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Aphids, the phloem sap feeders, probe into leaf tissues and activate a complex network of plant defense responses. Phytohormonal signaling plays a major role in this network; however, the dynamics of the signal spreading is yet to be clarified. Despite the growing knowledge about transcriptomic changes upon infestation, results often differ due to sampling, varying strongly between the tissues collected at the single feeding site, individual leaves, pooled infested leaves, or whole plant rosettes. This study focuses on activation of salicylic acid (SA) and jasmonic acid (JA) signals in Arabidopsis leaves during infestation by cabbage aphid (Brevicoryne brassicae) in high spatio-temporal resolution. We used genetically encoded fluorescent biosensors, histochemistry, and quantitative reverse transcription-PCR to precisely map activation of distinct branches of phytohormonal signaling. We found a rapid induction of SA and JA signaling markers in cells surrounding stylet puncture, colocalizing with callose deposition. For both PR1 and JAZ10, we detected activation at 24 h postinfestation (hpi), increasing and spreading along the veins until 72 hpi and, to a lesser extent, within the epidermal pavement cells. The SA signaling wave appeared in parallel with JA-associated signaling and continued to increase in time. Our results first show a local activation of SA- and JA-related responses after stylet penetration of Arabidopsis leaves and bring a detailed insight into the spatio-temporal complexity of plant defense activation during specialist aphid attack. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The integrity of the actin cytoskeleton is essential for plant immune signalling. Consequently, it is generally assumed that actin disruption reduces plant resistance to pathogen attack. Here, we demonstrate that actin depolymerization induced a dramatic increase in salicylic acid (SA) levels in Arabidopsis thaliana. Transcriptomic analysis showed that the SA pathway was activated due to the action of isochorismate synthase (ICS). The effect was also confirmed in Brassica napus. This raises the question of whether actin depolymerization could, under particular conditions, lead to increased resistance to pathogens. Thus, we explored the effect of pretreatment with actin-depolymerizing drugs on the resistance of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae, and on the resistance of an important crop Brassica napus to its natural fungal pathogen Leptosphaeria maculans. In both pathosystems, actin depolymerization activated the SA pathway, leading to increased plant resistance. To our best knowledge, we herein provide the first direct evidence that disruption of the actin cytoskeleton can actually lead to increased plant resistance to pathogens, and that SA is crucial to this process.

Background and Aims We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2. Methods To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. Key Results We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. Conclusions Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.

International audience; Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

International audience; Actin cytoskeleton is indispensable for plant cell integrity. Besides, increasing evidences illustrate its crucial role in plant responses to environment, including defence against pathogens. Recently, we have demonstrated that pre-treatment with actin disrupting drugs latrunculin B (latB) and cytochalasin E can enhance plant resistance against bacterial and fungal pathogens via activation of salicylic acid (SA) pathway. Here, we show that actin depolymerization in Arabidopsis thaliana seedlings not only triggers SA biosynthesis by ICS1, but also induces callose deposition via callose synthase PMR4. This effect is SA-independent since still present in mutants that do not accumulate SA. LatB also triggers the expression of several defence related genes. We could distinguish genes, induced in a SA-dependent manner (PR1, WRKY38, ICS1) and those that are SA-independent (PR2, PAD4, BAP1). As actin cytoskeleton is tightly connected with membrane trafficking, we assayed the effect of latB on mutant plants invalidated in phosphatidylinositol 4-kinase beta1 and beta2 (PI4Kβ1β2). Deficiency in PI4Kβ1β2 enhanced latB-triggered actin filaments depolymerisation. Yet, it did not lead to a stronger callose deposition or SA biosynthesis in response to latB. Surprisingly, introduction of NahG construct or pad4 mutation in pi4kß1ß2 background had much lower effect on SA induction and SA-dependent gene expression changes than in wild type. We can thus conclude that actin disruption itself triggers immune-like responses: there is an induction of SA via PAD4 coupled to ICS1; it leads to the induction of PR1 and WRKY38, and this requires a functional PI4Kβ1β2 to be properly regulated. However, an alternative, SA-independent, pathway also exists that leads to the enhanced expression of PR2 and to callose accumulation.

Phosphatidylinositol-4-kinases β1 and β2 (PI4Kβ1/PI4Kβ2), which are responsible for phosphorylation of phosphatidylinositol to phosphatidylinositol-4-phosphate, have important roles in plant vesicular trafficking. Moreover, PI4Kβ1/PI4Kβ2 negatively regulates biosynthesis of phytohormone salicylic acid (SA), a key player in plant immune responses. The study focused on the effect of PI4Kβ1/PI4Kβ2 deficiency and SA level on the proteome of microsomal fraction. For that purpose we used four Arabidopsis thaliana genotypes: wild type; double mutant with impaired function of PI4Kβ1/PI4Kβ2 (pi4kβ1/pi4kβ2) exhibiting high SA level; sid2 mutant with impaired SA biosynthesis depending on the isochorismate synthase 1 and triple mutant sid2/pi4kβ1/pi4kβ2. We identified 1797 proteins whose levels were changed between genotypes. We showed that increased SA concentration affected the levels of 473 proteins. This includes typical SA pathway markers but also points to connections between SA pathway and clathrin-independent endocytosis (flotillins) and exocytosis/protein secretion (syntaxins, tetraspanin) to be investigated in future. In contrast to SA, the absence of PI4Kβ1/PI4Kβ2 itself affected only 27 proteins. Among them we identified CERK1, a receptor for chitin. Although PI4Kβ1/PI4Kβ2 deficiency itself did not have a substantial impact on the proteome of the microsomal fraction, our data clearly show that it enhances proteome changes when SA pathway is modulated in parallel.

