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While diverse microbe‐ or damage‐associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP‐specific differences in Ca2+ signalling, defence gene expression and MAMP‐mediated growth arrest in Arabidopsis thaliana. Such MAMP‐specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca2+ elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1‐mediated signal amplification to attain MAMP/DAMP‐specific Ca2+ amplitudes in wild‐type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca2+ peak, demonstrating a feedback effect of ROS on Ca2+ signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca2+ response. Thus, fine‐tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up‐ or downstream of Ca2+ signalling.

Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis‐associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane‐binding and pore‐forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C‐terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24‐amino‐acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ.

Using a proteomics approach, a PP2C‐type phosphatase (renamed PIA1, for PP2C induced by AvrRpm1) was identified that accumulates following infection by Pseudomonas syringae expressing the type III effector AvrRpm1, and subsequent activation of the corresponding plant NB‐LRR disease resistance protein RPM1. No accumulation of PIA1 protein was seen following infection with P. syringae expressing AvrB, another type III effector that also activates RPM1, although PIA transcripts were observed. Accordingly, mutation of PIA1 resulted in enhanced RPM1 function in response to P. syringae pathover tomato (Pto) DC3000 (avrRpm1) but not to Pto DC3000 (avrB). Thus, PIA1 is a protein marker that distinguishes AvrRpm1‐ and AvrB‐dependent activation of RPM1. AvrRpm1‐induced expression of the pathogenesis‐related genes PR1, PR2 and PR3, and salicylic acid accumulation were reduced in two pia1 mutants. By contrast, expression of other defense‐related genes, including PR5 and PDF1.2 (plant defensin), was elevated in unchallenged pia1 mutants. Hence, PIA1 is required for AvrRpm1‐induced responses, and confers dual (both positive and negative) regulation of defense gene expression.

The endophytic fungus Piriformospora indica colonizes the roots of the model plant Arabidopsis thaliana and promotes its growth and seed production. The fungus can be cultivated in axenic culture without a host, and therefore this is an excellent system to investigate plant–fungus symbiosis.The growth of etr1, ein2 and ein3/eil1 mutant plants was not promoted or even inhibited by the fungus; the plants produced less seeds and the roots were more colonized compared with the wild‐type. This correlates with a mild activation of defence responses. The overexpression of ETHYLENE RESPONSE FACTOR1 constitutively activated defence responses, strongly reduced root colonization and abolished the benefits for the plants.Piriformospora indica‐mediated stimulation of growth and seed yield was not affected by jasmonic acid, and jasmonic acid‐responsive promoter β‐glucuronidase gene constructs did not respond to the fungus in Arabidopsis roots.We propose that ethylene signalling components and ethylene‐targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis. The results show that the restriction of fungal growth by ethylene signalling components is required for the beneficial interaction between the two symbionts.

Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal opening and seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggested to be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of the Protein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. The PP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus. Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhanced ABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in the opposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not being activated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABA treatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resulted in an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seed germination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genes such as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPK phosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression.

Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone‐responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2‐DE facilitated the discovery of low abundance proteins – enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA‐binding protein, and a C2‐domain‐containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA‐binding protein, involvement of PTM and post‐transcriptional regulation are implicated, respectively.

Calcium (Ca2+), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca2+] elevations are early events in the interaction between the plant growth‐promoting fungus Piriformospora indica and Arabidopsis thaliana . A cell wall extract (CWE) from the fungus promotes the growth of wild‐type seedlings but not of seedlings from P. indica ‐insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca2+ signalling‐related functions. The CWE induces a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum ) plants, as well as in BY‐2 suspension cultures expressing the Ca2+ bioluminescent indicator aequorin. Nuclear Ca2+ transients were also observed in tobacco BY‐2 cells. The Ca2+ response was more pronounced in roots than in shoots and involved Ca2+ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca2+ response by staurosporine and the refractory nature of the Ca2+ elevation suggest that a receptor may be involved. The CWE does not stimulate H2O2 production and the activation of defence gene expression, although it led to phosphorylation of mitogen‐activated protein kinases (MAPKs) in a Ca2+‐dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica . Thus, Ca2+ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.

In Arabidopsis thaliana, mitogen activated protein kinase (MAPK) signaling cascades that contain MPK3, MPK4 and MPK6 have been implicated in various aspects of developmental processes and stress responses. We identified an ethylene response factor (ERF104), which controls innate immunity, to be a specific substrate of MPK6 and showed that ethylene signaling regulates the release of the ERF104 substrate from its kinase. Implications and questions that arise from our findings are addressed. To promote discussions, previously unpublished data, that are rather confounding, are presented and possible explanation provided on how these may fit into our current model.

Mitogen-activated protein kinase (MAPK)–mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme–substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104–up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen.