Mitogen-activated Protein Kinases MPK6 Research Articles

Mitogen-activated protein kinases MPK3 and MPK6 phosphorylate receptor-like cytoplasmic kinase CDL1 to regulate soybean basal immunity.

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe), one of the most devastating soybean (Glycine max) pathogens, causes significant yield loss in soybean production. Nematode infection triggers plant defense responses; however, the components involved in the upstream signaling cascade remain largely unknown. In this study, we established that a mitogen-activated protein kinase (MAPK) signaling module, activated by nematode infection or wounding, is crucial for soybeans to establish SCN resistance. GmMPK3 and GmMPK6 directly interact with CDG1-LIKE1 (GmCDL1), a member of the receptor-like cytoplasmic kinase (RLCK) subfamily VII. These kinases phosphorylate GmCDL1 at Thr-372 to prevent its proteasome-mediated degradation. Functional analysis demonstrated that GmCDL1 positively regulates immune responses and promotes SCN resistance in soybeans. GmMPK3-mediated and GmMPK6-mediated phosphorylation of GmCDL1 enhances GmMPK3 and GmMPK6 activation and soybean disease resistance, representing a positive feedback mechanism. Additionally, 2 L-type lectin receptor kinases, GmLecRK02g and GmLecRK08g, associate with GmCDL1 to initiate downstream immune signaling. Notably, our study also unveils the potential involvement of GmLecRKs and GmCDL1 in countering other soybean pathogens beyond nematodes. Taken together, our findings reveal the pivotal role of the GmLecRKs-GmCDL1-MAPK regulatory module in triggering soybean basal immune responses.

Arabidopsis EXTRA-LARGE G PROTEIN 1 (XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity.

Pattern-triggered immunity (PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI. Of the three non-canonical EXTRA-LARGE G PROTEINs (XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity, but XLG1 was not considered to function in defense, based on the analysis of a weak xlg1 allele. In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern (PAMP)-triggered activation of mitogen-activated protein kinases (MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.

Extension of O-Linked Mannosylation in the Golgi Apparatus Is Critical for Cell Wall Integrity Signaling and Interaction with Host Cells in Cryptococcus neoformans Pathogenesis.

The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.

Phosphorylation of an ethylene response factor by MPK3/MPK6 mediates negative feedback regulation of pathogen-induced ethylene biosynthesis in Arabidopsis

Plants under pathogen attack produce high levels of the gaseous phytohormone ethylene to induce plant defense responses via the ethylene signaling pathway. The 1-aminocyclopropane-1-carboxylate synthase (ACS) is a critical rate-limiting enzyme of ethylene biosynthesis. Transcriptional and post-translational upregulation of ACS2 and ACS6 by the mitogen-activated protein kinases MPK3 and MPK6 are previously shown to be crucial for pathogen-induced ethylene biosynthesis in Arabidopsis. Here, we report that the fungal pathogen Botrytis cinerea-induced ethylene biosynthesis in Arabidopsis is under the negative feedback regulation by ethylene signaling pathway. The ethylene response factor ERF1A is further found to act downstream of ethylene signaling to negatively regulate the B. cinerea-induced ethylene biosynthesis via indirectly suppressing the expression of ACS2 and ACS6. Interestingly, ERF1A is shown to also upregulate defensin genes directly and therefore promote Arabidopsis resistance to B. cinerea. Furthermore, ERF1A is identified to be a substrate of MPK3 and MPK6, which phosphoactivate ERF1A to enhance its functions in suppressing ethylene biosynthesis and inducing defensin gene expression. Taken together, our data reveal that ERF1A and its phosphorylation by MPK3/MPK6 not only mediate the negative-feedback regulation of the B. cinerea-induced ethylene biosynthesis, but also upregulate defensin gene expression to increase Arabidopsis resistance to B. cinerea.

Multilayered synergistic regulation of phytoalexin biosynthesis by ethylene, jasmonate, and MAPK signaling pathways in Arabidopsis.

