Skip to main content

Main menu

  • For Authors
    • Submit a Manuscript
    • Instructions for Authors
  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
    • Focus Collections
    • Classics Collection
    • Upcoming Focus Issues
  • Advertisers
  • About
    • About the Journal
    • Editorial Board and Staff
  • Subscribers
  • Librarians
  • More
    • Alerts
    • Contact Us
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Plant Cell Teaching Tools
    • ASPB
    • Plantae

User menu

  • My alerts
  • Log in

Search

  • Advanced search
Plant Physiology
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Plant Cell Teaching Tools
    • ASPB
    • Plantae
  • My alerts
  • Log in
Plant Physiology

Advanced Search

  • For Authors
    • Submit a Manuscript
    • Instructions for Authors
  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
    • Focus Collections
    • Classics Collection
    • Upcoming Focus Issues
  • Advertisers
  • About
    • About the Journal
    • Editorial Board and Staff
  • Subscribers
  • Librarians
  • More
    • Alerts
    • Contact Us
  • Follow plantphysiol on Twitter
  • Visit plantphysiol on Facebook
  • Visit Plantae
Research ArticleArticles
You have accessRestricted Access

Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production

Juan Martín D’Ambrosio, Daniel Couto, Georgina Fabro, Denise Scuffi, Lorenzo Lamattina, Teun Munnik, Mats X. Andersson, María E. Álvarez, Cyril Zipfel, Ana M. Laxalt
Juan Martín D’Ambrosio
aInstituto de Investigaciones Biológicas IIB-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Mar del Plata, 7600 Mar del Plata, Argentina
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Daniel Couto
bSainsbury Laboratory, Norwich NR4 7UH, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Georgina Fabro
cCentro de Investigaciones en Química Biológica de Córdoba, UNC-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Córdoba, X5000HUA Cordoba, Argentina
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Denise Scuffi
aInstituto de Investigaciones Biológicas IIB-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Mar del Plata, 7600 Mar del Plata, Argentina
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lorenzo Lamattina
aInstituto de Investigaciones Biológicas IIB-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Mar del Plata, 7600 Mar del Plata, Argentina
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Lorenzo Lamattina
Teun Munnik
dSwammerdam Institute for Life Sciences, Section Plant Cell Biology, University of Amsterdam, 1098 XH Amsterdam, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Teun Munnik
Mats X. Andersson
eDepartment of Biological and Environmental Sciences, University of Gothenburg, SE-405 30 Gothenburg, Sweden
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Mats X. Andersson
María E. Álvarez
cCentro de Investigaciones en Química Biológica de Córdoba, UNC-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Córdoba, X5000HUA Cordoba, Argentina
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for María E. Álvarez
Cyril Zipfel
bSainsbury Laboratory, Norwich NR4 7UH, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Cyril Zipfel
Ana M. Laxalt
aInstituto de Investigaciones Biológicas IIB-Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Mar del Plata, 7600 Mar del Plata, Argentina
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Ana M. Laxalt
  • For correspondence: amlaxalt@mdp.edu.ar

Published October 2017. DOI: https://doi.org/10.1104/pp.17.00173

  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading
  • © 2017 American Society of Plant Biologists. All Rights Reserved.

Abstract

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC− and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi. However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Plants are constantly challenged by microbial pathogens, and to resist them, they exhibit various defense mechanisms. A first line of inducible defenses is triggered by the recognition of microbe-associated molecular patterns (MAMPs) by cell surface pattern recognition receptors (Antolín-Llovera et al., 2014). This recognition induces MAMP-triggered immunity (MTI), which confers resistance to multiple microbes (Couto and Zipfel, 2016). Adapted plant pathogens use secreted effector proteins to, among other things, interfere with MTI, resulting in the so-called effector-triggered susceptibility. Eventually, microbial effectors can become detected by intracellular nucleotide-binding Leu-rich repeat (NLR) proteins, triggering a second line of defense called effector-triggered immunity (ETI; Jones and Dangl, 2006).

After the recognition of MAMPs, a series of rapid responses are initiated, including an increase in cytosolic Ca2+, generation of apoplastic reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPKs) and Ca2+-dependent protein kinases (CDPKs), callose deposition, and stomatal closure (Boller and Felix, 2009; Segonzac and Zipfel, 2011). Among the best-studied responses to MAMPs are those triggered following the recognition of bacterial flagellin (or the derived peptide flg22) by the Arabidopsis (Arabidopsis thaliana) Leu-rich repeat receptor kinase (LRR-RK) FLAGELLIN SENSING2 (FLS2; Felix et al., 1999; Gómez-Gómez and Boller, 2000; Sun et al., 2013). Upon ligand recognition, FLS2 forms a complex with the LRR-RK BRASSINOSTEROID RECEPTOR1-ASSOCIATED KINASE1 (BAK1), also known as SOMATIC EMBRYOGENESIS-RELATED KINASE3 (SERK3; Chinchilla et al., 2007; Heese et al., 2007; Roux et al., 2011; Sun et al., 2013). This complex interacts with and phosphorylates the receptor-like cytoplasmic kinase BOTRYTIS INDUCED KINASE1 (BIK1; Veronese et al., 2006; Lu et al., 2010; Zhang et al., 2010). Upon activation, BIK1 phosphorylates the plasma membrane NADPH oxidase RBOHD, thus priming apoplastic ROS production (Kadota et al., 2014; Li et al., 2014).

Several lipids and lipid-derived metabolites have been shown to function in signal transduction pathways leading to the activation of plant defense responses (Laxalt and Munnik, 2002; Munnik and Vermeer, 2010; Hung et al., 2014; Hong et al., 2016). Specifically, phosphoinositide-specific phospholipase C (PI-PLC) is rapidly activated in plant cells after the recognition of different MAMPs, such as xylanase, flg22, and chitosan (van der Luit et al., 2000; Laxalt et al., 2007; Raho et al., 2011), or of pathogen effector proteins (de Jong et al., 2004; Andersson et al., 2006). PI-PLC catalyzes the hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5) bisphosphate (PIP2) to generate water-soluble inositol bisphosphate (IP2) or IP3 and diacylglycerol (DAG), which remains in the membrane. In plants, DAG produced by PI-PLC activity is phosphorylated by DAG kinase (DGK) to produce phosphatidic acid (PA), which regulates several protein targets (Arisz et al., 2009; Testerink and Munnik, 2011; Munnik, 2014). PA has been implicated specifically in the modulation of immune signaling components, such as MAPKs and PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1; Farmer and Choi, 1999; Lee et al., 2001; Szczegielniak et al., 2005; Anthony et al., 2006). In particular, PA binds to the NADPH oxidase isoforms RBOHD and RBOHF to induce ROS during abscisic acid (ABA)-mediated stomatal closure (Zhang et al., 2009). Additionally, it has been shown that PLC activity is required for ROS production during ETI responses (de Jong et al., 2004; Andersson et al., 2006).

In animals, IP3 triggers the release of Ca2+ from intracellular stores by activating a ligand-gated calcium channel at the endoplasmic reticulum. In plants, no clear homolog of the IP3-activated Ca2+ channel has been identified (Munnik and Testerink, 2009). Instead, IP2 and IP3 are further phosphorylated by inositolpolyphosphate kinase (Williams et al., 2015) to generate (1) IP6, which stimulates the release of Ca2+ from intracellular stores in guard cells (Lemtiri-Chlieh et al., 2000), affects gene transcription and mRNA export, and regulates the auxin receptor TIR1 (Tan et al., 2007; Lee et al., 2015); (2) IP5, which is part of the jasmonate receptor COI1 (Sheard et al., 2010); and (3) IP7 and IP8, which are involved in plant defense (Laha et al., 2015). In addition, PIP and PIP2, originally characterized as PLC substrates, do have signaling properties themselves, since many proteins involved in membrane trafficking and signal transduction have domains that bind to these lipids (Munnik and Nielsen, 2011; Delage et al., 2013; Heilmann, 2016).

The Arabidopsis genome contains nine genes encoding PI-PLCs (AtPLC1–AtPLC9; Mueller-Roeber and Pical, 2002). AtPLC2 (hereafter PLC2) is the most abundant PLC isoform, which is strongly and constitutively expressed and localizes to the plasma membrane (Pokotylo et al., 2014). PLC2 also is rapidly phosphorylated following flg22 recognition (Nühse et al., 2007). In this work, we analyzed the role of PLC2 in resistance to Pseudomonas syringae and Erysiphe pisi and in responses triggered upon flg22 perception. We found that PLC2 plays an important role in stomatal preinvasion immunity and nonhost resistance and that it associates with RBOHD, suggesting a potential regulation of the Arabidopsis NADPH oxidase and, consequently, of ROS-dependent processes by PLC2.

RESULTS

PLC2 Silencing by Artificial MicroRNA

To study the role of PLC2 in plant defense, we developed PLC2-silenced Arabidopsis plants by constitutively expressing a specific artificial microRNA (amiR). Expression analysis using quantitative PCR (qPCR) of PLC2 in leaves of T4 amiR-PLC2 homozygous plants showed that PLC2 was stably silenced (Fig. 1A). The expression of PLC7 (the closest homolog to PLC2), PLC4 (coexpressed with PLC2), and PLC1 (the second most abundant PLC; Pokotylo et al., 2014) was not altered in PLC2-silenced plants (Supplemental Fig. S1A). Western-blot analysis using a specific anti-PLC2 antibody (Otterhag et al., 2001) showed strongly reduced levels of PLC2 protein in amiR-silenced lines (Fig. 1B). The PLC2-silenced plants show similar morphological features to wild-type plants under standard growth conditions (Supplemental Fig. S1B).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

PLC2 silencing by artificial microRNAs in Arabidopsis. A, Total RNA was isolated from leaves of 4- to 5-week-old Columbia-0 (Col-0) or PLC2-silenced plants (T4 homozygous lines amiR-PLC2-4, amiR-PLC2-7, and amiR-PLC2-11). Relative transcript levels of PLC2 were determined by RT-qPCR. Transcript levels were normalized to ACT2. Error bars represent sd of three to nine individual plants. Different letters indicate significant differences (ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P < 0.001). B, PLC2 protein levels were analyzed by western blot using anti-AtPLC2 antibody in leaves of 4- to 5-week-old Col-0, empty vector (EV), and amiR-PLC2-11 and amiR-PLC2-4 independent silenced lines. Ponceau S staining of Rubisco subunit L was included as a loading control.

