Lennart Eschen-Lippold

Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) peak, demonstrating a feedback effect of ROS on Ca(2+) signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca(2+) response. Thus, fine-tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up- or downstream of Ca(2+) signalling.

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.

Salicylic Acid Is Important for Basal Defense of Solanum tuberosum Against Phytophthora infestans

The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.

PAMP‐induced defense responses in potato require both salicylic acid and jasmonic acid

To elucidate the molecular mechanisms underlying pathogen-associated molecular pattern (PAMP)-induced defense responses in potato (Solanum tuberosum), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep-13, a PAMP from Phytophthora, induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive-like cell death. We have previously shown that SA is required for Pep-13-induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12-oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F-box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep-13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep-13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA-deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans.

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.

Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression. In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.

A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1

BackgroundPlant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana.ResultsWe characterized here the “changed calcium elevation 5” (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.ConclusionsHence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction – perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components.

Unraveling Regulation of the Small Heat Shock Proteins by the Heat Shock Factor HvHsfB2c in Barley: Its Implications in Drought Stress Response and Seed Development

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved.

Activation of defense against Phytophthora infestans in potato by down‐regulation of syntaxin gene expression

The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed. Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane-localized SYNTAXIN-RELATED 1 (StSYR1) and SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively. Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1-RNAi plants, but not StSNAP33-RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans-infected StSYR1-RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines. The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1-RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose-containing papillae.