Mats X. Andersson Depar - Plant Physiology

36 Pages·2013·2.39 MB·English

Plant Physiology Preview. Published on August 26, 2013, as DOI:10.1104/pp.113.223503
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For Plant Physiology
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Running head: Phospholipase D in pathogen resistance signaling
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Correspondence to:
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Mats X. Andersson
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Department of Biological and Environmental sciences, University of Gothenburg
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Box 461
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SE-405 30 Göteborg
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Sweden
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Email: [email protected]
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Phone: [phone]
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Research area: Signaling and Response
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Arabidopsis phospholipase D is involved in basal defense and non-host resistance to
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powdery mildew fungi
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Francesco Pinosa1, Nathalie Buhot1, Mark Kwaaitaal2, Per Fahlberg1, Hans Thordal-
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Christensen2, Mats Ellerström1, Mats X. Andersson1*
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1.Department of Biological and Environmental Sciences, University of Gothenburg
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2.Department of Plant and Environmental Sciences, Faculty of Science University of Copenhagen
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*Correspondence to [email protected]
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One sentence summary:
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The Arabidopsis phospholipase D family was probed using reverse genetics for involvement in
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cell wall based defense to non-host powdery mildew and the PLD isoform was identified as a
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component in the defense reaction.
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Financial sources
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The financial support of the Swedish Council for Environment, Agricultural Sciences and Spatial
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Planning (project no. 2007-1563, 2009-888 and 2007-1051), the Carl Tryggers foundation, the
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Olle Engkvist Byggmästare and The Danish Council for Independent Research , Technology and
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Production Sciences (project no. 10-082292) foundation is gratefully acknowledged.
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Author addresses
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Francesco Pinosa, Per Fahlberg, Nathalie Buhot, Mats Ellerström and Mats X. Andersson:
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Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, SE-
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405 30 Göteborg, Sweden
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Mark Kwaaitaal and Hans Thordal-Christensen: Section for Plant and Soil Science, Department
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of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871
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Frederiksberg C, Denmark
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Correspondence to Mats X. Andersson, [email protected]
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Abstract
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Plants have evolved a complex array of defensive responses against pathogenic microorganisms.
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Recognition of microbes initiates signaling cascades that activate plant defenses. The membrane
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lipid phosphatidic acid (PA) produced by phospholipase D (PLD) has been shown to take part in
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both abiotic and biotic stress signaling. In this study, the involvement of PLD in the interaction
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between Arabidopsis thaliana and the barley powdery mildew fungus, Blumeria graminis f. sp.
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hordei (Bgh) was investigated. This non-adapted pathogen is normally resisted by a cell wall-
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based defense, which stops the fungal hyphae from penetrating the epidermal cell wall. Chemical
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inhibition of PA production by PLD increased the penetration rate of Bgh spores on wild-type
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leaves. The analysis of T-DNA knock-out lines for all Arabidopsis PLD genes revealed that
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PLDδ is involved in penetration resistance against Bgh, and chemical inhibition of PLDs in
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plants mutated in PLDδ indicated that this isoform alone is involved in Bgh resistance. In
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addition, we confirmed the involvement of PLDδ in penetration resistance against another non-
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adapted pea powdery mildew fungus, Erysiphe pisi. A GFP fusion of PLDδ localized to the
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plasma membrane at the Bgh attack site, where it surrounded the cell wall reinforcement.
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Furthermore, in the pld mutant transcriptional upregulation of early Microbe Associated
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Molecular Pattern (MAMP) response genes was delayed after chitin stimulation. In conclusion,
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we propose that PLD is involved in defense signaling in non-host resistance against powdery
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mildew fungi and put PLDδ forward as the main isoform participating in this process.
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Introduction
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A wide range of potentially pathogenic microbes are found in the environment of plants.
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Nevertheless, plants are able to resist the great majority of microbes they encounter and are
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susceptible to only a small number of specifically adapted pathogens. This capacity hinges
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primarily on a broad-range form of immunity called “non-host resistance” (NHR), which is
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defined as the immunity displayed by an entire plant species against all genetic variants of a
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pathogen (Thordal-Christensen, 2003). NHR against pathogens of distantly related host species
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is believed to be due to basal defense activated after recognition of microbe-associated molecular
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patterns (MAMPs) (Schulze-Lefert and Panstruga, 2011). While adapted pathogens have evolved
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effector proteins that target distinct plant mechanisms for instance to suppress plant defenses
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(Jones and Dangl, 2006), the effectors of non-adapted pathogens are not able to sufficiently
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suppress plant defenses. In this way, NHR is provided by multiple genes, which makes it
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effective and genetically robust (Schulze-Lefert and Panstruga, 2011). This form of resistance
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has thus gained particular interest for its agro-economic potential.
