Elsevier

Journal of Proteomics

Volume 71, Issue 2, 21 July 2008, Pages 148-159
Journal of Proteomics

The effect of the bacterial effector protein harpin on transcriptional profile and mitochondrial proteins of Arabidopsis thaliana

https://doi.org/10.1016/j.jprot.2008.04.002Get rights and content

Abstract

Here we report on the effect of the bacterial elicitor harpin from Pseudomonas syringae on Arabidopsis thaliana with an emphasis on transcriptional profiling and on changes of the mitochondrial proteome. Interestingly, of the currently about 400 identified mitochondrial proteins, transcriptional profiling by genome-wide DNA-microarray analyses revealed a total of 192 genes that showed significant changes in transcript abundance in response to the bacterial elicitor. The most dramatic changes were observed for the mitochondrial protein import apparatus of which 70% of all genes were induced. Proteomic analyses by 2D-PAGE and MALDI-TOF Mass Spectrometry for peptide fingerprint analysis confirmed the corresponding changes within the mitochondrial proteome in 28 cases. Strikingly, we found an accumulation of virtually all members of the TCA cycle. In sum, our results point to an involvement of mitochondria in the response of plant cells to the harpin elicitor, which becomes apparent both at the transcriptomic as well as proteomic level.

Introduction

Plant defense responses are frequently expressed in part as the so-called hypersensitive response (HR), which is characterized by necrosis at the sites of infection (resembling animal programmed cell death, PCD) and restriction of pathogen growth and spread. The HR is associated with the induction of defense-related genes which play important roles in containing pathogen growth either indirectly, by helping to reinforce the plant cell walls, or directly, by providing antimicrobial enzymes and phytoalexins [1], [2]. HR in plants is characterized by an increase of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and nitric oxide (NO) and is often followed by a systemic response called systemic acquired resistance (SAR) [3], [4], [5], [6].

Harpins are glycine-rich, cysteine-lacking, heat-stable proteins that are transported via the type III secretion system into cells of a host plant. Harpin can induce HR in non-host plants, but the role of harpin in host plants is not clear [7]. The structural properties of harpin from Pseudomonas ssp. resemble those of proteins assumed to interact with membranes, such as Yersinis enterocolitica YopB [8]. Harpin appears to bind to plasma membranes, and a receptor-mediated MAPK-dependent signaling pathway mediates the activation of plant defense responses [9]. It induces a strong pH shift and stimulates calcium influx across the plasma membrane of cells, which is essential for initiation of defense mechanisms [10]. Although harpin (and harpin-like orthologs) occur in specific bacteria only, it is regarded as pathogen-associated molecular patterns (PAMPs), which bind to pattern recognition receptors and thereby trigger the expression of immune response genes and the production of anti-microbial compounds [11]. The harpin elicitor from Erwinia and Pseudomonas species is the active ingredient in the plant activator Messenger®, which has been recently commercialized (Eden Bioscience) [12].

Intense investigations focus on how multifunctional elicitors affect the pathways that regulate plant growth and defense. The involvement of mitochondria in pathogen-induced defense responses and cell death has been demonstrated for several model systems. Mitochondrial reactive oxygen species contribute to oxidative stress and retrograde signaling [13]. In tobacco, mitochondria are involved in salicylic acid (SA)-induced plant resistance to viral pathogens [14]. Cyanide, an inhibitor of mitochondrial cytochrome c dependent respiration (but also of other heme-containing enzymes), induced formation of DNA laddering in cowpea [15]. In Arabidopsis, NO induced activation of alternative respiration and inhibition of cytochrome c respiration [16]. In tobacco treated with menadione as well as during agrobacterium-induced apoptosis in maize, cytochrome c release has been associated with cell death [17], [18]. More recently, a possible role of mitochondrial-derived ROS has been suggested in controlling apoptotic cell death in oats [19].

Mitochondria appear to play a key role in a plant's response to harpin. In Arabidopsis treated with harpin, both plant growth enhancement and induced resistance have been observed [20]. A growing body of evidence supports a central role of mitochondria in the mediation of harpin-induced responses in plants. Among the fast responses a decrease of the mitochondrial membrane potential ΔΨm and possibly as a direct consequence a decline of ATP production were observed [21]. For tobacco, infra-red thermography revealed a role for mitochondria in pre-symptomatic cooling during harpin-induced hypersensitive response [22]. In addition, harpin induces a rapid cytochrome c release from mitochondria into the cytosol, which is regarded as a hallmark of programmed cell death [3].

For this report, we analyzed the transcriptional changes of Arabidopsis cells treated with the bacterial elicitor protein harpin. Thanks to the well documented Arabidopsis mitochondrial proteome [23], [24] we also can provide proteomic data with an emphasis on mitochondria.

Section snippets

Cell culture growth

The Arabidopsis thaliana cell culture originates from the ecotype Col-O (Columbia) [25] and was maintained in the dark on an orbital shaker (120 rpm) at 27 °C and sub-cultured every 7 days by dilution in fresh growth medium. On average, 100 ml cell culture contained 35 g cells on a fresh weight basis. Media used for this cell culture was modified after [26], supplemented with 2% (w/v) sucrose as carbon source. Six days after sub-culturing (i.e. late logarithmic growth phase) 100 ml Arabidopsis

Respiratory oxygen uptake

Plant responses to harpin are characterized by an increase of reactive oxygen species (ROS) such as H2O2 and NO as well as by an enhanced accumulation of salicylic acid (SA) which is modulated by a strong pH shift [3], [4], [5], [6]. In a previous study we already reported a dramatic, transient increase of AOX in Arabidopsis suspension cells as well as a decline of mitochondrial transmembrane potential (ΔΨm) after harpin treatment [3]. Here we investigated the respiration of isolated and

Discussion

Previously, we and others reported on effects of the bacterial effector protein harpin regarding mitochondrial functions [3], [21], [22]. In animals and plants mitochondria play a central role in a variety of cellular processes, including ATP generation by oxidative phosphorylation and robust regulation of calcium homeostasis inside the cell, and also in production of toxic reactive oxygen species (ROS) as a by-product, and the initiation of cell death through the activation of the

Acknowledgement

This work was supported by a grant of Deutsche Forschungsgemeinschaft (DFG, SPP 1110 Innate Immunity).

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