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Background The utility of routine extensive molecular profiling of pediatric tumors is a matter of debate due to the high number of genetic alterations of unknown significance or low evidence and the lack of standardized and personalized decision support methods. Digital drug assignment (DDA) is a novel computational method to prioritize treatment options by aggregating numerous evidence-based associations between multiple drivers, targets, and targeted agents. DDA has been validated to improve personalized treatment decisions based on the outcome data of adult patients treated in the SHIVA01 clinical trial. The aim of this study was to evaluate the utility of DDA in pediatric oncology. Methods Between 2017 and 2020, 103 high-risk pediatric cancer patients ( Results Of the 100 cases with comprehensive molecular diagnostic data, 88 yielded WES and 12 panel sequencing results. DDA identified matching off-label targeted treatment options (actionability) in 72/100 cases (72%), while 57/100 (57%) showed potential drug resistance. Actionability reached 88% (29/33) by 2020 due to the continuous updates of the evidence database. MTB approved the clinical use of a DDA-top-listed treatment in 56 of 72 actionable cases (78%). The approved therapies had significantly higher aggregated evidence levels (AELs) than dismissed therapies. Filtering of WES results for targeted panels missed important mutations affecting therapy selection. Conclusions DDA is a promising approach to overcome challenges associated with the interpretation of extensive molecular profiling in the routine care of high-risk pediatric cancers. Knowledgebase updates enable automatic interpretation of a continuously expanding gene set, a “virtual” panel, filtered out from genome-wide analysis to always maximize the performance of precision treatment planning.

Precision oncology is currently based on pairing molecularly targeted agents (MTA) to predefined single driver genes or biomarkers. Each tumor harbors a combination of a large number of potential genetic alterations of multiple driver genes in a complex system that limits the potential of this approach. We have developed an artificial intelligence (AI)-assisted computational method, the digital drug-assignment (DDA) system, to prioritize potential MTAs for each cancer patient based on the complex individual molecular profile of their tumor. We analyzed the clinical benefit of the DDA system on the molecular and clinical outcome data of patients treated in the SHIVA01 precision oncology clinical trial with MTAs matched to individual genetic alterations or biomarkers of their tumor. We found that the DDA score assigned to MTAs was significantly higher in patients experiencing disease control than in patients with progressive disease (1523 versus 580, P = 0.037). The median PFS was also significantly longer in patients receiving MTAs with high (1000+ P = 0.044). Our results indicate that AI-based systems, like DDA, are promising new tools for oncologists to improve the clinical benefit of precision oncology.

Background: The anaplastic lymphoma kinase (ALK) gene fusion rearrangement is a potent oncogene, accounting for 2–7% of lung adenocarcinomas, with higher incidence (17–20%) in non-smokers. ALK-positive tumors are sensitive to ALK tyrosine kinase inhibitors (TKIs), thus ALK-positive non-small-cell lung cancer (NSCLC) is currently spearheading precision medicine in thoracic oncology, with three generations of approved ALK inhibitors in clinical practice. However, these treatments are eventually met with resistance. At the molecular level, ALK-positive NSCLC is of the lowest tumor mutational burden, which possibly accounts for the high initial response to TKIs. Nevertheless, TP53 co-mutations are relatively frequent and are associated with adverse outcome of crizotinib treatment, whereas utility of next-generation ALK inhibitors in TP53-mutant tumors is still unknown. Methods: We report the case of an ALK-positive, TP53-mutant NSCLC patient with about five years survival on ALK TKIs with continued next-generation regimens upon progression. Results: The tumor showed progression on crizotinib, but long tumor control was achieved following the incremental administration of next-generation ALK inhibitors, despite lack of evident resistance mechanisms. Conclusion: TP53 status should be taken into consideration when selecting ALK-inhibitor treatment for personalized therapies. In TP53-mutant tumors, switching TKI generations may overcome treatment exhaustion even in the absence of ALK-dependent resistance mechanisms.

Mitogen-activated protein kinase (MAPK) pathways are versatile signaling mechanisms in all eukaryotes. Their signaling outputs are defined by the protein substrates phosphorylated by MAPKs. An expanding list of substrates has been identified by high-throughput screens and targeted approaches in plants. The majority of these are phosphorylated by MPK3/6, and a few by MPK4, which are the best-characterized plant MAPKs, participating in the regulation of numerous biological processes. The identified substrates clearly represent the functional diversity of MAPKs: they are associated with pathogen defense, abiotic stress responses, ethylene signaling, and various developmental functions. Understanding their outputs is integral to unraveling the complex regulatory mechanisms of MAPK cascades. We review here methodological approaches and provide an overview of known MAPK substrates.