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A well-studied model system for this type of NHR is the interaction between Arabidopsis
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thaliana and non-adapted powdery mildew fungi (Micali et al., 2008). After germination on a
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leaf, fungal spores develop a specialized hyphal structure, called “appressorium”, which attaches
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to the epidermal cell wall. Subsequently, the fungus attempts to penetrate the cell from the
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attachment site. Here plants are thought to recognize fungal MAMPs and induce localized
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defense responses at the site of interaction. Fungal penetration is usually stopped by the
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formation of a multi-layered cell wall reinforcement known as a papilla. The papilla is rich in
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callose and other antimicrobial substances, which provides both a physical and a chemical barrier
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against the invading pathogen (Hardham et al., 2007; Hückelhoven, 2007). When powdery
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mildew fungi manage to evade this defense, they form a haustorium, a feeding structure that
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invades the host cell. Successfully penetrating spores are stopped either by a second level of
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defense: confinement of the haustorium within a callose encasement, or a third level of defense:
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a hypersensitive cell death response to stop further fungal development (Lipka et al., 2008).
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Thus, NHR against non-adapted powdery mildews and other biotrophic fungi can be
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conceptually divided into pre- and post-invasion defenses.
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It is well established that cell wall-based pathogen recognition in plants is mediated by trans-
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membrane pattern recognition receptors (PRRs) that recognize highly conserved MAMPs.
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Examples of fungal MAMPs are xylanase and chitin. (Boller and Felix, 2009; Monaghan and
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Zipfel, 2012). The corresponding PRRs, Ethylene Inducing-Xylanase (EIX)2 in tomato (Ron and
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Avni, 2004), Chitin Elicitor Receptor Kinase (CERK)1 in Arabidopsis (Miya et al., 2007) and
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Chitin elicitor binding protein (CEBiP) in rice (Shimizu et al., 2010) have been identified.
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Notably, the chitin receptor CERK1 has been found to be essential for resistance against a
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powdery mildew fungus in Arabidopsis (Wan et al., 2008). Several molecular and physiological
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changes are initially brought about by MAMP perception. These include fluxes of various ions
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including Ca2+ over the plasma membrane, protein phosphorylation, generation of reactive
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oxygen species and nitric oxide, activation of mitogen-activated protein kinase (MAPK), Ca2+-
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dependent protein kinase (CDPK) cascades and finally transcriptional changes in defense genes
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(Hückelhoven, 2007; Schwessinger and Zipfel, 2008; Boller and Felix, 2009). These early
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responses to MAMP detection are well studied and known to depend on one another (Ogasawara
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et al., 2008; Ma et al., 2009). However, thorough insight into the regulation and the genetic basis
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of these events remains fragmented.
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Three major components of pre-invasion defense were identified in forward genetic screens for
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Arabidopsis mutants with enhanced penetration frequencies of the non-adapted powdery mildew
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fungus, Blumeria graminis f. sp. hordei (Bgh) (Collins et al., 2003; Lipka et al., 2005; Stein et
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al., 2006). These correspond to the PENETRATION1 (PEN1), PEN2 and PEN3 genes, which
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encode a plasma membrane-localized syntaxin, a glycoside hydrolase and a pleiotropic drug
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resistance ATP binding cassette transporter, respectively. The current model predicts existence
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of a PEN1-defined pathway where this syntaxin is involved in polar secretion of material at sites
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of attempted fungal ingress, together with an ADP ribosylation factor (ARF) GTPase and the
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corresponding, GTP exchange factor, GNOM (Böhlenius et al., 2010; Nielsen et al., 2012). In a
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second pathway, PEN2 is required for production of an antifungal glucosinolate product,
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secreted into the apoplast by PEN3 (Bednarek et al., 2009). The three PEN proteins accumulate
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at the incipient fungal entry site: PEN1 in exosomes in the papilla body (Meyer et al., 2009;
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Nielsen et al., 2012), PEN2 in peroxisomes in the cytoplasmic area surrounding the papilla
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(Lipka et al., 2005) and PEN3 at the plasma membrane (Stein et al., 2006).