PLC2-Silenced Plants Are More Susceptible to the Bacterial P. syringae pv tomato DC3000 hrcC− Strain and to the Nonadapted Fungus E. pisi

To investigate the role of PLC2 in plant innate immunity, we tested the interaction of PLC2-silenced plants with two different pathogens. First, we selected Pseudomonas syringae pv tomato (Pst) strain DC3000 as a hemibiotrophic pathogen that infects Arabidopsis (Xin and He, 2013). The virulence of Pst DC3000 on Arabidopsis depends on the type III secretion system (TTSS), which allows MTI suppression (Block and Alfano, 2011). Thus, proliferation of the Pst DC3000 mutant strain hrcC− lacking a functional TTSS is restricted in this plant (Hauck et al., 2003). We used Pst DC3000 hrcC− to evaluate MTI in PLC2-silenced plants. After spraying adult plants with this bacterium, pathogen proliferation was assessed 1 and 3 d postinoculation. PLC2-silenced plants were more susceptible to Pst DC3000 hrcC− than wild-type plants (Fig. 2A). Under natural conditions, Pst enters host plants, through wounds or natural openings such as stomata, and then spreads and multiplies in intercellular spaces (Beattie and Lindow, 1995). The infiltration of bacteria with a syringe bypasses the first steps of the natural infection process. When Pst DC3000 hrcC− was infiltrated, no significant difference was detected between PLC2-silenced and nonsilenced plants (Fig. 2B). These experiments indicate that PLC2 is likely involved in stomata-related MTI responses.

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

Growth of P. syringae and E. pisi in Arabidopsis PLC2-silenced plants. Wild-type (Col-0), empty vector (EV), and PLC2-silenced lines (amiR-PLC2-11 and amiR-PLC2-4) were used. A, PLC2-silenced plants are more susceptible to the Pst DC3000 hrcC− mutant. Bacteria were inoculated by spray at OD600 = 0.1, and the number of colony-forming units (CFU) per cm2 of leaf extracts was determined. Data from three biological replicates each with three technical replicates were averaged (n = 9), and ANOVA was performed considering each replicate as a factor. Error bars represents se. Different letters indicate significant differences between genotypes (ANOVA, P < 0.001, posthoc Tukey’s test). dpi, Days postinoculation. B, PLC2-silenced plants do not show increased susceptibility to the Pst DC3000 hrcC− mutant when the bacteria are syringe inoculated into the leaf apoplast. Bacterial suspension was inoculated at OD600 = 0.0001, and the number of CFU per cm2 of leaf extracts was determined. Data from three biological replicates each with three technical replicates were averaged (n = 9), and ANOVA was performed considering each replicate as a factor. No significant differences were observed between genotypes. Error bars represent se. C and D, PLC2-silenced lines showed no differences in susceptibility to virulent (C) and avirulent (D) Pst DC3000 infections. Pst DC3000 (virulent) and Pst DC3000:AvrRpm1 (avirulent) were inoculated by infiltration at OD600 = 0.0002, and CFU per cm2 of leaf was calculated. A representative experiment of four biological replicates is depicted. No significant differences were observed regarding the EV control according to Student’s t test (P < 0.05). E, PLC2-silenced plants are more susceptible to the nonadapted pea powdery mildew E. pisi. The penetration rate at 3 d after inoculation was calculated as the percentage of successful penetration of at least 50 germinated spores on three independent leaves. Error bars represent se. Different letters indicate significant differences (multiple comparison using one-way ANOVA, posthoc Tukey’s test at P < 0.05). One representative experiment of four biologically independent replicates is depicted.

We further studied the growth of the virulent wild-type Pst DC3000, whose TTSS effectors do interfere with MTI (Block and Alfano, 2011). No significant difference in the proliferation of this adapted pathogen was detected between both plant genotypes (Fig. 2C), indicating that PLC2 silencing does not have an effect when the virulent pathogen is infiltrated.

In order to study if PLC2 also played a role during ETI, we infiltrated Arabidopsis leaves with an avirulent strain of Pst DC3000 expressing the type III secreted effector AvrRpm1, which is recognized by the NLR RPM1 (Block and Alfano, 2011). PLC2-silenced plants showed the same ability as the wild type in constraining the growth of this strain (Fig. 2D), indicating that the lack of PLC2 does not affect AvrRpm1 recognition-triggered growth restriction. Moreover, the hypersensitive response (HR) cell death measured by ion leakage induced by Pst DC3000 AvrRpm1 was identical in wild-type and PLC2-silenced plants (Supplemental Fig. S2A). The effect of PLC2 silencing on HR also was tested by ion leakage using Arabidopsis plants expressing AvrRpm1 under the control of a dexamethasone-inducible promoter (DEX::AvrRpm1; Andersson et al., 2006). As a negative control, AvrRpm1 was expressed in an RPM1 knockout background (rpm1-3; DEX::AvrRpm1/rpm1-3; Mackey et al., 2002, 2003). We stably silenced PLC2 by transforming both backgrounds with ubi::amiR-PLC2 (Supplemental Fig. S2C). Leaf discs from DEX::AvrRpm1/Col-0 or DEX::AvrRpm1/rpm1-3 silenced and nonsilenced plants were induced with dexamethasone, and ion leakage was measured at different time points. This experiment demonstrated no significant difference between the PLC2-silenced plants and wild-type plants (Supplemental Fig. S2B), confirming that PLC2 is not required for AvrRpm1-induced HR.

Finally, we tested the ability of PLC2-silenced plants to restrict the entry of the nonadapted pathogen E. pisi, the causal agent of pea (Pisum sativum) powdery mildew. Arabidopsis displays nonhost resistance toward E. pisi (Kuhn et al., 2016), whose spores are restricted from penetrating the epidermal cell wall. This resistance relies on basal defenses and MAMP recognition that function also against powdery mildews adapted to Arabidopsis (Kuhn et al., 2016). We assayed the epidermal penetration of the pathogen on wild-type and PLC2-silenced plants (Fig. 2E). We observed a significantly increased success in penetration of the epidermis by E. pisi spores on PLC2-silenced plants compared with wild-type plants, indicating that PLC2 is involved in nonhost resistance. Altogether, the above-presented results suggest that PLC2 might play a role in MTI establishment.

PLC2 Is Involved in ROS-Regulated Processes during MTI

In order to study the function of PLC2 in MTI, we used the MAMP flg22, a 22-amino acid sequence of the conserved N-terminal part of flagellin that is recognized by the FLS2 receptor (Gómez-Gómez and Boller, 2000), and studied the two distinct MAPK- and ROS-dependent branches of MTI signaling (Bigeard et al., 2015).

Flg22-induced activation of a particular MAPK cascade is an early event that regulates transcriptional reprogramming, which finally results in resistance (Bethke et al., 2012). Western-blot analysis of Arabidopsis wild-type seedlings treated with flg22 using an antibody directed against the conserved phosphorylated motif on the activation loop of MAPKs recognized three immunoreactive bands 15 min after treatment corresponding to at least MPK6, MPK3, and MPK4/11 (Bethke et al., 2012; Supplemental Fig. S3). PLC2-silenced lines showed a similar MAPK activation to wild-type plants (Supplemental Fig. S3). Similarly, flg22-induced expression of FRK1, PHI1, and WRKY33, which are MAPK- and CDPK-dependent MAMP-activated immune marker genes (Boudsocq et al., 2010), showed no significant differences between wild-type and PLC2-silenced seedlings (Supplemental Fig. S4). These results suggest that PLC2 is not required for this particular branch of MTI signaling.

Since oxidative burst is a MAPK-independent signaling event occurring after flg22 recognition in plant immunity (Zhang et al., 2007; Segonzac and Zipfel, 2011; Xu et al., 2014), we further studied the role of PLC2 in ROS-dependent processes. First, we analyzed flg22-induced callose deposition (Luna et al., 2011). To this end, leaves were infiltrated with flg22 and, 18 h later, stained with Aniline Blue for callose visualization. PLC2-silenced lines showed significantly less callose deposition upon flg22 treatment compared with leaves of control plants, which were either transformed with the empty vector or nontransformed wild-type plants (Fig. 3).

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

PLC2-silenced plants exhibit impaired flg22-induced callose deposition. Leaves from 4- to 5-week-old Col-0 or amiR-PLC2 plants were infiltrated with 1 µm flg22 or water as a control and incubated for 18 h, and callose deposition was measured as dots per area. Six different microscopic areas (1 mm2) were taken per leaf. Two different leaves per individual were analyzed. Three independent plants were analyzed per line per experiment. Three independent experiments were performed. Error bars represent se. Different letters indicate significant differences (ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P < 0.001). EV, Empty vector.