Background: We present the case of a 50-year-old female whose metastatic pancreatic neuroendocrine tumor (pNET) diagnosis was delayed by the COVID-19 pandemic. The patient was in critical condition at the time of diagnosis due to the extensive tumor burden and failing liver functions. The clinical dilemma was to choose between two registered first-line molecularly-targeted agents (MTAs), sunitinib or everolimus, or to use chemotherapy to quickly reduce tumor burden. Methods: Cell-free DNA (cfDNA) from liquid biopsy was analyzed by next generation sequencing (NGS) using a comprehensive 591-gene panel. Next, a computational method, digital drug-assignment (DDA) was deployed for rapid clinical decision support. Results: NGS analysis identified 38 genetic alterations. DDA identified 6 potential drivers, 24 targets, and 79 MTAs. Everolimus was chosen for first-line therapy based on supporting molecular evidence and the highest DDA ranking among therapies registered in this tumor type. The patient’s general condition and liver functions rapidly improved, and CT control revealed partial response in the lymph nodes and stable disease elsewhere. Conclusion: Deployment of precision oncology using liquid biopsy, comprehensive molecular profiling, and DDA make personalized first-line therapy of advanced pNET feasible in clinical settings.

The development of leaf primordia is subject to light control of meristematic activity. Light regulates the expression of thousands of genes with roles in cell proliferation, organ development, and differentiation of photosynthetic cells. Previous work has highlighted roles for hormone homeostasis and the energy-dependent Target of Rapamycin (TOR) kinase in meristematic activity, yet a picture of how these two regulatory mechanisms depend on light perception and interact with each other has yet to emerge. Their relevance beyond leaf initiation also is unclear. Here, we report the discovery that the dark-arrested meristematic region of Arabidopsis (Arabidopsis thaliana) experiences a local energy deprivation state and confirm previous findings that the PIN1 auxin transporter is diffusely localized in the dark. Light triggers a rapid removal of the starvation state and the establishment of PIN1 polar membrane localization consistent with auxin export, both preceding the induction of cell cycle- and cytoplasmic growth-associated genes. We demonstrate that shoot meristematic activity can occur in the dark through the manipulation of auxin and cytokinin activity as well as through the activation of energy signaling, both targets of photomorphogenesis action, but the organ developmental outcomes differ: while TOR-dependent energy signals alone stimulate cell proliferation, the development of a normal leaf lamina requires photomorphogenesis-like hormonal responses. We further show that energy signaling adjusts the extent of cell cycle activity and growth of young leaves non-cellautonomously to available photosynthates and leads to organs constituted of a greater number of cells developing under higher irradiance. This makes energy signaling perhaps the most important biomass growth determinant under natural, unstressed conditions.

Plant growth flexibly adapts to environmental conditions, implying cross‐talk between environmental signalling and developmental regulation. Here, we show that the PIN auxin efflux carrier family possesses three highly conserved putative mitogen‐activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well‐characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6. In contrast to AGC kinases, MAPK‐mediated phosphorylation of PIN1 at adjacent sites leads to a partial loss of the plasma membrane localisation of PIN1. MAPK‐mediated modulation of PIN trafficking may participate in environmental adjustment of plant growth.

Summary Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6. PIN6 localizes to endomembrane domains in tissues with low PIN6 expression levels such as roots, but localizes at the plasma membrane (PM) in tissues with increased PIN6 expression such as the inflorescence stem and nectary glands. We provide evidence that this dual localization is controlled by PIN6 phosphorylation and demonstrate that PIN6 is phosphorylated by mitogen-activated protein kinases (MAPKs) MPK4 and MPK6. The analysis of transgenic plants expressing PIN6 at PM or in endomembrane domains reveals that PIN6 subcellular localization is critical for Arabidopsis inflorescence stem elongation post-flowering (bolting). In line with a role for PIN6 in plant bolting, inflorescence stems elongate faster in pin6 mutant plants than in wild-type plants. We propose that PIN6 subcellular localization is under the control of developmental signals acting on tissue-specific determinants controlling PIN6-expression levels and PIN6 phosphorylation.