We show that both abscisic acid (ABA) and salicylic acid (SA) inhibit in vivo basal PI-PLC activity in Arabidopsis suspension cells. However, SA also activates in vivo phosphatidylinositol phosphorylation, which ABA does not. The transcriptome response of Arabidopsis suspension cells to a 4 h ABA treatment was compared to that of cells treated with U73122 or wortmannin, inhibitors of phospholipase C (PLC) and of the phosphatidylinositol-4-kinase that provides substrates to PLC, respectively. A clear over representation of genes for which ABA and these inhibitors have similar effects can be observed. Furthermore, 28% of ABA-induced genes were also induced by SA, while 40% of ABA-repressed genes were also repressed by SA. A similarity search in publically accessible transcriptome data confirms that those genes are also similarly regulated in response to ABA and SA in seedlings. We have shown that some of the SA responsive genes responded via an increase in phosphorylated phosphatidylinositol (phosphoinositides), while others did so via the activation of a phospholipase D pathway, and that the SA response of other genes was mimicked by the inhibition of PI-PLC. Interestingly, the pool of genes similarly regulated by SA and ABA is impoverished in genes responsive to SA via phosphoinositides but is enriched in those for which the response to SA is mimicked by the inhibition of PI-PLC. The increase in phosphoinositide appears to be an important process explaining the SA-specific response, while inhibition of a basal PI-PLC might contribute to the common ABA and SA transcriptomic responses.

The phytohormone salicylic acid (SA) has a crucial role in plant physiology. Its role is best described in the context of plant response to pathogen attack. During infection, SA is rapidly accumulated throughout the green tissues and is important for both local and systemic defences. However, some genetic/metabolic variations can also result in SA overaccumulation in plants, even in basal conditions. To date, more than forty Arabidopsis thaliana mutants have been described as having enhanced endogenous SA levels or constitutively activated SA signalling pathways. In this study, we established a collection of mutants containing different SA levels due to diverse genetic modifications and distinct gene functions. We chose prototypic SA-overaccumulators (SA-OAs), such as bon1-1, but also &ldquo, non-typical&rdquo, ones such as exo70b1-1, the selection of OA is accompanied by their crosses with SA-deficient lines. Here, we extensively studied the plant development and SA level/signalling under various growth conditions in soil and in vitro, and showed a strong negative correlation between rosette size, SA content and PR1/ICS1 transcript signature. SA-OAs (namely cpr5, acd6, bon1-1, fah1/fah2 and pi4k&beta, 1&beta, 2) had bigger rosettes under high light conditions, whereas WT plants did not. Our data provide new insights clarifying a link between SA and plant behaviour under environmental stresses. The presented SA mutant collection is thus a suitable tool to shed light on the mechanisms underlying trade-offs between growth and defence in plants.

Brassinosteroids (BRs) are steroid hormones regulating various aspects of plant metabolism, including growth, development and stress responses. However, little is known about the mechanism of their impact on antioxidant systems and phospholipid turnover. Using tobacco plants overexpressing H+/Ca2+vacuolar Arabidopsis antiporter CAX1, we showed the role of Ca2+ ion balance in the reactive oxygen species production and rapid phosphatidic acid accumulation induced by exogenous BR. Combination of our experimental results with public transcriptomic and proteomic data revealed a particular role of Ca2+-dependent phospholipid metabolizing enzymes in BR signaling. Here we provide novel insights into the role of calcium balance and lipid-derived second messengers in plant responses to exogenous BRs and propose a complex model integrating BR-mediated metabolic changes with phospholipid turnover.

Introductory paragraphThe integrity of the actin cytoskeleton is essential for plant immune signalling1. Consequently, it is generally assumed that actin disruption reduces plant resistance to pathogen attack2-4. However, in a previous study, it was shown that actin depolymerisation triggers the salicylic acid (SA) signalling pathway5, which is interesting because increased SA is associated with enhanced plant resistance to pathogen attack6,7. Here, we attempt to resolve this seeming inconsistency by showing that the relationship between actin depolymerization and plant resistance is more complex than currently thought. We investigate the precise nature of this relationship using two completely different plant pathosystems: i) a model plant (Arabidopsis thaliana) and a bacterial pathogen (Pseudomonas syringae), and ii) an important crop (Brassica napus) and a fungal pathogen (Leptosphaeria maculans). We demonstrate that actin depolymerization induces a dramatic increase in SA levels and that the increased SA is biosynthesized by the isochorismate synthase pathway. In both pathosystems, this phenomenon leads to increased plant resistance.

Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.