Camalexin, an indolic antimicrobial metabolite, is the major phytoalexin in Arabidopsis thaliana, and plays a crucial role in pathogen resistance. Our previous studies revealed that the Arabidopsis mitogen-activated protein kinases MPK3 and MPK6 positively regulate pathogen-induced camalexin biosynthesis via phosphoactivating the transcription factor WRKY33. Here, we report that the ethylene and jasmonate (JA) pathways act synergistically with the MPK3/MPK6-WRKY33 module at multiple levels to induce camalexin biosynthesis in Arabidopsis upon pathogen infection. The ETHYLENE RESPONSE FACTOR1 (ERF1) transcription factor integrates the ethylene and JA pathways to induce camalexin biosynthesis via directly upregulating camalexin biosynthetic genes. ERF1 also interacts with and depends on WRKY33 to upregulate camalexin biosynthetic genes, indicating that ERF1 and WRKY33 form transcriptional complexes to cooperatively activate camalexin biosynthetic genes, thereby mediating the synergy of ethylene/JA and MPK3/MPK6 signaling pathways to induce camalexin biosynthesis. Moreover, as an integrator of the ethylene and JA pathways, ERF1 also acts as a substrate of MPK3/MPK6, which phosphorylate ERF1 to increase its transactivation activity and therefore further cooperate with the ethylene/JA pathways to induce camalexin biosynthesis. Taken together, our data reveal the multilayered synergistic regulation of camalexin biosynthesis by ethylene, JA, and MPK3/MPK6 signaling pathways via ERF1 and WRKY33 transcription factors in Arabidopsis.

The monothiol glutaredoxin Grx4 influences thermotolerance, cell wall integrity, and Mpk1 signaling in Cryptococcus neoformans.

Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here, we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37°C and 39°C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white (CFW), and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the CWI pathway in cells grown at elevated temperature or upon treatment with CFW, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ, and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall, and other stresses. In summary, we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans.

Dual-specificity protein phosphatase Msg5 controls cell wall integrity and virulence in Fusarium oxysporum

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules controlling development and virulence in fungal pathogens. Down-regulation of MAPK activity by protein phosphatases provides a critical layer of control during desensitization or adaptation to stimuli. In Saccharomyces cerevisiae, the dual-specificity phosphatase Msg5 dephosphorylates target threonine and tyrosine residues in the two MAPKs Mpk1 and Fus3, which regulate the cell wall integrity (CWI) and pheromone responses, respectively. Here we studied the role of the Msg5 ortholog in Fusarium oxysporum, a soilborne phytopathogen that infects host plants through the roots to cause vascular wilt and plant death. F. oxysporum mutants lacking Msg5 showed constitutively high levels of Mpk1 phosphorylation and increased sensitivity to the cell wall targeting compound Calcofluor White. Moreover, these mutants displayed reduced colony growth and conidiation. Importantly, msg5Δ mutants were impaired in hyphal chemotropism towards host plant roots and in virulence on tomato plants. These results reveal a key role of Msg5 in regulation of the CWI MAPK cascade of F. oxysporum as well as in infection-related signaling of this important fungal pathogen.

Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis.

Camalexin is a major phytoalexin that plays a crucial role in disease resistance in Arabidopsis (Arabidopsis thaliana). We previously characterized the regulation of camalexin biosynthesis by the mitogen-activated protein kinases MPK3 and MPK6 and their downstream transcription factor WRKY33. Here, we report that the pathogen-responsive CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5) and CPK6 also regulate camalexin biosynthesis in Arabidopsis. Chemically induced expression of constitutively active CPK5 or CPK6 variants was sufficient to induce camalexin biosynthesis in transgenic Arabidopsis plants. Consistently, the simultaneous mutation of CPK5 and CPK6 compromised camalexin production in Arabidopsis induced by the fungal pathogen Botrytis cinerea Moreover, we identified that WRKY33 functions downstream of CPK5/CPK6 to activate camalexin biosynthetic genes, thereby inducing camalexin biosynthesis. CPK5 and CPK6 interact with WRKY33 and phosphorylate its Thr-229 residue, leading to an increase in the DNA binding ability of WRKY33. By contrast, the MPK3/MPK6-mediated phosphorylation of WRKY33 on its N-terminal Ser residues enhances the transactivation activity of WRKY33. Furthermore, both gain- and loss-of-function genetic analyses demonstrated the cooperative regulation of camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6. Taken together, these findings indicate that WRKY33 functions as a convergent substrate of CPK5/CPK6 and MPK3/MPK6, which cooperatively regulate camalexin biosynthesis via the differential phospho-regulation of WRKY33 activity.

Mitogen-activated protein kinases MPK3 and MPK6 are required for stem cell maintenance in the Arabidopsis shoot apical meristem.

