The effect of the bacterial effector protein harpin on transcriptional profile and mitochondrial proteins of Arabidopsis thaliana
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).
References (45)
Resistance response physiology and signal transduction
Curr Opin Plant Biol
(1998)- et al.
Innate immunity in plants and animals: emerging parallels between recognition of general elicitors and pathogen-associated molecular patterns
Curr Opin Plant Biol
(2002) - et al.
Cytochrome c release and caspase activation during menadione-induced apoptosis in plants
FEBS Lett
(1999) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
Purification of the NADH:ubiquinone oxidoreductase (complex I) of the respiratory chain from the inner mitochondrial membrane of Solanum tuberosum
J Biol Chem
(1994) - et al.
The plant mitochondrial proteome
Trends Plant Sci
(2005) - et al.
New insights into protein S-nitrosylation: Mitochondria as a model system
J Biol Chem
(2004) - et al.
Protein oxidation in plant mitochondria detected as oxidized tryptophan
Free Radic Biol Med
(2006) - et al.
Nitrosylation: the prototypic redox-based signaling mechanism
Cell
(2001) - et al.
Plant pathogens and integrated defence responses to infection
Nature
(2001)
Harpin inactivates mitochondria in Arabidopsis suspension cells
Mol Plant Microb Interact
Induction of systemic acquired resistance in cucumber by Pseudomonas syringae pv. syringae 61 HrpZ-Pss protein
Plant J
Harpin an elicitor of the hypersensitive response in tobacco caused by Erwinia-amylovora elicits active oxygen production in suspension cells
Plant Physiol (Rockville)
Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures
Biochem J
Type III secretion machines: bacterial devices for protein delivery into host cells
Science
A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity
Plant Cell
Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley
Plant Cell
Innate immunity in plants and animals: striking similarities and obvious differences
Immunol Rev
Mitochondrial reactive oxygen species. Contribution to oxidative stress and interorganellar signaling
Plant Physiol
Cyanide restores N gene-mediated resistance to tobacco mosaic virus in transgenic tobacco expressing salicylic acid hydroxylase
Plant Cell
Cleavage of nuclear DNA into oligonucleosomal fragments during cell death induced by fungal infection or by abiotic treatments
Plant Cell
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