Mitogen-activated protein kinase (MAPK) pathways are central cellular signalling mechanisms in all eukaryotes. They are key regulators of the cell cycle and stress responses, yet evolution of MAPK families took markedly different paths in the animal and plant kingdoms. Instead of the characteristic divergence of MAPK types in animals, in plants an expanded network of ERK-like MAPKs has emerged. To gain insight into the early evolution of the plant MAPK family we identified and analysed MAPKs in 13 representative species across green algae, a large and diverse early-diverging lineage within the plant kingdom. Our results reveal that the plant MAPK gene family emerged from three types of progenitor kinases, which are ubiquitously present in algae, implying their formation in an early ancestor. Low number of MAPKs is characteristic across algae, the few losses or duplications are associated with genome complexity rather than habitat ecology, despite the importance of MAPKs in environmental signalling in flowering plants. ERK-type MAPKs are associated with cell cycle regulation in opisthokont models, yet in plants their stress-signalling function is more prevalent. Unicellular microalgae offer an excellent experimental system to study the cell cycle, and MAPK gene expression profiles show CDKB-like peaks around S/M phase in synchronised Chlamydomonas reinhardtii cultures, suggesting their participation in cell cycle regulation, in line with the notion that the ancestral eukaryotic MAPK was a cell cycle regulator ERK-like kinase. Our work also highlights the scarcity of signalling knowledge in microalgae, in spite of their enormous ecological impact and emerging economic importance.

Squamous cell carcinomas (SCCs) are among the most frequent solid tumors in humans. SCCs, related or not to the human papillomavirus, share common molecular features. Immunotherapies, and specifically immune checkpoint inhibitors, have been shown to improve overall survival in multiple cancer types, including SCCs. However, only a minority of patients experience a durable response with immunotherapy. Epigenetic modulation plays a major role in escaping tumor immunosurveillance and confers resistance to immune checkpoint inhibitors. Preclinical evidence suggests that modulating the epigenome might improve the efficacy of immunotherapy. We herein review the preclinical and the clinical rationale for combining immunotherapy with an epidrug, and detail the design of PEVOsq, a basket clinical trial combining pembrolizumab with vorinostat, a histone deacetylase inhibitor, in patients with SCCs of different locations. Sequential blood and tumor sampling will be collected in order to identify predictive and pharmacodynamics biomarkers of efficacy of the combination. We also present how clinical and biological data will be managed with the aim to enable the development of a prospective integrative platform to allow secure and controlled access to the project data as well as further exploitations., Highlights • Squamous cell carcinomas (SCCs) of different locations share molecular features. • Immunotherapy improves overall survival in several cancers including SCCs. • Epigenetic modulation plays a major role in resistance to immunotherapy. • PEVOsq evaluates pembrolizumab/vorinostat combination in SCCs of several locations. • An integrative platform ensures secured optimal management of generated data.

3642 Background: Precision oncology requires the identification of individual molecular pathomechanisms to find optimal personalized treatment strategies for every cancer patient. Incorporation of complex molecular information into routine clinical practice remains a significant challenge due to the lack of a reproducible, standardized process of clinical decision making. Methods: To provide a standardized process for molecular interpretation, we develop a precision oncology decision support system, the Realtime Oncology Molecular Treatment Calculator (MTC). MTC is a rule-based medical knowledge engine that dynamically aggregates and ranks relevant scientific and clinical evidence using currently 26,000 evidence-based associations and reproducible algorithm scoring of drivers, molecular targets to match molecular alterations to efficient therapies. To validate this novel method and system, we used data of the SHIVA01 trial of molecularly targeted therapy (Lancet Oncol 2015 16:1324-34). Molecular profiles of participants were uploaded to MTC and aggregated evidence level (AEL) values of associated targeted treatments were calculated, including those used in the SHIVA01 trial. Results: The MTC output provided a prioritized list of drugs associated with the driver alterations in the patient molecular profile, where ranking is based on AEL values. Of 113 patients who received targeted therapy with available clinical best response data, disease control was experienced in 63 cases (PR: 5, SD: 58), while disease progression occurred in 50 cases. The average AEL score for the therapies applied was significantly higher in the responsive group than in the non-responsive group (1512 and 614, respectively (p = 0.049)). In 94 cases, drugs other than those used for therapy were ranked higher by the MTC. The average AEL difference between the top-ranked and the used drugs was in an inverse correlation with clinical response, i.e. smaller differences associated with a better outcome. Conclusions: Results indicate that the aggregation of evidence-based tumor-driver-target-drug associations using standardized mathematical algorithms of this computational tool is a promising novel approach to improve clinical decisions in precision oncology. Further validation based on the results of other targeted clinical trials and real-life data using more detailed molecular profiles is warranted to explore the full clinical potential of this novel medical solution.

