.