An earlier response of active immunity at the preinvasive level is the closure of the stomata upon MAMP perception, which is also a ROS-dependent defense response (Mersmann et al., 2010; Kadota et al., 2014; Li et al., 2014). In order to evaluate if stomatal closure was affected in PLC2-silenced plants, epidermal peels were treated with flg22. As shown in Figure 4, flg22-mediated induction of stomatal closure was impaired in epidermal peels of PLC2-silenced plants, whereas ABA-induced stomatal closure was unaffected. Together, these results imply that PLC2 is required for the full activation of stomatal ROS-dependent immune responses.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

PLC2-silenced plants exhibit impaired flg22-induced stomatal closure. Epidermal peels from Col-0 and PLC2-silenced plants were incubated in opening buffer under light for 3 h. The peels were treated with water, 1 µm flg22, 50 µm ABA, or 50 µm ABA + 1 µm flg22 for 1 h. The results show means of 90 to 120 stomata measured from three independent experiments. Error bars represent se. Different letters denote statistical differences (ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P < 0.05). EV, Empty vector.

PLC2 Is Involved in the flg22-Induced ROS Burst

Flg22 perception triggers a fast and transient increase of apoplastic ROS (Felix et al., 1999). Using a luminol/peroxidase-based method, apoplastic ROS levels were quantified in flg22-treated leaf discs. A representative experiment is shown in Figure 5A, indicating that in PLC2-silenced line 11 (amiR-PLC2-11), ROS accumulation had similar kinetics but significantly lower levels than in control plants. To estimate such reduction, we quantified apoplastic ROS in additional independent experiments including three different silenced lines as well as a control line carrying an empty vector. All PLC2-silenced lines showed a reduction in ROS levels in response to flg22 (40%–75% compared with control plants; Fig. 5B). Thus, our results demonstrated that PLC2 is required for the full ROS accumulation following flg22 recognition in Arabidopsis leaf discs.

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5.

PLC2-silenced plants exhibit impaired flg22-induced oxidative burst. The production of ROS was measured with a luminol-based assay in Col-0 or amiR-PLC2 plants. A, Leaf discs from 4- to 5-week-old plants were incubated with 100 nm flg22, and the luminescence was measured every 1 min for 30 min and expressed as relative light units (RLU). A representative experiment is shown using wild-type (Col-0) and a PLC2-silenced line (amiR-PLC2-11) plants. B, Total ROS production was calculated integrating the areas under the curves and referring to the Col-0 wild type treated with flg22 as 100%. Averages of four independent experiments are shown. Error bars represent se. Asterisks indicate statistically significant differences compared with the flg22-treated Col-0 plant (ANOVA for unbalanced samples, multiple comparisons versus control group posthoc Dunnett’s method at P < 0.05). EV, Empty vector.

PLC2 Associates with RBOHD

The flg22-induced ROS burst is generated via activation of the plasma membrane NADPH oxidase RBOHD (Nühse et al., 2007; Zhang et al., 2007). Our results show that PLC2 is required for the flg22-mediated ROS burst that is generated via RBOHD activation (Fig. 5). As mentioned earlier, PLC2 is localized at the plasma membrane, where RBOHD also exists in a complex with FLS2 and BIK1 (Kadota et al., 2014; Li et al., 2014). To investigate whether PLC2 associates with RBOHD, we immunoprecipitated N-terminally FLAG-tagged RBOHD (stably expressed in Arabidopsis under the control of its own promoter) using anti-FLAG affinity beads. In three independent biological experiments, PLC2 coimmunoprecipitated with RBOHD in planta (Fig. 6; Supplemental Fig. S5). PLC2 could not be immunoprecipitated in wild-type plants that did not express FLAG-RBOHD. Notably, the brassinosteroid receptor BRI1 (used here as an unrelated plasma membrane-located protein control) was not detected in anti-FLAG immunoprecipitates (Fig. 6). In addition, experiments in the presence of flg22 revealed that the association was independent of the ligand binding to FLS2, since the same amount of PLC2 was immunoprecipitated in treated as in nontreated plants (Fig. 6).

Figure 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 6.

PLC2 associates with RBOHD. Coimmunoprecipitation of PLC2 and ROBHD was performed in stable transgenic Arabidopsis seedlings (T3) expressing FLAG-RBOHD (pRBOHD:FLAG-RBOHD) treated (+) or not (−) with 1 µm flg22 for 15 min. Total protein extracts (Input) were subjected to immunoprecipitation (IP) with anti-FLAG beads followed by immunoblot analysis with anti-PLC2 (α-PLC2) and anti-FLAG (α-FLAG) antibodies as indicated. Protein extracts of Col-0 plants were used as negative controls. Anit-BRI1 (α-BRI1) antibodies were used as plasma membrane protein not associated with RBOHD. CBB, Coomassie Brilliant Blue. These experiments were performed three times with similar results.

DISCUSSION

The participation of PI-PLC activity in signaling after the recognition of different MAMPs such as xylanase, flg22, and chitosan (van der Luit et al., 2000; Laxalt et al., 2007; Raho et al., 2011) or pathogen effector proteins (de Jong et al., 2004; Andersson et al., 2006) has been described previously (Laxalt and Munnik, 2002; Munnik, 2014). Here, we show genetic evidence that PLC2 is particularly involved in MTI signaling. The molecular details of PI-PLC signaling in plants are still unclear, but there is evidence that (1) phosphatidylinositol 4-phosphate and PIP2 are most likely the substrates and (2) the phosphorylated products of IP2, IP3, and DAG, including various inositol polyphosphates, PA, and DAG pyrophosphate, have roles as secondary messengers (Munnik, 2014). PA is involved in the modulation of immune signaling components, such as MAPKs, PDK1, and RBOHD (Farmer and Choi, 1999; Lee et al., 2001; Szczegielniak et al., 2005; Anthony et al., 2006; Zhang et al., 2009). Unfortunately, in Arabidopsis, we were not able to detect in vivo flg22-induced PA increase by radiolabeling with 32Pi on seedlings, cotyledons, or leaf discs (Supplemental Fig. S6). We can envisage different plausible explanations: (1) flg22 triggers a very local increase of PA in specialized cells or tissues, so when we measure overall in vivo production in organs with different tissues, the signal gets diluted; (2) PA may be rapidly produced and metabolized and, thus, difficult to detect; or (3) 32Pi labeling conditions are not sensitive enough to distinguish small PA differences in Arabidopsis. However, those undetectable changes in the levels of signaling lipids can have strong effects on plant responses. We cannot exclude that IP2 and IP3 can be very rapidly phosphorylated to IP6 and, thus, probably increase Ca2+ in the cytosol or also participate in auxin signaling via TIR1 and COI1-JA signaling, among others (Xue et al., 2009; Munnik, 2014; Williams et al., 2015). Indeed, mutants with altered inositol polyphosphate levels showed altered defense responses (Murphy et al., 2008; Donahue et al., 2010; Mosblech et al., 2011; Hung et al., 2014; Laha et al., 2015). Whether these compounds are generated downstream of PLC2 remains to be demonstrated.

PLC2 Is Required for Full Activation of Plant Immunity

In tomato (Solanum lycopersicum), using virus induced-gene silencing of different PLCs, SlPLC4 was found to be involved specifically in the HR upon AVR4 perception, while SlPLC6 is required for multiple NLR-mediated responses (Vossen et al., 2010), suggesting that, in tomato, both PLCs participate in ETI responses. Similarly, overexpression of SlPLC3 enhanced the Cf-4/Avr4-triggered HR (Abd-El-Haliem et al., 2016). Further studies in tomato showed that SlPLC2 is required for xylanase-induced gene expression, ROS production, and plant susceptibility against Botrytis cinerea (Gonorazky et al., 2014, 2016).

Here, we assayed three different strains of the hemibiotrophic pathogen Pst DC3000: the virulent wild-type strain to study the role of PLC2 in effector-triggered susceptibility, the avirulent strain expressing AvrRpm1 to determine if PLC2 played a role during ETI, and the hrcC− strain mutated in the type III secretion system to investigate if PLC2 was required for MTI. PLC2-silenced plants showed increased susceptibility to Pst DC3000 hrcC− but not to the virulent or avirulent strain, suggesting that this protein is mostly involved in MTI. When the hrcC− strain was infiltrated, no differences in susceptibility were found between PLC2-silenced and wild-type plants, indicating that the differences found when the strain was sprayed could be explained by the role of stomata closure during infection. In order to further corroborate that PLC2 does not affect MTI final output when the bacteria are syringe infiltrated into the apoplast, we studied whether flg22 leads to induced plant resistance in PLC2-silenced plants. Supplemental Figure S7 shows that treatment of plants with flg22 triggers resistance to syringe-infiltrated Pst DC3000 in wild-type as well as PLC2-silenced plants. These results suggest that PLC2 controls stomatal preinvasive but not postinvasive immunity. Accordingly, PLC2-silenced plants are impaired in stomatal closure upon flg22 treatment. These results suggest a role of PLC2 in stomatal immunity.

Further studies also indicated that basal resistance against the nonadapted pathogen pea powdery mildew, E. pisi, was impaired in PLC2-silenced plants. In this nonhost interaction with Arabidopsis, the first line of defense is the recognition of MAMPs, such as chitin by the LYK5-CERK1 receptor complex, triggering a series of immune responses including MAPK activation and ROS burst mediated by NADPH oxidases (Kuhn et al., 2016).

PLC2 Participates in RBOHD-Dependent Plant Defense Responses

Callose accumulation is an MTI response that requires RBOHD (Luna et al., 2011), and flg22-induced callose deposition is reduced in PLC2-silenced plants. Another RBOHD-dependent response is flg22-induced stomatal closure (Mersmann et al., 2010; Kadota et al., 2014; Li et al., 2014). The restriction of microbial entry by stomatal closure is one of the first MTI responses (Melotto et al., 2006). fls2 mutant plants are impaired in stomatal closure in response to flg22 and show increased susceptibility to Pst DC3000 when sprayed onto the leaf surface but not when infiltrated into leaves (Gómez-Gómez et al., 2001; Zipfel et al., 2004; Chinchilla et al., 2006; Zeng and He, 2010). Importantly, the action of ABA on stomatal immunity seems to occur downstream or independently of the pattern recognition receptor complex, because fls2, bik1, and rbohD mutants exhibit wild-type stomatal closure in response to exogenous ABA (Macho et al., 2012; Kadota et al., 2014). Accordingly, we demonstrate that PLC2 is involved in flg22-induced stomatal closure, whereas ABA-dependent stomatal closure is unaffected. These results show that PLC2 is involved in callose deposition and stomatal closure following flg22 perception in Arabidopsis plants.

PLC2 Acts Upstream of RBOHD Activation

We have demonstrated that PLC2 is required for the full activation of flg22-induced ROS production. ROS production upon flg22 perception in Arabidopsis is dependent on the NADPH oxidase RBOHD (Kadota et al., 2014; Li et al., 2014). Posttranslational regulation of RBOHD activation involves Ca2+ via direct binding to EF hand motifs and phosphorylation by Ca2+-dependent (i.e. CPKs) and Ca2+-independent (i.e. BIK1) protein kinases (Logan et al., 1997; Boudsocq et al., 2010; Kadota et al., 2014, 2015; Li et al., 2014). By using PLC inhibitors, PLC activation has been suggested to be required for ROS production upon xylanase, chitosan, and Avr4 (de Jong et al., 2004; Laxalt et al., 2007; Raho et al., 2011). PA also has been shown to interact directly with RBOHD and enhance ROS production (Zhang et al., 2009). Upon cryptogein treatment of tobacco (Nicotiana tabacum) BY2 cells, PLC and DGK inhibitors or silencing of the cluster III of the tobacco DGK family resulted in reduced PA and ROS production (Cacas et al., 2017). Therefore, it could be speculated that the second messengers derived from PLC2 activation, PA and/or increased cytosolic Ca2+ via IP6, for example, could positively regulate the NADPH oxidase activity, since PLC2-silenced plants showed reduced ROS production in response to flg22.

Flg22 activates MAPK signaling pathways leading to the induction of immune gene expression. MPK3, MPK4/11, and MPK6 activation act independently of the RBOHD-mediated ROS burst (Zhang et al., 2012; Xu et al., 2014). Flg22-treated PLC2-silenced plants showed similar levels of MAPK activation and immune gene expression as the wild type, suggesting that MAPK signaling is independent of PLC2.

RBOHD exists in a complex with the receptor kinase FLS2, interacting directly with BIK1 (Kadota et al., 2014; Li et al., 2014). Our results show that PLC2 is associated with RBOHD and that this association is ligand independent. In Arabidopsis, the receptor complex FLS2-BAK1 perceives flg22 and activates the downstream kinases BIK1 and PBL1 by phosphorylation, which induce an influx of extracellular Ca2+ in the cytosol (Li et al., 2014; Ranf et al., 2014). PLC2 contains a Ca2+-dependent phospholipid-binding domain (C2) and EF hand domains (Otterhag et al., 2001). In addition, PLC2 is localized to the plasma membrane and is rapidly phosphorylated upon flg22 treatment (Niittylä et al., 2007; Nühse et al., 2007). One can envisage that PLC2 is part of the FLS2, BIK1, and RBOHD complex and that BIK1 or another component of the receptor complex phosphorylates PLC2, leading to the generation of second messengers like PA or IP6, which, in turn, positively regulate or are required to sustain/reinforce the activity of RBOHD.

Other Roles of PLC2

Seeking knockout mutant plants for PLC2, we could not recover homozygous mutants and, therefore, decided to silence PLC2. Nevertheless, further characterization showed that this gene is expressed during early megagametogenesis and in the embryo after fertilization, being required for both reproductive and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes (Li et al., 2015; Di Fino et al., 2017). The fact that we were able to obtain PLC2-silenced lines could be related to (1) low expression levels of the 35S::amiR-PLC2 in the reproductive organs and embryos or (2) the silencing not being fully effective, with low levels of PLC2 in the gametophyte and/or embryos being sufficient for correct development. The requirement for PLC2 during development suggests that the mechanisms for PLC2 activation and/or its downstream targets, such as RBOHD, could be similar in both the sporophyte during flg22 perception and the gametophyte during development. Arabidopsis has five SERK proteins. SERK3/BAK1 and SERK4/BKK1 associate with FLS2 and BIK1 (Chinchilla et al., 2007; Lu et al., 2010; Zhang et al., 2010; Roux et al., 2011). SERK1 and SERK2 are crucial in regulating male fertility and are expressed in the ovule, female gametophyte, early embryos, and vascular cells (Hecht et al., 2001; Albrecht et al., 2005; Colcombet et al., 2005; Kwaaitaal et al., 2005). We speculate that PLC2 has a role in gametogenesis and embryo development, probably by signaling downstream of LRR-RKs like SERKs. Nonetheless, whether PLC2 is specific for the FLS2-BAK1-BIK1 receptor complex or participates in the signaling of other receptor complexes, like LYK5-CERK1, as suggested by the results obtained with E. pisi, remains to be elucidated.

CONCLUSION

The activity of PI-PLC in signaling after the recognition of different MAMPs was described earlier. The Arabidopsis genome contains nine PI-PLC genes; however, until this work, it was not known which one was specifically linked to the plant defense response. We here present genetic evidence that PLC2 participates in MTI. PLC2 is required for the full activation of ROS production and ROS-dependent responses elicited by the MAMP flg22. PLC2 associates with RBOHD, suggesting a positive regulation of the Arabidopsis NADPH oxidase activity by PLC2.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds from Arabidopsis (Arabidopsis thaliana Col-0) transformed with an artificial microRNA targeting specifically PLC2 (amiR-PLC2) under the control of the cauliflower mosaic virus 35S promoter or with the empty vector were germinated in soil (soil:vermiculite:perlite, 3:1:1) and kept at 4°C for 2 d. Then, they were grown at 25°C using a 16-h-light/8-h-dark photoperiod. In the case of infections (bacterial and fungal), plants were grown at 22°C in an 8-h-light/16-h-dark photoperiod.

For ion leakage experiments, Col-0 or rpm1.3 mutant plants transformed with the coding sequence for Pseudomonas syringae pv tomato AvrRpm1 under the control of a dexamethasone-inducible promoter (Aoyama and Chua, 1997) were grown as described at 22°C in an 8-h-light/16-h-dark cycle. Both backgrounds were transformed with amiR-PLC2 under the control of the Ubiquitin10 promoter (pUBQ10).

amiR-PLC2-Silencing Constructs

AtPLC2 (At3g08510) silencing was performed using a specific artificial microRNA designed with WMD3 Web microRNA designer (http://wmd3.weigelworld.org). Arabidopsis miR319 was used as a template, and the cloning strategy was according to Ossowski et al. (2008).

Primers for Artificial MicroRNA Cloning

The following primers were used: PLC2 miR-s, 5′-gaTTAAACACTCAGTAATTGCGCtctctcttttgtattcc-3′; PLC2 miR-a, 5′-gaGCGCAATTACTGAGTGTTTAAtcaaagagaatcaatga-3′; PLC2 miR*s, 5′-gaGCACAATTACTGACTGTTTATtcacaggtcgtgatatg-3′; and PLC2 miR*a, 5′-gaATAAACAGTCAGTAATTGTGCtctacatatatattcct-3′. Uppercase letters denote AtPLC2-targeted sites. The amiR-PLC2 was cloned into pCHF3 vector (kanamycin resistance in plants) driven by the cauliflower mosaic virus 35S promoter or into pUBQ10 destination vector driven by the Ubiquitin10 promoter (Basta resistance in plants).

Arabidopsis Transformation

Arabidopsis plants were transformed using the floral dip method (Zhang et al., 2006). T1 plants were sown on Murashige and Skoog (MS) agar (MS medium with Gamborg’s vitamins and 1% agar) plates with kanamycin (50 µg mL−1 for pCHF3:amiR-PLC2) or BASTA (10 µg mL−1 for pUBQ10:amiR-PLC2). After 2 weeks, resistant plants were transferred to soil. T3 or T4 homozygous plants on which silencing levels were checked by qPCR were used for experiments.

Expression Analysis by RT-qPCR

Total RNA was extracted from 10-d-old seedlings or leaves from 4- to 5-week-old plants using the Trizol method according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized on 1 µg of total RNA by MMLV reverse transcriptase from Promega using oligo(dT) primer in a final volume of 20 µL. The cDNA was diluted to a final volume of 100 µL, and 2.5 µL was used for qPCR. The Fast Universal SYBR Green Master Mix from Roche was employed, using a Step-One Real-Time PCR machine from Applied Biosystems. The standard amplification program was used. The expression levels of the gene of interest were normalized to those of the constitutive ACT2 (At3g18780) gene by subtracting the cycle threshold value of ACT2 from the cycle threshold value of the gene (ΔCT). The nucleotide sequences of the specific primers for qPCR analysis are listed in Supplemental Table S1. The annealing temperature for each primer was 60°C. LinRegPCR was the program employed for the analysis of real-time qPCR data (Ruijter et al., 2009).

Western-Blot Analysis

Polyclonal antibodies were prepared as described (Otterhag et al., 2001). A peptide, KDLGDEEVWGREVPSFIQR, corresponding to residues 266 to 284 of AtPLC2 was synthesized. One rabbit was immunized at 2-week intervals, and serum was collected after the second boost. Protein extraction buffer (100 mm NaPi, pH 7.5, 150 mm NaCl, 1 mm EDTA, and Sigma-Aldrich proteinase inhibitor cocktail) was added to an equal volume of 4- to 5-week-old ground leaf tissue, mixed, and centrifuged for 10 min at 10,000g. Protein concentration in the supernatant was determined. Samples were loaded onto a 10% SDS-polyacrylamide gel, blotted onto nitrocellulose membranes, and stained with Ponceau S for a loading control. Membranes were incubated overnight in phosphate-buffered saline plus Tween 20 containing polyclonal anti-PLC2 antibody (1:2,000). The blot was washed three times with phosphate-buffered saline plus Tween 20 and revealed using a secondary anti-rabbit IgG antibody coupled to alkaline phosphatase according to the manufacturer’s instructions (Sigma-Aldrich).

Bacterial Infection Assays

Six- to 8-week-old plants were used for bacterial inoculations. Strains Pst DC3000 (virulent), Pst DC3000 AvrRpm1 (avirulent), and Pst DC3000 hrcC− mutant were maintained on solid Pseudomonas agar F (King’s B medium; Biolife) supplemented with 50 mg L−1 rifampicin (for Pst DC3000 hrcC−) or plus 50 mg L−1 kanamycin (for virulent and avirulent Pst strains). Bacterial suspensions of virulent and avirulent strains were inoculated into the abaxial side of leaves with a needleless syringe (10 mm MgCl2, OD600 = 0.00002). The bacteria were extracted at 1 or 3 d postinfiltration, and the number of colony-forming units was determined after serial dilution and plating as described (Johansson et al., 2014). The strain Pst DC3000 hrcC− was inoculated either by spraying (10 mm MgCl2, OD600 = 0.1; 0.02% Silwet) or by infiltration with a needless syringe (10 mm MgCl2, OD600 = 0.0001). Following spray inoculations, plants were kept covered with a transparent lid for 6 h. For spray- and syringe-inoculated plants, samples were taken at day 0, 1, or 3 postinoculation with a number 1 cork borer. Bacterial growth was evaluated as described previously (Katagiri et al., 2002). Data shown in Figure 2, A, C, D, and E, correspond to one experiment representative of four independent biological assays performed. Again, in Figure 2B, one of three biological replicates that showed similar results is depicted. For all cases, each value in the graphs represents the average ± sd of three technical replicates (three pools of four leaf discs collected from four independent plants at each time point) that were ground, diluted, and plated separately.

Ion Leakage

Ion leakage was measured in leaf discs after infiltration of Pst DC3000 AvrRpm1 (OD600 = 0.1), as well as in leaf discs of Col-0 plants expressing the coding sequence of P. syringae AvrRpm1, under the control of a dexamethasone-inducible promoter (Andersson et al., 2006) as described (Johansson et al., 2014). Leaf discs from 4- to 5-week-old empty vector or PLC2-silenced plants in the wild-type or rpm1-3 background were placed in deionized water during 1 to 2 h and then washed and transferred to six-well cultivation plates containing 10 mL of water (four discs per well). For the dexamethasone-inducible AvrRpm1 plants, leaf discs were treated with 20 µm dexamethasone. The release of electrolytes from the leaf discs was determined every 30 min for 5 h using a conductivity meter (Orion; Thermo Scientific) as described (Johansson et al., 2014). The experiment was repeated twice.

Fungal Inoculation and Scoring of Fungal Penetration

The nonhost powdery mildew fungus Erysiphe pisi (isolate CO-01) was propagated on pea (Pisum sativum ‘Kelvedon Wonder’) plants. Inoculations were carried out by powdering spores on leaves of 4-week-old Arabidopsis wild-type and PLC2-silenced plants. At 3 d postinoculation, leaves were stained with Trypan Blue as described (Koch and Slusarenko, 1990). The penetration rate after inoculation was calculated as the percentage of successful penetration attempts (penetration ending in plant cell death) as described (Pinosa et al., 2013) on at least 50 germinated spores on three independent leaves per genotype. The experiment was repeated four times.

MAPK Activation

MAPK assays were performed on six 2-week-old seedlings grown in liquid MS medium (including vitamins; Duchefa) and 1% Suc. Seedlings were elicited with 1 mm flg22 for 5, 15, or 30 min and frozen in liquid nitrogen. MAPK activation was monitored by western blot with antibodies that recognize the dual phosphorylation of the activation loop of MAPK (pTEpY). Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr-204) rabbit monoclonal antibodies from Cell Signaling were used according to the manufacturer’s protocol (1:5,000). Blots were stained with Coomassie Brilliant Blue to verify equal loading.

Callose Deposition

Leaves from 4- to 5-week-old plants were fully infiltrated with 1 µm flg22 or water for 18 h. Leaves were then incubated in 96% ethanol until all tissue was transparent, washed in 0.07 m phosphate buffer (pH 9), and incubated for 2 h in 0.07 m phosphate buffer containing 0.01% Aniline Blue. Observations were performed with an epifluorescence microscope with UV filter (excitation, 365/10 nm; emission, 460/50 nm). The number of callose dots was calculated using ImageJ software (Schneider et al., 2012). Six different microscopic areas (1 mm2) were taken per leaf. Two different leaves per individual were analyzed. Three independent plants were analyzed per line per experiment. Three independent experiments were performed.

Epidermal Peel Preparation and Stomatal Aperture Measurement

Epidermal peels were obtained from the abaxial surface of fully expanded leaves. The peels were preincubated in opening buffer (10 mm MES, pH 6.1 [MES titrated to its pKa with KOH], and 10 mm KCl) under white light at 25°C to promote stomatal opening. After 3 h of preincubation, flg22 (1 µm) or ABA (50 µm; Sigma-Aldrich) was added to the opening buffer and incubated for 1 h. Stomatal apertures were measured from digital images taken with a Nikon Coolpix 990 camera coupled to an optical microscope (Nikon Eclipse 2000). Then, the stomatal pore width was digitally determined using the image-analysis software ImageJ. Aperture values are means of 90 to 120 stomata measured from at least three independent experiments.

ROS Detection

Leaf discs from 4- to 5-week-old plants were placed on 96-well black plates floating in 200 µL of deionized water overnight. ROS production was triggered with 100 nm flg22 (N-QRLSTGSRINSAKDDAAGLQIA-C; Genbiotech) applied together with 20 mm luminol (Sigma-Aldrich; catalog no. A8511) and 0.02 mg mL−1 horseradish peroxidase (Sigma-Aldrich; catalog no. P6782). Luminescence was measured with a luminometer (Thermo Scientific Luminoskan Ascent Microplate). Each plate contained 36 leaf discs for flg22 treatment and 12 leaf discs for mock treatment of the same Arabidopsis line. Every plate was measured over a period of 30 min with an interval of 1 min and repeated in four independent experiments.

Seedling Protein Extraction and Immunoprecipitation

For immunoprecipitation studies in seedlings, Arabidopsis rbohd/pRBOHD::FLAG-RBOHD (Kadota et al., 2014) seeds were surface sterilized with chlorine gas and germinated on plates containing MS medium (with Gamborg’s vitamins; Duchefa) and 1% Suc and 0.8% agar for the first 7 d at 22°C and with a 16-h light period. Seedlings were transferred to liquid MS medium supplemented with 1% Suc and grown under the same conditions for an additional 7 d.

Two-week-old seedlings were treated with flg22 (1 µm) or water and ground to a fine powder in liquid nitrogen with sand (Sigma-Aldrich). Proteins were isolated in extraction buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 5 mm DTT, 1 mm NaF, 1 mm Na2MoO4∙2H2O, 1% phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich), 1% (v/v) P9599 protease inhibitor cocktail (Sigma-Aldrich), 100 μm phenylmethylsulfonyl fluoride, and 1% (v/v) IGEPAL CA-630 (Sigma-Aldrich). Extracts were incubated 30 min at 4°C and centrifuged for 20 min at 16,000g at 4°C. Supernatants were incubated for 1 to 2 h at 4°C with ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) and washed five times with extraction buffer. Beads were heated at 55°C in SDS loading buffer for 20 min to release proteins. For immunoblotting, antibodies were used at the following dilutions: α-PLC2 (1:5,000), α-FLAG-HRP (Sigma-Aldrich; 1:5,000), α-rabbit-HRP (Sigma-Aldrich; 1:10,000), and anti-BRI1 (1:5,000).

Accession Numbers

Accession numbers are as follows: AtPLC2 (At3g08510), AtRBOHD (At5G47910), and AtBRI1 (At4g39400).

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. PLC2 silencing specificity and phenotypes of silenced plants.

  • Supplemental Figure S2. PLC2 is not involved in programmed cell death during ETI upon recognition of AvrRpm1 from P. syringae.

  • Supplemental Figure S3. PLC2 is not required for flg22-induced MAPK activation.

  • Supplemental Figure S4. MAMP-activated gene expression is not deregulated in PLC2-silenced seedlings.

  • Supplemental Figure S5. PLC2 associates with RBOHD.

  • Supplemental Figure S6. Effect of flg22 on the formation of PA

  • Supplemental Figure S7. Flg22-induced resistance.

  • Supplemental Table S1. Primer sequences.

Acknowledgments

We thank Alexandra Leschnin and Dr. Oskar Johansson for helping with ion leakage measurements and bacterial and fungal infections, Dr. Jesús Nuñez for statistical analysis, Sergio Batista for assistance in the greenhouse, and Teresa Quattrini for technical assistance.

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.17.00173

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ana M. Laxalt (amlaxalt{at}mdp.edu.ar).

  • A.M.L. conceived the original research plans; A.M.L., M.X.A., M.E.Á., and C.Z. designed and supervised the experiments and analyzed the data; J.M.D. performed most of the experiments and analyzed the data; D.C., G.F., and D.S. performed some of the experiments; A.M.L. conceived the project and wrote the article with contributions of all the authors; L.L. and T.M. supervised and complemented the writing.

  • ↵1 This work was supported by UNMdP, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; PIP 1142010010 0219), by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2010 No 574, PICT 2012 No 2117, PICT 2014 No 1621, and PICT 2014 No 3255), by EMBO Short Term Fellowship (ASTF 477 - 2015) and by the Carl Trygger Foundation for Scientific Research to M.X.A. C.Z. was funded by the Gatsby Charitable Foundation and the European Research Council (PHOSPHinnATE). T.M. was funded by the Netherlands Organization for Scientific Research (867.15.020).

Glossary

MAMP
microbe-associated molecular pattern
MTI
MAMP-triggered immunity
ETI
effector-triggered immunity
NLR
nucleotide-binding Leu-rich repeat
ROS
reactive oxygen species
DAG
diacylglycerol
PA
phosphatidic acid
ABA
abscisic acid
qPCR
quantitative PCR
TTSS
type III secretion system
HR
hypersensitive response
Col-0
Columbia-0
MS
Murashige and Skoog
  • Received February 7, 2017.
  • Accepted August 18, 2017.
  • Published August 21, 2017.

REFERENCES

  1. ↵
    1. Abd-El-Haliem AM,
    2. Vossen JH,
    3. van Zeijl A,
    4. Dezhsetan S,
    5. Testerink C,
    6. Seidl MF,
    7. Beck M,
    8. Strutt J,
    9. Robatzek S,
    10. Joosten MH
    (2016) Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity. Biochim Biophys Acta 1861: 1365–1378
    OpenUrl
  2. ↵
    1. Albrecht C,
    2. Russinova E,
    3. Hecht V,
    4. Baaijens E,
    5. de Vries S
    (2005) The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis. Plant Cell 17: 3337–3349
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Andersson MX,
    2. Kourtchenko O,
    3. Dangl JL,
    4. Mackey D,
    5. Ellerström M
    (2006) Phospholipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana. Plant J 47: 947–959
    OpenUrlCrossRefPubMed
  4. ↵
    1. Anthony RG,
    2. Khan S,
    3. Costa J,
    4. Pais MS,
    5. Bögre L
    (2006) The Arabidopsis protein kinase PTI1-2 is activated by convergent phosphatidic acid and oxidative stress signaling pathways downstream of PDK1 and OXI1. J Biol Chem 281: 37536–37546
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Antolín-Llovera M,
    2. Petutsching EK,
    3. Ried MK,
    4. Lipka V,
    5. Nürnberger T,
    6. Robatzek S,
    7. Parniske M
    (2014) Knowing your friends and foes: plant receptor-like kinases as initiators of symbiosis or defence. New Phytol 204: 791–802
    OpenUrlCrossRefPubMed
  6. ↵
    1. Aoyama T,
    2. Chua NH
    (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11: 605–612
    OpenUrlCrossRefPubMed
  7. ↵
    1. Arisz SA,
    2. Testerink C,
    3. Munnik T
    (2009) Plant PA signaling via diacylglycerol kinase. Biochim Biophys Acta 1791: 869–875
    OpenUrlCrossRefPubMed
  8. ↵
    1. Beattie GA,
    2. Lindow SE
    (1995) The secret life of foliar bacterial pathogens on leaves. Annu Rev Phytopathol 33: 145–172
    OpenUrlCrossRefPubMed
  9. ↵
    1. Bethke G,
    2. Pecher P,
    3. Eschen-Lippold L,
    4. Tsuda K,
    5. Katagiri F,
    6. Glazebrook J,
    7. Scheel D,
    8. Lee J
    (2012) Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22. Mol Plant Microbe Interact 25: 471–480
    OpenUrlCrossRefPubMed
  10. ↵
    1. Bigeard J,
    2. Colcombet J,
    3. Hirt H
    (2015) Signaling mechanisms in pattern-triggered immunity (PTI). Mol Plant 8: 521–539
    OpenUrlCrossRefPubMed
  11. ↵
    1. Block A,
    2. Alfano JR
    (2011) Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys? Curr Opin Microbiol 14: 39–46
    OpenUrlCrossRefPubMed
  12. ↵
    1. Boller T,
    2. Felix G
    (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60: 379–406
    OpenUrlCrossRefPubMed
  13. ↵
    1. Boudsocq M,
    2. Willmann MR,
    3. McCormack M,
    4. Lee H,
    5. Shan L,
    6. He P,
    7. Bush J,
    8. Cheng SH,
    9. Sheen J
    (2010) Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464: 418–422
    OpenUrlCrossRefPubMed
  14. ↵
    1. Cacas JL,
    2. Gerbeau-Pissot P,
    3. Fromentin J,
    4. Cantrel C,
    5. Thomas D,
    6. Jeannette E,
    7. Kalachova T,
    8. Mongrand S,
    9. Simon-Plas F,
    10. Ruelland E
    (2017) Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein. Plant Cell Environ 40: 585–598
    OpenUrl
  15. ↵
    1. Chinchilla D,
    2. Bauer Z,
    3. Regenass M,
    4. Boller T,
    5. Felix G
    (2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18: 465–476
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Chinchilla D,
    2. Zipfel C,
    3. Robatzek S,
    4. Kemmerling B,
    5. Nürnberger T,
    6. Jones JD,
    7. Felix G,
    8. Boller T
    (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448: 497–500
    OpenUrlCrossRefPubMed
  17. ↵
    1. Colcombet J,
    2. Boisson-Dernier A,
    3. Ros-Palau R,
    4. Vera CE,
    5. Schroeder JI
    (2005) Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation. Plant Cell 17: 3350–3361
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Couto D,
    2. Zipfel C
    (2016) Regulation of pattern recognition receptor signalling in plants. Nat Rev Immunol 16: 537–552
    OpenUrlCrossRefPubMed
  19. ↵
    1. de Jong CF,
    2. Laxalt AM,
    3. Bargmann BO,
    4. de Wit PJ,
    5. Joosten MH,
    6. Munnik T
    (2004) Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction. Plant J 39: 1–12
    OpenUrlCrossRefPubMed
  20. ↵
    1. Delage E,
    2. Puyaubert J,
    3. Zachowski A,
    4. Ruelland E
    (2013) Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: convergences and divergences among eukaryotic kingdoms. Prog Lipid Res 52: 1–14
    OpenUrlCrossRefPubMed
  21. ↵
    1. Di Fino LM,
    2. D’Ambrosio JM,
    3. Tejos R,
    4. van Wijk R,
    5. Lamattina L,
    6. Munnik T,
    7. Pagnussat GC,
    8. Laxalt AM
    (2017) Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development. Planta 245: 717–728
    OpenUrl
  22. ↵
    1. Donahue JL,
    2. Alford SR,
    3. Torabinejad J,
    4. Kerwin RE,
    5. Nourbakhsh A,
    6. Ray WK,
    7. Hernick M,
    8. Huang X,
    9. Lyons BM,
    10. Hein PP, et al.
    (2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppression of cell death. Plant Cell 22: 888–903
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Farmer PK,
    2. Choi JH
    (1999) Calcium and phospholipid activation of a recombinant calcium-dependent protein kinase (DcCPK1) from carrot (Daucus carota L.). Biochim Biophys Acta 1434: 6–17
    OpenUrlCrossRefPubMed
  24. ↵
    1. Felix G,
    2. Duran JD,
    3. Volko S,
    4. Boller T
    (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18: 265–276
    OpenUrlCrossRefPubMed
  25. ↵
    1. Gómez-Gómez E,
    2. Isabel M,
    3. Roncero G,
    4. Di Pietro A,
    5. Hera C
    (2001) Molecular characterization of a novel endo-beta-1,4-xylanase gene from the vascular wilt fungus Fusarium oxysporum. Curr Genet 40: 268–275
    OpenUrlPubMed
  26. ↵
    1. Gómez-Gómez L,
    2. Boller T
    (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5: 1003–1011
    OpenUrlCrossRefPubMed
  27. ↵
    1. Gonorazky G,
    2. Guzzo MC,
    3. Abd-El-Haliem AM,
    4. Joosten MH,
    5. Laxalt AM
    (2016) Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea. Mol Plant Pathol 17: 1354–1363
    OpenUrl
  28. ↵
    1. Gonorazky G,
    2. Ramirez L,
    3. Abd-El-Haliem A,
    4. Vossen JH,
    5. Lamattina L,
    6. ten Have A,
    7. Joosten MH,
    8. Laxalt AM
    (2014) The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase. J Plant Physiol 171: 959–965
    OpenUrl
  29. ↵
    1. Hauck P,
    2. Thilmony R,
    3. He SY
    (2003) A Pseudomonas syringae type III effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis plants. Proc Natl Acad Sci USA 100: 8577–8582
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Hecht V,
    2. Vielle-Calzada JP,
    3. Hartog MV,
    4. Schmidt ED,
    5. Boutilier K,
    6. Grossniklaus U,
    7. de Vries SC
    (2001) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol 127: 803–816
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Heese A,
    2. Hann DR,
    3. Gimenez-Ibanez S,
    4. Jones AM,
    5. He K,
    6. Li J,
    7. Schroeder JI,
    8. Peck SC,
    9. Rathjen JP
    (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA 104: 12217–12222
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Heilmann I
    (2016) Phosphoinositide signaling in plant development. Development 143: 2044–2055
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Hong Y,
    2. Zhao J,
    3. Guo L,
    4. Kim SC,
    5. Deng X,
    6. Wang G,
    7. Zhang G,
    8. Li M,
    9. Wang X
    (2016) Plant phospholipases D and C and their diverse functions in stress responses. Prog Lipid Res 62: 55–74
    OpenUrl
  34. ↵
    1. Hung CY,
    2. Aspesi P Jr.,
    3. Hunter MR,
    4. Lomax AW,
    5. Perera IY
    (2014) Phosphoinositide-signaling is one component of a robust plant defense response. Front Plant Sci 5: 267
    OpenUrlCrossRefPubMed
  35. ↵
    1. Johansson ON,
    2. Fahlberg P,
    3. Karimi E,
    4. Nilsson AK,
    5. Ellerstrom M,
    6. Andersson MX
    (2014) Redundancy among phospholipase D isoforms in resistance triggered by recognition of the Pseudomonas syringae effector AvrRpm1 in Arabidopsis thaliana. Front Plant Sci 5: 639
    OpenUrl
  36. ↵
    1. Jones JD,
    2. Dangl JL
    (2006) The plant immune system. Nature 444: 323–329
    OpenUrlCrossRefPubMed
  37. ↵
    1. Kadota Y,
    2. Shirasu K,
    3. Zipfel C
    (2015) Regulation of the NADPH oxidase RBOHD during plant immunity. Plant Cell Physiol 56: 1472–1480
    OpenUrlCrossRefPubMed
  38. ↵
    1. Kadota Y,
    2. Sklenar J,
    3. Derbyshire P,
    4. Stransfeld L,
    5. Asai S,
    6. Ntoukakis V,
    7. Jones JD,
    8. Shirasu K,
    9. Menke F,
    10. Jones A, et al.
    (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54: 43–55
    OpenUrlCrossRefPubMed
  39. ↵
    1. Katagiri F,
    2. Thilmony R,
    3. He SY
    (2002) The Arabidopsis thaliana-Pseudomonas syringae interaction. The Arabidopsis Book 1: e0039, doi/10.1199/tab.0039
    OpenUrlCrossRef
  40. ↵
    1. Koch E,
    2. Slusarenko A
    (1990) Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell 2: 437–445
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Kuhn H,
    2. Kwaaitaal M,
    3. Kusch S,
    4. Acevedo-Garcia J,
    5. Wu H,
    6. Panstruga R
    (2016) Biotrophy at its best: novel findings and unsolved mysteries of the Arabidopsis-powdery mildew pathosystem. The Arabidopsis Book 14: e0184, doi/10.1199/tab.0184
    OpenUrl
  42. ↵
    1. Kwaaitaal MA,
    2. de Vries SC,
    3. Russinova E
    (2005) Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis. Protoplasma 226: 55–65
    OpenUrlCrossRefPubMed
  43. ↵
    1. Laha D,
    2. Johnen P,
    3. Azevedo C,
    4. Dynowski M,
    5. Weiß M,
    6. Capolicchio S,
    7. Mao H,
    8. Iven T,
    9. Steenbergen M,
    10. Freyer M, et al.
    (2015) VIH2 regulates the synthesis of inositol pyrophosphate InsP8 and jasmonate-dependent defenses in Arabidopsis. Plant Cell 27: 1082–1097
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Laxalt AM,
    2. Munnik T
    (2002) Phospholipid signalling in plant defence. Curr Opin Plant Biol 5: 332–338
    OpenUrlCrossRefPubMed
  45. ↵
    1. Laxalt AM,
    2. Raho N,
    3. Have AT,
    4. Lamattina L
    (2007) Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells. J Biol Chem 282: 21160–21168
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Lee HS,
    2. Lee DH,
    3. Cho HK,
    4. Kim SH,
    5. Auh JH,
    6. Pai HS