Plant growth flexibly adapts to environmental conditions. Growth initiation itself may be conditional to a suitable environment, while the most common response of plants to adverse conditions is growth inhibition. Most of our understanding about environmental growth inhibition comes from studies on various plant hormones, while less is known about the signaling mechanisms involved. The mitogen-activated protein kinase (MAPK) cascades are central signal transduction pathways in all eukaryotes and their roles in plant stress responses is well-established, while increasing evidence points to their involvement in hormonal and developmental processes. Here we show that the MKK7-MPK6 module is a suppressor of meristem activity using genetic approaches. Shoot apical meristem activation during light-induced de-etiolation is accelerated in mpk6 and mkk7 seedlings, whereas constitutive or induced overexpression of MKK7 results in meristem defects or collapse, both in the shoot and the root apical meristems. These results underscore the role of stress-activated MAPK signaling in regulating growth responses at the whole plant level, which may be an important regulatory mechanism underlying the environmental plasticity of plant development.

35 Background: Most tumours harbor multiple driver genetic alterations and many driver alterations are linked to multiple targeted therapies with various level of evidence. In addition, a specific treatment can be linked to multiple genetic alterations in the same tumor. Several public and private databases and software solutions are available to link driver alterations to treatments options, but in clinical practice of precision oncology we need a solution to select the right treatment for our patients based on the highest level of evidence also in case of complex molecular profiles. Methods: We have developed an AI oncology algorithm and rule-engine to prioritise treatment options for every cancer patient based on the individual molecular of their tumor. This IT solution can now prioritise 1200 compounds in clinical use or clinical development based on the computing of 24,000 evidence-based associations (“rules”) between drivers, targets and compounds. The software calculates a numeric score, the “aggregated evidence level” for each driver alterations and compounds. We have linked this decision support software to a dynamic patient case management system, which records responds to therapy to create learning system to provide dynamic decision support through several lines of therapies of each patient and to use real-life evidence to further improve the algorithm. Results: Our first results indicate that system allows individualised decision of diagnostic option between single gene tests to comprehensive 600 gene NGS panels and identification of actionable alterations in 83% of cancer cases. Conclusions: This system can be a first working solution to standardise clinical decisions precision oncology, which also helps the real-life evaluation of novel multigene molecular diagnostic tests and therapies to find their best indications and accelerate their reimbursement by insurance companies and national health funds.

Background While increasing number of diagnostic laboratories perform new generation sequencing on tumor DNA at a rapidly decreasing cost, beyond companion diagnostics, multi-gene panels are still underutilized due to lack of reimbursement and value based technical assessment. Besides financial reasons, prioritizing between diagnostic tests, multiple genetic alterations and multiple on-label or off-label targeted treatment options linked to these alterations can be a major challenge without having a reproducible, standardized and effective information service system in hand that a molecular tumor board could safely rely on. Here, we present an easily clinically adaptable, dynamic treatment decision support program (Protocol), that makes tumor molecular profile-based oncology care feasible and potentially reimbursed by public insurance funds. Methods Our Protocol was developed based on 4868 cancer cases with 429 different histology, 2367 genes with 36492 variants. Our program utilizes multidisciplinary molecular tumor board (MTB) supported by Realtime Oncology Treatment CalculatorTM (Calculator) with the idea of integrating molecular and clinical data to provide genomics-driven treatment strategy plan. MTB mobilizes multiple sub-specialties for managing patients and tumor samples. The Calculator under constant upgrade is currently operating based on 24000 rules, 4000+ curated evidence, 101 registered and 618 targeted drugs under development. Results This medical procedure (protocol) was endorsed by the Hungarian National Health Insurance Fund based on QUALY gain calculation and value-based technology assessment. Conclusions It is crucial to have an established protocol in the community of practice to indicate effective and eliminate ineffective genomics-driven treatments based on molecular profiling. The technology assessment provided evidence that standardized decision support of the molecular tumor board (MTB) to select diagnostic tests and treatments can assure clinical utility and cost effectiveness of multigene testing (NGS). Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure I. Petak: Leadership role, ownership: Oncompass Medicine Hungary Ltd. C. Hegedus: Full / Part-time employment: Oncompass Medicine Hungary Ltd. E. Varkondi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. Z. Farkas: Full / Part-time employment: Oncompass Medicine Hungary Ltd. D. Tihanyi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. R. Doczi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. D. Mathiasz: Full / Part-time employment: Oncompass Medicine Hungary Ltd. G. Pajkos: Full / Part-time employment: Oncompass Medicine Hungary Ltd. R. Schwab: Advisory / Consultancy, ownership: Oncompass Medicine Hungary Ltd. J. Deri: Full / Part-time employment: Oncompass Medicine Hungary Ltd. All other authors have declared no conflicts of interest.