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Several lipids and lipid metabolites have been shown to function in the signal transduction
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pathway leading to activation of plant defense responses (Laxalt and Munnik, 2002; Kachroo and
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Kachroo, 2009). A prominent role has been postulated for phosphatidic acid (PA), whose levels
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increase within minutes in plant cells recognizing different MAMPs (van der Luit et al., 2000) or
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upon recognition of pathogen effector proteins (De Jong et al., 2004; Andersson et al., 2006;
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Kirik and Mudgett, 2009). PA has been implicated in the modulation of MAPK activity, Ca2+
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influx from the apoplast, and oxidative burst during abiotic and biotic stresses (Testerink and
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Munnik, 2011). In plants, PA can be formed via two main pathways: the direct hydrolysis of
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phospholipids by phospholipase D (PLD) or the combined action of phospholipase C (PLC) and
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diacylglycerol kinase (Wang, 2004). Both pathways are involved in the early signaling events of
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plant-pathogen interactions and contribute to the activation of plant defenses. Specifically, PLD
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activity has been found to be required for the hypersensitive response induced by recognition of
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bacterial effector proteins (Andersson et al., 2006; Kirik and Mudgett, 2009).
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The twelve different PLD isoforms encoded in the Arabidopsis genome are classified into six
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groups (α, β, γ, δ, ε, ζ), based on sequence similarity and in vitro activity. Two HxKxxxxD
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(HKD) motifs that interact with each other are essential for lipase activity in all eukaryotic PLD
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isoforms (Bargmann and Munnik, 2006). Although an increase in PLD activity upon treatment
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with various MAMPs has been reported for different cell culture systems (van der Luit et al.,
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2000; Yamaguchi et al., 2005; Suzuki et al., 2007), genetic evidence for the involvement of
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specific PLD isoforms in pathogen defense is so far missing. Here, we reveal a role for PLD
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activity in Arabidopsis cell wall-based defense against powdery mildew fungi and genetically
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identify PLD as the responsible PLD isoform. We also report that this PLD is involved in
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MAMP signaling at a more general level.
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Results
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Inhibition of PLD-generated PA causes increased Bgh penetration in Arabidopsis
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The development of Bgh spores on Arabidopsis leaves is synchronous and penetration attempts
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occur in a narrow time frame around 12 hours-post-inoculation (hpi) (Assaad et al., 2004). It is
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thus possible to score and compare fungal penetration on different plants at a certain time point.
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To test a possible contribution of PLD-generated PA to plant resistance against Bgh penetration,
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we exploited the transphosphatidylation activity of PLD, which uses primary alcohols as
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substrates to form an artificial phosphatidyl alcohol. The preferential formation of this compound
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impairs PA production (Munnik et al., 1995). Thus, we employed n-butanol to inhibit PA
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formation by PLD. Leaves were infiltrated with different n-butanol or tert-butanol concentrations
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3 hours before inoculation and penetration was scored at 3 days-post-inoculation (dpi).
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Increasing n-butanol concentrations caused progressively higher Bgh penetration rates, from
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16% (SD 1.0) for a water control to 38% (SD 7.5) for leaves treated with 0.6% n-butanol (Fig.
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1A). In contrast, the penetration rate for leaves infiltrated with tert-butanol, an alcohol with no
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inhibitory effects on PA formation, was not significantly different compared to that of the water
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control. The results thus indicate the involvement of PLD and PA in cell wall-based defense
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against Bgh. We next attempted to measure PLD activity and PA accumulation during the
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defense response. To this end, leaf explants were labeled with 33PO over-night before
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inoculation with Bgh spores. However, no increase in PA label could be measured at 4, 8, 12 and
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24 hpi (Figure S1).
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PLDδ is the only PLD isoform involved in penetration resistance against Bgh
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We set out to identify which, if any, of the twelve known Arabidopsis PLD isoforms contributes
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to penetration resistance against Bgh. For this purpose, we identified at least one T-DNA
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insertion line for each PLD gene. The insertion site of each T-DNA line was verified by
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sequencing (Table S I and Fig. S2). Homozygous plants for T-DNA insertion lines not
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previously published were tested by RT-PCR for the presence of the corresponding PLD
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transcript using primers flanking the insertion site (Fig. S3 and Table S II). Absence of
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transcripts confirmed that the T-DNA lines indeed are knock-out (KO) mutants of the PLD
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genes. Absence of transcript and/or protein in the pldα1, pldα3, pldζ1 and pldζ2 mutants was
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described in previous publications (Zhang et al., 2004; Li et al., 2006; Hong et al., 2008). The
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different PLD single KO lines and two PLD double KO lines (pldβ1-2 pldβ2 and pldζ1 pldζ2)
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were scored for Bgh penetration resistance by counting interaction events in leaves stained by
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trypan blue two days after inoculation with Bgh spores. Among all, only the PLDδ KO line
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(pld ) displayed a significantly higher Bgh penetration rate compared to wild-type (Fig. 1B). The
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penetration rate in this mutant was approximately doubled compared to wild-type, representing
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an increase from 12% (SD 0.8) penetration in wild-type to over 23% (SD 2.3) in pldδ. None of
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the PLD mutants investigated was affected in the cell death response to successfully penetrating
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spores (Fig. S4). Furthermore, none of the lines displayed any visible growth or other phenotype
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under our cultivation regime (Fig. S5).
