UDP-glucosyltransferase HvUGT13248 confers type II resistance to Fusarium graminearum in barley.

Fusarium head blight (FHB) of barley (Hordeum vulgare) causes yield losses and accumulation of trichothecene mycotoxins (e.g. deoxynivalenol [DON]) in grains. Glucosylation of DON to the nontoxic DON-3-O-glucoside (D3G) is catalyzed by UDP-glucosyltransferases (UGTs), such as barley UGT13248. We explored the natural diversity of UGT13248 in 496 barley accessions and showed that all carried potential functional alleles of UGT13248, as no genotypes showed strongly increased seedling sensitivity to DON. From a TILLING population, we identified 2 mutant alleles (T368I and H369Y) that, based on protein modeling, likely affect the UDP-glucose binding of UGT13248. In DON feeding experiments, DON-to-D3G conversion was strongly reduced in spikes of these mutants compared to controls, and plants overexpressing UGT13248 showed increased resistance to DON and increased DON-to-D3G conversion. Moreover, field-grown plants carrying the T368I or H369Y mutations inoculated with Fusarium graminearum showed increased FHB disease severity and reduced D3G production. Barley is generally considered to have type II resistance that limits the spread of F. graminearum from the infected spikelet to adjacent spikelets. Point inoculation experiments with F. graminearum showed increased infection spread in T368I and H369Y across the spike compared to wild type, while overexpression plants showed decreased spread of FHB symptoms. Confocal microscopy revealed that F. graminearum spread to distant rachis nodes in T368I and H369Y mutants but was arrested at the rachis node of the inoculated spikelet in wild-type plants. Taken together, our data reveal that UGT13248 confers type II resistance to FHB in barley via conjugation of DON to D3G.

Measuring Pectin Properties to Track Cell Wall Alterations During Plant-Pathogen Interactions.

Plant cell walls act both as a barrier to pathogen entry and as a source of signaling molecules that can modulate plant immunity. Cell walls consist mainly of three polymeric sugars: cellulose, pectin, and hemicellulose (Mohnen et al., Biomass Recalcitrance: deconstructing the plant cell wall for bioenergy, 2008). In Arabidopsis more than 50% of the primary cell wall is pectin (Zablackis et al., Plant Physiol 107:1129-1138, 1995). There are various types of pectin, but all pectins contain galacturonic acid subunits in their backbone (Harholt et al., Plant Physiol 153:384-395, 2010; Mohnen, Curr Opin Plant Biol 11:266-277, 2008). Many pathogens secrete pectin-degrading enzymes as part of their infection strategy (Espino et al., Proteomics 10:3020-3034, 2010; ten Have et al., Mol Plant-Microbe Interact 11:1009-1016, 1998). Pectin is synthesized in a highly esterified fashion and is de-esterified in the cell wall by pectin methylesterases (Harholt et al., Plant Physiol 153:384-395, 2010; Mohnen, Curr Opin Plant Biol 11:266-277, 2008). During plant-pathogen interactions, both the amount and the patterns of pectin methylesterification in the wall can be altered (Bethke et al., Plant Physiol 164:1093-1107, 2014; Lionetti et al., J Plant Physiol 169:1623-1630, 2012). Pectin methylesterifications influence mechanical properties of pectin, and pectins must be at least partially de-methylesterified to be substrates for pectin-degrading enzymes (Levesque-Tremblay et al., Planta 242:791-811, 2015). Additionally, alterations of pectin methylesterification or pectin content affect pathogen growth (Bethke et al., Plant Physiol 164:1093-1107, 2014; Lionetti et al., J Plant Physiol 169:1623-1630, 2012; Bethke et al., Plant Cell 28:537-556, 2016; Raiola et al., Mol Plant-Microbe Interact 24:432-440, 2011; Vogel et al., Plant Cell 14:2095-2106, 2002; Vogel et al., Plant J 40:968-978, 2004; Wietholter et al., Mol Plant-Microbe Interact 16:945-952, 2003). This chapter explains a simple protocol that can be used in any molecular biology laboratory to estimate total pectin content using a colorimetric assay and pectin composition using antibodies raised against specific pectin components.