Background Cancer immunotherapy based on immune checkpoint inhibitors (ICIs) is approved in several indications, in most of them independently of biomarker status. Yet, in the era of precision oncology, detailed tumor molecular profiling is a prerequisite of informed therapeutic decisions. Methods Tumor molecular profiles of 159 patients were analyzed by NGS (595 cancer-associated genes), PD-L1 IHC and MSI assays. Molecular alterations and targeted therapy drugs were classified and ranked by relevance based on scientific evidence by our in-house developed algorithm, the Realtime Oncology Molecular Treatment Calculator. Results Of the 159 samples 20 (13%) originated from tumors where at least one ICI is approved without biomarker status. 10 (50%) of these were positive to at least one immunotherapy-related biomarker (TMB-H, MSI-H or PD-L1 positive). In the remaining samples biomarker positivity was detected in 38 cases (27%). Directly targetable driver mutations were detected in 60% of the biomarker-positive and 49% of the biomarker-negative samples. Directly targetable mutations were detected in 60% of the biomarker-independently ICI-approved tumors, and in 51% of the other tumor types. JAK1/2 and STK11 mutations are known to lead to resistance to ICI treatments. Deleterious JAK1/2 mutations were detected in 9 patients, 1 in the biomarker-independent ICI group, while both two detected STK11 mutations were present in this group. Conclusions We found that ICI-biomarkers are more common in tumor types where ICIs are approved irrespectively of biomarker status, suggesting that these tumor types are more prone to immune evasion. However, in line with the increased mutational burden, biomarker positive tumors also tend to harbor somewhat more directly targetable driver mutations. We computationally ranked driver-drug relations according to the relevant evidence database and found that in 8 of the 29 ICI-biomarker and actionable driver double positive cases targeting the drivers was ranked with higher evidence scores than ICI therapy. These results highlight that strong drivers may favor targeted inhibition, and the importance of use of an evidence-based standardized algorithm to support driver versus ICI therapy decisions in precision oncology. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure R. Doczi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. D. Tihanyi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. P. Filotas: Full / Part-time employment: Oncompass Medicine Hungary Ltd. A. Dirner: Full / Part-time employment: Oncompass Medicine Hungary Ltd. B. Palhazi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. E. Varkondi: Full / Part-time employment: Oncompass Medicine Hungary Ltd. Z. Farkas: Full / Part-time employment: Oncompass Medicine Hungary Ltd. J. Deri: Full / Part-time employment: Oncompass Medicine Hungary Ltd. C. Hegedus: Full / Part-time employment: Oncompass Medicine Hungary Ltd. I. Petak: Leadership role, ownership: Oncompass Medicine Hungary Ltd. All other authors have declared no conflicts of interest.