CLV3p-mediated phosphorylation of MPK3 and MPK6 occurs via CLV1 and BAM1 receptors to regulate the maintenance of SAM development. The CLAVATA peptide-receptor (CLV3p-CLV1) pathway modulates a homeodomain master regulator WUSCHEL (WUS) transcription factor in the shoot apical meristem (SAM) with poorly defined signaling mechanisms. Here, we report that mitogen-activated protein kinases (MAPKs, also known as MPKs in plants) act in an intracellular signaling cascade to play an important role in the maintenance of SAM development. Interestingly, the application of exogenous CLV3p triggers rapid signaling in the SAM via dynamic activation of MPK3 and MPK6, which are positively regulated by both CLV1 and BARELY ANY MERISTEM 1 (BAM1) receptors. Surprisingly, the timing of MAPK activation is tightly correlated with the transcriptional repression of WUS expression in the SAM, indicating a fast CLV3p-CLV1/BAM1 signaling event. Furthermore, conditional mpk3,6 double mutants exhibited CLV3p insensitivity in stem cell maintenance manifested by the persistent SAM growth in the presence of exogenous CLV3p signals, as well as elevated WUS expression and repressed WUS-specific target genes. Taken together, these results suggest that MPK3 and MPK6 activated by CLV3p signals through mainly CLV1 and BAM1 receptors are key regulators controlling stem cell homeostasis in the SAM.

Receptor-Like Cytoplasmic Kinases Directly Link Diverse Pattern Recognition Receptors to the Activation of Mitogen-Activated Protein Kinase Cascades in Arabidopsis.

Plants deploy numerous cell surface-localized pattern-recognition receptors (PRRs) to perceive host- and microbe-derived molecular patterns that are specifically released during infection and activate defense responses. The activation of the mitogen-activated protein kinases MPK3, MPK4, and MPK6 (MPK3/4/6) is a hallmark of immune system activation by all known PRRs and is crucial for establishing disease resistance. The MAP kinase kinase kinase (MAPKKK) MEKK1 controls MPK4 activation, but the MAPKKKs responsible for MPK3/6 activation downstream of diverse PRRs and how the perception of diverse molecular patterns leads to the activation of MAPKKKs remain elusive. Here, we show that two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens in Arabidopsis thaliana The receptor-like cytoplasmic kinases VII (RLCK VII), which act downstream of PRRs, directly phosphorylate MAPKKK5 Ser-599, which is required for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance. Surprisingly, MPK6 further phosphorylates MAPKKK5 Ser-682 and Ser-692 to enhance MPK3/6 activation and disease resistance, pointing to a positive feedback mechanism. Finally, MEKK1 Ser-603 is phosphorylated by both RLCK VII and MPK4, which is required for pattern-triggered MPK4 activation. These findings illustrate central mechanisms by which multiple PRRs activate MAPK cascades and disease resistance.

Role of the phosphatase Ptc1 in stress responses mediated by CWI and HOG pathways in Fusarium oxysporum

Reversible protein phosphorylation is crucial for cell signal transduction in stress response. Fusarium oxysporum is a soil inhabiting fungus that can adapt to a wide range of ecological niches and environmental conditions. Three mitogen activated protein kinase (MAPK) cascades have been shown to orchestrate the response of the fungus to external insults such as high temperature, cell wall, oxidative or hyperosmotic stress in F. oxysporum. However, the protein phosphatases that fine-tune phosphorylation levels of different MAPKs in this fungus are unknown. In this study we show that the serine/threonine phosphatase Ptc1 regulates phosphorylation of the high osmolarity glycerol response (HOG) MAPK Hog1 and the cell wall integrity (CWI) MAPK Mpk1. A Δptc1 mutant showed decreased phosphorylation levels of the Mpk1 and was more sensitive to cell wall damaging agents in comparison to the wild type strain. In contrast, this mutant exhibited higher phosphorylation levels of the p38 MAPK Hog1, increased tolerance to osmotic stress compounds and higher expression of genes induced by osmotic stress. Moreover, Δptc1 contained fragmented vacuoles even in absence of the osmotic stressor, supporting the involvement of Ptc1 in the HOG pathway.

Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases.

Genetic dissection of Arabidopsis MAP kinase phosphatase 1-dependent PAMP-induced transcriptional responses.

Plant immunity is initiated by extracellular detection of pathogen-associated molecular patterns (PAMPs) through surface-localized pattern recognition receptors (PRRs). PRR activation induces many responses including the activation of mitogen-activated protein kinases (MAPKs) that ultimately limit bacterial growth. Previous work identified Arabidopsis MAP kinase phosphatase 1 (MKP1) as a negative regulator of signaling pathways required for some, but not all, of PAMP-initiated responses. Specifically, loss of MAPK MPK6 in an mkp1 background suppressed a subset of the mkp1-dependent biological phenotypes, indicating the requirement for MPK6 in MKP1-dependent signaling. To further genetically separate the outputs of PAMP-responsive signaling pathways, we performed a transcriptome analysis in Arabidopsis wild type, mkp1 and mkp1 mpk6 seedlings treated with the bacterially derived PAMP elf26 for 0, 30, and 90 min. Using differential genetic and temporal clustering analyses between and within genotypes, we identified and separated 6963 elf26-responsive transcripts based on both genetic requirements of MKP1 (with or without a requirement for MPK6) and temporal transcriptional accumulation patterns, and some of these novel response markers were validated by qRT-PCR over a more extended time course. Taken together, our transcriptome analysis provides novel information for delineating PAMP signaling pathways.