Different Modes of Negative Regulation of Plant Immunity by Calmodulin-Related Genes.

Plant immune responses activated through the perception of microbe-associated molecular patterns, leading to pattern-triggered immunity, are tightly regulated. This results in low immune responses in the absence of pathogens and a rapid return to the resting state following an activation event. Here, we show that two CALMODULIN-LIKE genes, CML46 and CML47, negatively regulate salicylic acid accumulation and immunity in Arabidopsis (Arabidopsis thaliana). The double mutant cml46 cml47 is highly resistant to the pathogen Pseudomonas syringae pv maculicola (Pma). The effects of cml46 cml47 on Pma growth are genetically additive to that of cbp60a, a known negative regulator in the CALMODULIN-BINDING PROTEIN60 (CBP60) family. Transcriptome profiling revealed the effects of cbp60a and cml46 cml47 on both common and separate sets of genes, with the majorities of these differentially expressed genes being Pma responsive. CBP60g, a positive regulator of immunity in the CBP60 family, was found to be transcriptionally regulated by CBP60a, CML46, and CML47 Analysis of the flg22-induced mRNA levels of CBP60g in cbp60a and cml46 cml47 revealed that cml46 cml47 plants have higher induced expression while cbp60a plants retain elevated levels longer than wild-type plants. Assays for the effect of flg22 treatment on Pma growth showed that the effect is stronger in cml46 cml47 plants and lasts longer in cbp60a plants. Thus, the expression pattern of CBP60g is reflected in flg22-induced resistance to Pma.

WRKY70 prevents axenic activation of plant immunity by direct repression of SARD1.

SARD1 is an activator of plant immunity that promotes production of the hormone salicylic acid (SA) and activation of defense gene expression. SARD1 itself is strongly inducible by infection. Here, we investigated the transcriptional control of SARD1. We used yeast one-hybrid assays to identify WRKY70. The WRKY70 binding site was defined using electrophoretic mobility shift assays, and its importance was investigated using an Arabidopsis thaliana protoplast system. The effect of wrky70 mutations was studied by measurements of pathogen growth, SA concentrations, and gene expression by RNA-seq. WRKY70 binds to a GACTTTT motif in the SARD1 promoter in yeast and Arabidopsis protoplasts. Plants with wrky70 mutations have elevated expression of SARD1 in the absence of pathogens, but not when infected. Expression profiling revealed that WRKY70 represses many pathogen-inducible genes in the absence of pathogens, yet is required for activation of many other pathogen-inducible genes in infected plants. The GACTTTT motif is enriched in the promoters of both these gene sets, and conserved in SARD1 orthologs within the Brassicaceae. WRKY70 represses SARD1 by binding the motif GACTTTT in the absence of pathogens. Conservation of the WRKY70 binding among the Brassicaceae suggests that WRKY70 repression of SARD1 is important for fitness.

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana.

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

The receptor-like cytoplasmic kinase PCRK1 contributes to pattern-triggered immunity against Pseudomonas syringae in Arabidopsis thaliana.

In this paper we describe PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) of Arabidopsis thaliana, an RLCK that is important for defense against the pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326). We examined defense responses such as bacterial growth, production of reactive oxygen species (ROS) and callose deposition in pcrk1 mutant plants to determine the role of PCRK1 during pathogen infection. Expression of PCRK1 was induced following pathogen infection. Pathogen growth was significantly higher in pcrk1 mutant lines than in wild-type Col-0. Mutant pcrk1 plants showed reduced pattern-triggered immunity (PTI) against Pma ES4326 after pretreatment with peptides derived from flagellin (flg22), elongation factor-Tu (elf18), or an endogenous protein (pep1). Deposition of callose was reduced in pcrk1 plants, indicating a role of PCRK1 in activation of early immune responses. A PCRK1 transgene containing a mutation in a conserved lysine residue important for phosphorylation activity of kinases (K118E) failed to complement a pcrk1 mutant for the Pma ES4326 growth phenotype. Our study shows that PCRK1 plays an important role during PTI and that a conserved lysine residue in the putative kinase domain is important for PCRK1 function.

The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2.

Multi-layered defense responses are activated in plants upon recognition of invading pathogens. Transmembrane receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and activate MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune responses. For example, Arabidopsis MAP kinase 4 (MPK4) regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with PAT1, a component of the mRNA decapping machinery. PAT1 is also phosphorylated by MPK4 and, upon flagellin PAMP treatment, PAT1 accumulates and localizes to cytoplasmic processing (P) bodies which are sites for mRNA decay. Pat1 mutants exhibit dwarfism and de-repressed immunity dependent on the immune receptor SUMM2. Since mRNA decapping is a critical step in mRNA turnover, linking MPK4 to mRNA decay via PAT1 provides another mechanism by which MPK4 may rapidly instigate immune responses.

The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of 'VQ-motif'-containing proteins to regulate immune responses.

Mitogen-activated protein kinase (MAPK) cascades play key roles in plant immune signalling, and elucidating their regulatory functions requires the identification of the pathway-specific substrates. We used yeast two-hybrid interaction screens, in vitro kinase assays and mass spectrometry-based phosphosite mapping to study a family of MAPK substrates. Site-directed mutagenesis and promoter-reporter fusion studies were performed to evaluate the impact of substrate phosphorylation on downstream signalling. A subset of the Arabidopsis thaliana VQ-motif-containing proteins (VQPs) were phosphorylated by the MAPKs MPK3 and MPK6, and renamed MPK3/6-targeted VQPs (MVQs). When plant protoplasts (expressing these MVQs) were treated with the flagellin-derived peptide flg22, several MVQs were destabilized in vivo. The MVQs interact with specific WRKY transcription factors. Detailed analysis of a representative member of the MVQ subset, MVQ1, indicated a negative role in WRKY-mediated defence gene expression - with mutation of the VQ-motif abrogating WRKY binding and causing mis-regulation of defence gene expression. We postulate the existence of a variety of WRKY-VQP-containing transcriptional regulatory protein complexes that depend on spatio-temporal VQP and WRKY expression patterns. Defence gene transcription can be modulated by changing the composition of these complexes - in part - through MAPK-mediated VQP degradation.

Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae.

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.

Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana.

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

The CALMODULIN-BINDING PROTEIN60 family includes both negative and positive regulators of plant immunity.

Two members of the eight-member CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1), encode positive regulators of plant immunity that promote the production of salicylic acid (SA) and affect the expression of SA-dependent and SA-independent defense genes. Here, we investigated the other six family members in Arabidopsis (Arabidopsis thaliana). Only cbp60a mutations affected growth of the bacterial pathogen Pseudomonas syringae pv maculicola ES4326. In contrast to cbp60g and sard1 mutations, cbp60a mutations reduced pathogen growth, indicating that CBP60a is a negative regulator of immunity. Bacterial growth was increased by cbp60g only in the presence of CBP60a, while the increase in growth due to sard1 was independent of CBP60a, suggesting that the primary function of CBP60g may be to counter the repressive effect of CBP60a. In the absence of pathogen, levels of SA as well as of several SA-dependent and SA-independent pathogen-inducible genes were higher in cbp60a plants than in the wild type, suggesting that the enhanced resistance of cbp60a plants may result from the activation of immune responses prior to pathogen attack. CBP60a bound calmodulin, and the calmodulin-binding domain was defined at the C-terminal end of the protein. Transgenes encoding mutant versions of CBP60a lacking the ability to bind calmodulin failed to complement null cbp60a mutations, indicating that calmodulin-binding ability is required for the immunity-repressing function of CBP60a. Regulation at the CBP60 node involves negative regulation by CBP60a as well as positive regulation by CBP60g and SARD1, providing multiple levels of control over the activation of immune responses.

Pattern-triggered immunity suppresses programmed cell death triggered by fumonisin b1.