Background Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo. The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Phosphorylation status of epitope-tagged proteins is subsequently detected by high-resolution capillary isoelectric focusing coupled with nanofluidic immunoassay, which is capable of detecting subtle changes in isoform distribution. Results The concept is validated by showing phosphorylation of the known mitogen-activated protein kinase (MAPK) substrate, ACS6, by MPK6. Next, we demonstrate that two transcription factors, WUS and AP2, both of which are shown to be master regulators of plant development by extensive genetic studies, exist in multiple isoforms in plant cells and are phosphorylated by activated MAPKs. Conclusion As plant development flexibly responds to environmental conditions, phosphorylation of developmental regulators by environmentally-activated kinases may participate in linking external cues to developmental regulation. As a counterpart of advances in unbiased screening methods to identify potential protein kinase substrates, such as phosphoproteomics and computational predictions, our results expand the candidate-based experimental toolkit for kinase research and provide an alternative in vivo approach to existing in vitro methodologies. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0894-1) contains supplementary material, which is available to authorized users.

The evolutionarily conserved mitogen-activated protein kinase (MAPK) signaling network comprises connected protein kinases arranged in MAPK modules. In this Opinion article, we analyze MAPK signaling components in evolutionarily representative species of the plant lineage and in Naegleria gruberi, a member of an early diverging eukaryotic clade. In Naegleria, there are two closely related MAPK kinases (MKKs) and a single conventional MAPK, whereas in several species of algae, there are two distinct MKKs and multiple MAPKs belonging to different groups. This suggests that the formation of multiple MAPK modules began early during plant evolution. The expansion of MAPK signaling components through gene duplications and the evolution of interaction motifs could have contributed to the highly connected complex MAPK signaling network that we know in Arabidopsis.

* Mitogen activated protein kinase (MAPK) pathways are signal transduction modules with layers of protein kinases having c. 120 genes in Arabidopsis, but only a few have been linked experimentally to functions. * We analysed microarray expression data for 114 MAPK signalling genes represented on the ATH1 Affymetrix arrays; determined their expression patterns during development, and in a wide range of time-course microarray experiments for their signal-dependent transcriptional regulation and their coregulation with other signalling components and transcription factors. * Global expression correlation of the MAPK genes with each of the represented 21 692 Arabidopsis genes was determined by calculating Pearson correlation coefficients. To group MAPK signalling genes based on similarities in global regulation, we performed hierarchical clustering on the pairwise correlation values. This should allow inferring functional information from well-studied MAPK components to functionally uncharacterized ones. Statistical overrepresentation of specific gene ontology (GO) categories in the gene lists showing high expression correlation values with each of the MAPK components predicted biological themes for the gene functions. * The combination of these methods provides functional information for many uncharacterized MAPK genes, and a framework for complementary future experimental dissection of the function of this complex family.

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant's response to B. cinerea.

The drought-inducible DS2 genes of potatoes are members of the ASR (abscisic acid, stress and ripening) gene family. Previously it was shown that expression of DS2 genes is highly dehydration-specific in potato leaves, is not inducible by cold, heat, salt, hypoxia or oxidative stresses, and is independent of abscisic acid (ABA). Now it is shown that StDS2 does not respond either to sucrose or any plant hormones. Conservation of DS2 genes with this unique mode of regulation was studied in the solanaceous species with different relationships to potatoes. DS2 orthologues were identified by DNA sequence alignment in the closely related Lycopersicon and Capsicum species but not in the more distantly related Nicotiana sp. DNA and RNA gel blot analysis revealed the presence of a gene highly homologous to the potato gene StDS2 in tomato (LeDS2) with the same desiccation-specific expression in leaves and organ-specific expression in flowers and green fruits. The LeDS2 promoter was isolated and found to be almost identical in sequence with the promoter of StDS2, except for a 45-bp insertion in tomato. In contrast, no gene highly similar to StDS2 was detected in Nicotiana species on DNA gel blots. Neither StDS2 nor LeDS2 promoter regions were able to confer expression for the beta-glucuronidase (GUS) reporter gene in transgenic tobacco plants indicating that the trans regulatory factors necessary for DS2 expression are not conserved either in Nicotiana tabacum. These data suggest a narrow species-specificity and late evolution of the DS2-type genes within the family Solanaceae.