    (2015) InsP6-sensitive variants of the Gle1 mRNA export factor rescue growth and fertility defects of the ipk1 low-phytic-acid mutation in Arabidopsis. Plant Cell 27: 417–431
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Lee S,
    2. Hirt H,
    3. Lee Y
    (2001) Phosphatidic acid activates a wound-activated MAPK in Glycine max. Plant J 26: 479–486
    OpenUrlCrossRefPubMed
  48. ↵
    1. Lemtiri-Chlieh F,
    2. MacRobbie EAC,
    3. Brearley CA
    (2000) Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells. Proc Natl Acad Sci USA 97: 8687–8692
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Li L,
    2. He Y,
    3. Wang Y,
    4. Zhao S,
    5. Chen X,
    6. Ye T,
    7. Wu Y,
    8. Wu Y
    (2015) Arabidopsis PLC2 is involved in auxin-modulated reproductive development. Plant J 84: 504–515
    OpenUrl
  50. ↵
    1. Li L,
    2. Li M,
    3. Yu L,
    4. Zhou Z,
    5. Liang X,
    6. Liu Z,
    7. Cai G,
    8. Gao L,
    9. Zhang X,
    10. Wang Y, et al.
    (2014) The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe 15: 329–338
    OpenUrlCrossRefPubMed
  51. ↵
    1. Logan H,
    2. Basset M,
    3. Very A,
    4. Sentenac H
    (1997) Plasma membrane transport systems in higher plants: from black boxes to molecular physiology. Physiol Plant 100: 1–15
    OpenUrlCrossRef
  52. ↵
    1. Lu D,
    2. Wu S,
    3. Gao X,
    4. Zhang Y,
    5. Shan L,
    6. He P
    (2010) A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc Natl Acad Sci USA 107: 496–501
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Luna E,
    2. Pastor V,
    3. Robert J,
    4. Flors V,
    5. Mauch-Mani B,
    6. Ton J
    (2011) Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact 24: 183–193
    OpenUrlCrossRefPubMed
  54. ↵
    1. Macho AP,
    2. Boutrot F,
    3. Rathjen JP,
    4. Zipfel C
    (2012) Aspartate oxidase plays an important role in Arabidopsis stomatal immunity. Plant Physiol 159: 1845–1856
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Mackey D,
    2. Belkhadir Y,
    3. Alonso JM,
    4. Ecker JR,
    5. Dangl JL
    (2003) Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell 112: 379–389
    OpenUrlCrossRefPubMed
  56. ↵
    1. Mackey D,
    2. Holt BF III.,
    3. Wiig A,
    4. Dangl JL
    (2002) RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108: 743–754
    OpenUrlCrossRefPubMed
  57. ↵
    1. Melotto M,
    2. Underwood W,
    3. Koczan J,
    4. Nomura K,
    5. He SY
    (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126: 969–980
    OpenUrlCrossRefPubMed
  58. ↵
    1. Mersmann S,
    2. Bourdais G,
    3. Rietz S,
    4. Robatzek S
    (2010) Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiol 154: 391–400
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Mosblech A,
    2. Thurow C,
    3. Gatz C,
    4. Feussner I,
    5. Heilmann I
    (2011) Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana. Plant J 65: 949–957
    OpenUrlCrossRefPubMed
  60. ↵
    1. Mueller-Roeber B,
    2. Pical C
    (2002) Inositol phospholipid metabolism in Arabidopsis: characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C. Plant Physiol 130: 22–46
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. X Wang
    1. Munnik T
    (2014) PI-PLC: phosphoinositide-phospholipase C in plant signaling. In X Wang, ed, Phospholipases in Plant Signalling, Vol 20. Springer-Verlag, Berlin, pp 27–54
    OpenUrl
  62. ↵
    1. Munnik T,
    2. Nielsen E
    (2011) Green light for polyphosphoinositide signals in plants. Curr Opin Plant Biol 14: 489–497
    OpenUrlCrossRefPubMed
  63. ↵
    1. Munnik T,
    2. Testerink C
    (2009) Plant phospholipid signaling: “in a nutshell”. J Lipid Res (Suppl) 50: S260–S265
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Munnik T,
    2. Vermeer JE
    (2010) Osmotic stress-induced phosphoinositide and inositol phosphate signalling in plants. Plant Cell Environ 33: 655–669
    OpenUrlCrossRefPubMed
  65. ↵
    1. Murphy AM,
    2. Otto B,
    3. Brearley CA,
    4. Carr JP,
    5. Hanke DE
    (2008) A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens. Plant J 56: 638–652
    OpenUrlCrossRefPubMed
  66. ↵
    1. Niittylä T,
    2. Fuglsang AT,
    3. Palmgren MG,
    4. Frommer WB,
    5. Schulze WX
    (2007) Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis. Mol Cell Proteomics 6: 1711–1726
    OpenUrlAbstract/FREE Full Text
  67. ↵
    1. Nühse TS,
    2. Bottrill AR,
    3. Jones AM,
    4. Peck SC
    (2007) Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J 51: 931–940
    OpenUrlCrossRefPubMed
  68. ↵
    1. Ossowski S,
    2. Schwab R,
    3. Weigel D
    (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53: 674–690
    OpenUrlCrossRefPubMed
  69. ↵
    1. Otterhag L,
    2. Sommarin M,
    3. Pical C
    (2001) N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana. FEBS Lett 497: 165–170
    OpenUrlCrossRefPubMed
  70. ↵
    1. Pinosa F,
    2. Buhot N,
    3. Kwaaitaal M,
    4. Fahlberg P,
    5. Thordal-Christensen H,
    6. Ellerström M,
    7. Andersson MX
    (2013) Arabidopsis phospholipase Dδ is involved in basal defense and nonhost resistance to powdery mildew fungi. Plant Physiol 163: 896–906
    OpenUrlAbstract/FREE Full Text
  71. ↵
    1. Pokotylo I,
    2. Kolesnikov Y,
    3. Kravets V,
    4. Zachowski A,
    5. Ruelland E
    (2014) Plant phosphoinositide-dependent phospholipases C: variations around a canonical theme. Biochimie 96: 144–157
    OpenUrlCrossRefPubMed
  72. ↵
    1. Raho N,
    2. Ramirez L,
    3. Lanteri ML,
    4. Gonorazky G,
    5. Lamattina L,
    6. ten Have A,
    7. Laxalt AM
    (2011) Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide. J Plant Physiol 168: 534–539
    OpenUrlCrossRefPubMed
  73. ↵
    1. Ranf S,
    2. Eschen-Lippold L,
    3. Fröhlich K,
    4. Westphal L,
    5. Scheel D,
    6. Lee J
    (2014) Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1. BMC Plant Biol 14: 374
    OpenUrlCrossRefPubMed
  74. ↵
    1. Roux M,
    2. Schwessinger B,
    3. Albrecht C,
    4. Chinchilla D,
    5. Jones A,
    6. Holton N,
    7. Malinovsky FG,
    8. Tör M,
    9. de Vries S,
    10. Zipfel C
    (2011) The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23: 2440–2455
    OpenUrlAbstract/FREE Full Text
  75. ↵
    1. Ruijter JM,
    2. Ramakers C,
    3. Hoogaars WM,
    4. Karlen Y,
    5. Bakker O,
    6. van den Hoff MJ,
    7. Moorman AF
    (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37: e45
    OpenUrlCrossRefPubMed
  76. ↵
    1. Schneider CA,
    2. Rasband WS,
    3. Eliceiri KW
    (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675
    OpenUrlCrossRefPubMed
  77. ↵
    1. Segonzac C,
    2. Zipfel C
    (2011) Activation of plant pattern-recognition receptors by bacteria. Curr Opin Microbiol 14: 54–61
    OpenUrlCrossRefPubMed
  78. ↵
    1. Sheard LB,
    2. Tan X,
    3. Mao H,
    4. Withers J,
    5. Ben-Nissan G,
    6. Hinds TR,
    7. Kobayashi Y,
    8. Hsu FF,
    9. Sharon M,
    10. Browse J, et al.
    (2010) Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468: 400–405
    OpenUrlCrossRefPubMed
  79. ↵
    1. Sun Y,
    2. Li L,
    3. Macho AP,
    4. Han Z,
    5. Hu Z,
    6. Zipfel C,
    7. Zhou JM,
    8. Chai J
    (2013) Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science 342: 624–628
    OpenUrlAbstract/FREE Full Text
  80. ↵
    1. Szczegielniak J,
    2. Klimecka M,
    3. Liwosz A,
    4. Ciesielski A,
    5. Kaczanowski S,
    6. Dobrowolska G,
    7. Harmon AC,
    8. Muszyńska G
    (2005) A wound-responsive and phospholipid-regulated maize calcium-dependent protein kinase. Plant Physiol 139: 1970–1983
    OpenUrlAbstract/FREE Full Text
  81. ↵
    1. Tan X,
    2. Calderon-Villalobos LI,
    3. Sharon M,
    4. Zheng C,
    5. Robinson CV,
    6. Estelle M,
    7. Zheng N
    (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446: 640–645
    OpenUrlCrossRefPubMed
  82. ↵
    1. Testerink C,
    2. Munnik T
    (2011) Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J Exp Bot 62: 2349–2361
    OpenUrlCrossRefPubMed
  83. ↵
    1. van der Luit AH,
    2. Piatti T,
    3. van Doorn A,
    4. Musgrave A,
    5. Felix G,
    6. Boller T,
    7. Munnik T
    (2000) Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate. Plant Physiol 123: 1507–1516
    OpenUrlAbstract/FREE Full Text
  84. ↵
    1. Veronese P,
    2. Nakagami H,
    3. Bluhm B,
    4. Abuqamar S,
    5. Chen X,
    6. Salmeron J,
    7. Dietrich RA,
    8. Hirt H,
    9. Mengiste T
    (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell 18: 257–273
    OpenUrlAbstract/FREE Full Text
  85. ↵
    1. Vossen JH,
    2. Abd-El-Haliem A,
    3. Fradin EF,
    4. van den Berg GC,
    5. Ekengren SK,
    6. Meijer HJ,
    7. Seifi A,
    8. Bai Y,
    9. ten Have A,
    10. Munnik T, et al.
    (2010) Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance. Plant J 62: 224–239
    OpenUrlCrossRefPubMed
  86. ↵
    1. Williams SP,
    2. Gillaspy GE,
    3. Perera IY
    (2015) Biosynthesis and possible functions of inositol pyrophosphates in plants. Front Plant Sci 6: 67
    OpenUrlPubMed
  87. ↵
    1. Xin XF,
    2. He SY
    (2013) Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51: 473–498
    OpenUrlCrossRefPubMed
  88. ↵
    1. Xu J,
    2. Xie J,
    3. Yan C,
    4. Zou X,
    5. Ren D,
    6. Zhang S
    (2014) A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase-mediated oxidative burst are two independent signaling events in plant immunity. Plant J 77: 222–234
    OpenUrlCrossRefPubMed
  89. ↵
    1. Xue HW,
    2. Chen X,
    3. Mei Y
    (2009) Function and regulation of phospholipid signalling in plants. Biochem J 421: 145–156
    OpenUrlAbstract/FREE Full Text
  90. ↵
    1. Zeng W,
    2. He SY
    (2010) A prominent role of the flagellin receptor FLAGELLIN-SENSING2 in mediating stomatal response to Pseudomonas syringae pv tomato DC3000 in Arabidopsis. Plant Physiol 153: 1188–1198
    OpenUrlAbstract/FREE Full Text
  91. ↵
    1. Zhang X,
    2. Henriques R,
    3. Lin SS,
    4. Niu QW,
    5. Chua NH
    (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1: 641–646
    OpenUrlCrossRefPubMed
  92. ↵
    1. Zhang J,
    2. Li W,
    3. Xiang T,
    4. Liu Z,
    5. Laluk K,
    6. Ding X,
    7. Zou Y,
    8. Gao M,
    9. Zhang X,
    10. Chen S, et al.
    (2010) Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7: 290–301
    OpenUrlCrossRefPubMed
  93. ↵
    1. Zhang J,
    2. Shao F,
    3. Li Y,
    4. Cui H,
    5. Chen L,
    6. Li H,
    7. Zou Y,
    8. Long C,
    9. Lan L,
    10. Chai J, et al.
    (2007) A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immunity in plants. Cell Host Microbe 1: 175–185
    OpenUrlCrossRefPubMed
  94. ↵
    1. Zhang Y,
    2. Zhu H,
    3. Zhang Q,
    4. Li M,
    5. Yan M,
    6. Wang R,
    7. Wang L,
    8. Welti R,
    9. Zhang W,
    10. Wang X
    (2009) Phospholipase Dα1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. Plant Cell 21: 2357–2377
    OpenUrlAbstract/FREE Full Text
  95. ↵
    1. Zhang Z,
    2. Wu Y,
    3. Gao M,
    4. Zhang J,
    5. Kong Q,
    6. Liu Y,
    7. Ba H,
    8. Zhou J,
    9. Zhang Y
    (2012) Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2. Cell Host Microbe 11: 253–263
    OpenUrlCrossRefPubMed
  96. ↵
    1. Zipfel C,
    2. Robatzek S,
    3. Navarro L,
    4. Oakeley EJ,
    5. Jones JD,
    6. Felix G,
    7. Boller T
    (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428: 764–767
    OpenUrlCrossRefPubMed
PreviousNext
Back to top

Table of Contents

Print
Download PDF
Email Article

Thank you for your interest in spreading the word on Plant Physiology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production
(Your Name) has sent you a message from Plant Physiology
(Your Name) thought you would like to see the Plant Physiology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production
Juan Martín D’Ambrosio, Daniel Couto, Georgina Fabro, Denise Scuffi, Lorenzo Lamattina, Teun Munnik, Mats X. Andersson, María E. Álvarez, Cyril Zipfel, Ana M. Laxalt
Plant Physiology Oct 2017, 175 (2) 970-981; DOI: 10.1104/pp.17.00173

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production
Juan Martín D’Ambrosio, Daniel Couto, Georgina Fabro, Denise Scuffi, Lorenzo Lamattina, Teun Munnik, Mats X. Andersson, María E. Álvarez, Cyril Zipfel, Ana M. Laxalt
Plant Physiology Oct 2017, 175 (2) 970-981; DOI: 10.1104/pp.17.00173
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • RESULTS
    • DISCUSSION
    • CONCLUSION
    • MATERIALS AND METHODS
    • Acknowledgments
    • Footnotes
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

In this issue

Plant Physiology: 175 (2)
Plant Physiology
Vol. 175, Issue 2
Oct 2017
  • Table of Contents
  • Table of Contents (PDF)
  • Cover (PDF)
  • About the Cover
  • Index by author
View this article with LENS

More in this TOC Section

Articles

  • Developmental Programming of Thermonastic Leaf Movement
  • BRASSINOSTEROID-SIGNALING KINASE5 Associates with Immune Receptors and Is Required for Immune Responses
  • Deetiolation Enhances Phototropism by Modulating NON-PHOTOTROPIC HYPOCOTYL3 Phosphorylation Status
Show more Articles

SIGNALING AND RESPONSE

  • Pinstatic Acid Promotes Auxin Transport by Inhibiting PIN Internalization
  • Hypermorphic SERK1 Mutations Function via a SOBIR1 Pathway to Activate Floral Abscission Signaling
  • Multi-omics Analysis Reveals Sequential Roles for ABA during Seed Maturation
Show more SIGNALING AND RESPONSE

Similar Articles

Our Content

  • Home
  • Current Issue
  • Plant Physiology Preview
  • Archive
  • Focus Collections
  • Classic Collections
  • The Plant Cell
  • Plant Direct
  • Plantae
  • ASPB

For Authors

  • Instructions
  • Submit a Manuscript
  • Editorial Board and Staff
  • Policies
  • Recognizing our Authors

For Reviewers

  • Instructions
  • Journal Miles
  • Policies

Other Services

  • Permissions
  • Librarian resources
  • Advertise in our journals
  • Alerts
  • RSS Feeds

Copyright © 2021 by The American Society of Plant Biologists

Powered by HighWire