Programmed cell death (PCD) is a crucial process for plant innate immunity and development. In plant innate immunity, PCD is believed to prevent the spread of pathogens from the infection site. Although proper control of PCD is important for plant fitness, we have limited understanding of the molecular mechanisms regulating plant PCD. Plant innate immunity triggered by recognition of effectors (effector-triggered immunity, ETI) is often associated with PCD. However pattern-triggered immunity (PTI), which is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs), is not. Therefore we hypothesized that PTI might suppress PCD. Here we report that PCD triggered by the mycotoxin fumonisin B1 (FB1) can be suppressed by PTI in Arabidopsis. FB1-triggered cell death was suppressed by treatment with the MAMPs flg22 (a part of bacterial flagellin) or elf18 (a part of the bacterial elongation factor EF-Tu) but not chitin (a component of fungal cell walls). Although plant hormone signaling is associated with PCD and PTI, both FB1-triggered cell death and suppression of cell death by flg22 treatment were still observed in mutants deficient in jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) signaling. The MAP kinases MPK3 and MPK6 are transiently activated and inactivated within one hour during PTI. We found that FB1 activated MPK3 and MPK6 about 36-48 hours after treatment. Interestingly, this late activation was attenuated by flg22 treatment. These results suggest that PTI suppression of FB1-triggered cell death may involve suppression of MPK3/MPK6 signaling but does not require JA/ET/SA signaling.

Metabolite profiling of Arabidopsis inoculated with Alternaria brassicicola reveals that ascorbate reduces disease severity.

The interaction between the pathogenic ascomycete Alternaria brassicicola and Arabidopsis was investigated by metabolite profiling. The effect of A. brassicicola challenge on metabolite levels was substantial, with nearly 50% of detected compounds undergoing significant changes. Mutations blocking ethylene, jasmonic acid, or ethylene signaling had little effect on metabolite levels. The effects of altering levels of some metabolites were tested by exogenous application during A. brassicicola inoculation. Gamma amino-butyric acid (GABA) or xylitol promoted, while trehalose and ascorbate inhibited, disease severity. GABA promoted, and ascorbate strongly inhibited, fungal growth in culture. Arabidopsis vtc1 and vtc2 mutants, that have low levels of ascorbate, were more susceptible to A. brassicicola. Ascorbate levels declined following A. brassicicola inoculation while levels of dehydroascorbate increased, resulting in a shift of the redox balance between these compounds in the direction of oxidation. These results demonstrate that ascorbate is an important component of resistance to this pathogen.

MPK11-a fourth elicitor-responsive mitogen-activated protein kinase in Arabidopsis thaliana.

Recognition of pathogen attack or elicitation with pathogen-associated molecular patterns (PAMPs) leads to defense signaling that includes activation of the three mitogen-activated protein kinases (MPKs), MPK3, MPK4 and MPK6 in Arabidopsis. Recently, we demonstrated the activation of a fourth MPK, MPK11, after treatment with flg22, a 22 amino acid PAMP derived from bacterial flagellin. Here, we extended the study by examining elicitation with two other PAMPs, elf18 (derived from bacterial elongation factor EF-Tu) and ch8 (N-acetylchitooctaose derived from fungal chitin). Both PAMPs led to rapid MPK11 transcript accumulation and increased MPK11 kinase activity, suggesting that multiple PAMPs (or stresses) can activate MPK11. However, probably due to functional redundancies, bacteria-induced phytoalexin accumulation does not absolutely require MPK11.

An efficient Agrobacterium-mediated transient transformation of Arabidopsis.

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a ß-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.

Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22.

Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK.

The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression.

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.

Ethylene signalling and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis between the endophytic fungus Piriformospora indica and Arabidopsis thaliana.

*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 beta-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.

A protein phosphatase 2C, responsive to the bacterial effector AvrRpm1 but not to the AvrB effector, regulates defense responses in Arabidopsis.

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.

Sometimes new results raise new questions: the question marks between mitogen-activated protein kinase and ethylene signaling.

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.