Environmental stresses induce the expression of several plant genes via multiple and cross-talking signalling pathways. Previously it was shown that ScDS2 , a gene of the wild potato species, Solanum chacoense , is highly inducible by dehydration but not by abscisic acid (ABA), the mediator of many plant stress responses. Herein it is shown that ScDS2 -related genes are present in the cultivated potato, Solanum tuberosum ( StDS2 ) and also in the non-tuberizing Solanum species, Solanum brevidens ( SbDS2 ). We show that expression of StDS2 is dehydration-specific, is not inducible by cold, heat, salt, hypoxia or oxidative stresses, and is independent of ABA. Signalling of StDS2 induction, however, is dependent on the synthesis of novel proteins because cycloheximide can block StDS2 expression. To analyse the promoter region of StDS2 a genomic library of Solanum tuberosum was established and 1140 and 498 bp regions of the StDS2 promoter were isolated. The promoter fragments were fused to the β -glucuronidase ( GUS ) reporter gene and tested in transgenic potato plants. Both promoter fragments were able to induce GUS activity in response to dehydration. This result suggests that drought-specific cis -elements are located within 498 bp upstream to the StDS2 coding sequence.

Mitogen-activated protein (MAP) kinase pathways are conserved in eukaryotes and transmit a plethora of stimuli. MAP kinases (MAPKs) are part of signalling modules that consist of three to four tiers of protein kinases in a phosphorylation cascade. MAPKs are known to phosphorylate specific substrates at specific sites at a -threonine or serine residue followed by proline, but the surrounding amino acids of the phosphorylation site and docking interactions are also important for substrate recognition. MAPK activity can be assayed by detecting their phosphotransferase activity, their activation state, or detecting the switching on or off reaction of specific genes, or cellular responses. Prior to the kinase assay, specific MAPK proteins can be immunoprecipitated either by MAPK-specific antibodies or by the introduction of C-terminal epitope tags and expression of the fusion proteins in planta or transiently in protoplasts. Protoplasts derived from Arabidopsis thaliana cell cultures or leaves provide a valuable tool to co-express multiple gene constructs, thus in this system MAPKs can be co-expressed with upstream regulatory components or downstream targets. In protoplasts, the signalling activity through MAPK pathways can also be monitored by -co-transforming reporter genes fused to target promoters. Furthermore, components of the MAPK -signalling pathways can be silenced by co-transformation of RNAi or amiRNA constructs, and the impact of silencing on MAPK activation or gene expression can thus be determined.

Mitogen-activated protein (MAP) kinase pathways are conserved in eukaryotes and transmit a plethora of stimuli. MAP kinases (MAPKs) are part of signalling modules that consist of three to four tiers of protein kinases in a phosphorylation cascade. MAPKs are known to phosphorylate specific substrates at specific sites at a -threonine or serine residue followed by proline, but the surrounding amino acids of the phosphorylation site and docking interactions are also important for substrate recognition. MAPK activity can be assayed by detecting their phosphotransferase activity, their activation state, or detecting the switching on or off reaction of specific genes, or cellular responses. Prior to the kinase assay, specific MAPK proteins can be immunoprecipitated either by MAPK-specific antibodies or by the introduction of C-terminal epitope tags and expression of the fusion proteins in planta or transiently in protoplasts. Protoplasts derived from Arabidopsis thaliana cell cultures or leaves provide a valuable tool to co-express multiple gene constructs, thus in this system MAPKs can be co-expressed with upstream regulatory components or downstream targets. In protoplasts, the signalling activity through MAPK pathways can also be monitored by -co-transforming reporter genes fused to target promoters. Furthermore, components of the MAPK -signalling pathways can be silenced by co-transformation of RNAi or amiRNA constructs, and the impact of silencing on MAPK activation or gene expression can thus be determined.

The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) and the downstream MAPKs MPK4 and MPK6 were isolated by functional complementation of osmosensitive yeast mutants. In Arabidopsis protoplasts, MKK2 was specifically activated by cold and salt stress and by the stress-induced MAPK kinase kinase MEKK1. Yeast two-hybrid, in vitro, and in vivo protein kinase assays revealed that MKK2 directly targets MPK4 and MPK6. Accordingly, plants overexpressing MKK2 exhibited constitutive MPK4 and MPK6 activity, constitutively upregulated expression of stress-induced marker genes, and increased freezing and salt tolerance. In contrast, mkk2 null plants were impaired in MPK4 and MPK6 activation and were hypersensitive to salt and cold stress. Full genome transcriptome analysis of MKK2-overexpressing plants demonstrated altered expression of 152 genes involved in transcriptional regulation, signal transduction, cellular defense, and stress metabolism. These data identify a MAP kinase signaling cascade mediating cold and salt stress tolerance in plants.