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Absence of the PLDδ−specific transcript in pldδ was verified with a primer pair annealing
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downstream of the insertion site in addition to the T-DNA flanking pair (Fig. 2A-B). To confirm
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that the penetration phenotype of pldδ is caused by the specific loss of the PLDδ protein, the
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mutant was complemented with PLDδ coding sequence fused to GFP under the transcriptional
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regulation of a 1.4 kb PLDδ promoter region. This restored wild-type levels of Bgh penetration
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(Fig. 2C). In addition, pldδ plants were transformed with a mutated version of the pPLDδ:PLDδ-
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GFP transgene, which carried a point mutation turning a histidine into aspartic acid residue
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(Η707D) in one of the catalytically active HKD sites of PLDδ. This H to D mutation was
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previously shown to abolish the enzymatic activity of a PLD isoform in cabbage (Brassica
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oleracea) (Lerchner et al., 2006). Transgenic pldδ plants expressing the pPLDδ:PLDδΗ707D-
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GFP construct displayed Bgh penetration levels similar to the untransformed pldδ (Fig. 2B-C),
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further supporting that PLDδ activity is essential for normal penetration resistance.
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To verify that PLDδ is the only PLD isoform that plays a role in penetration resistance against
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Bgh, the pldδ mutant was treated with n-butanol before inoculation with Bgh. This treatment
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caused no further increase in Bgh penetration rate (Fig. 3A). The transcripts of PLDα1 and PLDδ
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are the most abundant among the PLD genes in green tissues (Li et al., 2006), while PLDβ1 and
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PLDβ2 are the two isoforms closest related to PLDδ (Qin and Wang, 2002). Bgh penetration was
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scored in pldα1 pldδ double- and pldβ1-2 pldβ2 pldδ triple-mutants, but none of these displayed
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higher levels than the pldδ single mutant (Fig. 3B and C). This, together with the n-butanol
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treatment of pldδ, verified that PLDδ is the sole PLD isoform involved in penetration resistance.
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Double mutants combining the pldδ mutation with the pen1-1 or the pen3-1 mutations, did not
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display higher Bgh penetration frequencies than the respective parent pen mutants (Fig. 3D).
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PLDδ contributes to penetration resistance against the non-host pea powdery mildew
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To investigate the conservation of the involvement of PLDδ in penetration resistance against
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powdery mildew fungi, we used the Arabidopsis non-host fungal pathogen, Erysiphe pisi (Ep),
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which is an adapted powdery mildew fungus on pea plants. Ep penetration rate on wild-type
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Arabidopsis leaves was higher than that of Bgh, suggesting a higher level of adaptation to
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Arabidopsis. The pldδ mutant displayed a significantly higher penetration rate compared to wild-
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type for Ep (Fig. 4). Penetration success rate increased from 18% (SD 3.1) of the germinated
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spores on wild-type leaves to 35% (SD 4.0) on the pldδ mutant. This, thus clearly demonstrates
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that the PLDδ gene is involved in penetration resistance against this fungus as well.
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PLDδ in chitin-induced MAMP-response gene activation
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Since knockout of the PLDδ gene caused decreased penetration resistance against two different
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non-host powdery mildew fungi, and since the gene appears not specifically linked to either
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PEN1- or PEN2/PEN3-defined penetration resistance pathways, we hypothesized that the
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activity of the PLDδ isoform might have a more general role in plant pathogen defense. In
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particular, it seems reasonable to suggest that the gene might be involved in signaling after
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recognition of MAMPs. It would be interesting to test the response of the pldδ mutant to
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MAMPs, such as chitin, since a chitin receptor mutant has been previously found to have
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reduced powdery mildew resistance (Wan et al., 2008). For this purpose, wild-type and
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pldδ mutant seedlings were treated with a chitin extract and transcriptional changes of the early
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MAMP-induced genes, FRK1, NHL10 and PHI-1 (Boudsocq et al., 2010; Kwaaitaal et al., 2011),
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were evaluated by QRT-PCR (Fig. 5). The transcript levels of all three genes rapidly increased
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following treatment with chitin in both the wild-type and mutant plants. PHI1 and NHL10 both
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reached a peak of transcript accumulation 30 min after treatment, whereas FRK1 transcript
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increased throughout the experiment. However, the pldδ mutant showed a trend for an
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approximately 50% reduction in transcript accumulation after 30 min for PHI1 and NHL10. For
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all three genes, a trend for a delayed chitin-induced accumulation of transcripts was observed.
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