Phosphatidylinositol 3,5-bisphosphate is involved in methylglyoxal-induced activation of the Mpk1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and this 2-oxoaldehyde has been implicated in some diseases including diabetes. However, the physiological significance of MG for cellular functions is yet to be fully elucidated. We previously reported that MG activates the Mpk1 (MAPK) cascade in the yeast Saccharomyces cerevisiae To gain further insights into the cellular functions and responses to MG, we herein screened yeast-deletion mutant collections for susceptibility to MG. We found that mutants defective in the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) are more susceptible to MG. PtdIns(3,5)P2 levels increased following MG treatment, and vacuolar morphology concomitantly changed to a single swollen shape. MG activated the Pkc1-Mpk1 MAPK cascade in which a small GTPase Rho1 plays a crucial role, and the MG-induced phosphorylation of Mpk1 was impaired in mutants defective in the PtdIns(3,5)P2 biosynthetic pathway. Of note, heat shock-induced stress also provoked Mpk1 phosphorylation in a Rho1-dependent manner; however, PtdIns(3,5)P2 was dispensable for the heat shock-stimulated activation of this signaling pathway. Our results suggest that PtdIns(3,5)P2 is specifically involved in the MG-induced activation of the Mpk1 MAPK cascade and in the cellular adaptation to MG-induced stress.

Corrigendum

New PhytologistVolume 215, Issue 4 p. 1623-1624 CorrigendumFree Access Corrigendum This article corrects the following: The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of ‘VQ-motif’-containing proteins to regulate immune responses Pascal Pecher, Lennart Eschen-Lippold, Siska Herklotz, Katja Kuhle, Kai Naumann, Gerit Bethke, Joachim Uhrig, Martin Weyhe, Dierk Scheel, Justin Lee, Volume 203Issue 2New Phytologist pages: 592-606 First Published online: April 22, 2014 First published: 24 July 2017 https://doi.org/10.1111/nph.14708 Author for correspondence: Justin Lee Tel: +49 345 5582 1410 Email: [email protected] AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL New Phytologist203 (2014), 592–606. Since its publication, it has been drawn to our attention that there are errors in Fig. 3 of Pecher et al. (2014). A number of yeast colonies were mistakenly duplicated and used to depict different interaction pairs. Several of the errors have similar-looking numbers (e.g. MVQ5 with WRKY33 or WRKY13; MVQ7 with WRKY26 or WRKY28; MVQ9 with WRKY58 or WRKY28) and since the yeast colonies were not always perfectly aligned on the original plates, the error presumably arose during the assembly of the figure through the thousands (24 × 59 times) of ‘cut & paste’ steps. To validate the reported data, the authors have repeated the complete set of the yeast 2-hybrid (Y2H) assay two further times. In order to minimize the potential for error during figure assembly, the colonies were plated – within the space limitation of a standard 12-cm square plate – in the depicted sequential format (in blocks of 10–12 × 12 colonies using a 12-channel pipette). Transformation efficiency was monitored on selection drop-out (SD) media lacking leucine and tryptophan (SD-LW) and protein interaction assessed after selection on SD-LWAH (lacking leucine, tryptophan, adenine and histidine) or SD-LWAH supplemented with the indicated concentrations of 3-amino-1,2,4-triazole (+3AT). A complete set of the experiment showing the growth of yeast on the different selection plates can be viewed in in Figshare (doi: https://doi.org/10.6084/m9.figshare.5086942). Figure 3Open in figure viewerPowerPoint Yeast-two-hybrid analysis of WRKY-MVQ interactions. (MVQ, mitogen-activated protein kinase 3/6 (MPK3/6)-targeted VQ-motifcontaining protein). Y2H analysis was carried out as described in Fig 1c and positive interactions are indicated by the yeast growth on -His/-Ade selection media. The top half of the panel depicts interactions with wild-type MVQs while the lower half covers the MVQs with the “VQ” residues mutated to “DL” (MVQDL). Note that substantial auto-activation in the Y2H assay precluded the assessment of whether MVQ9 (= VQ14 or IKU1) interacted with WRKYs. pDEST22/32 are the corresponding empty vector controls for the WRKYs and VQ-motif-containing proteins (VQPs), respectively. The majority of the previously reported interaction (mainly between MVQs with Group I and Group IIc WRKYs) could be confirmed. Where there are discrepancies to the previously data, selected pairs were re-tested in a third transformation study and furthermore, identities of the plasmid clones used for transformation were re-verified by DNA restriction or sequencing analysis. Thus, the current figure represents a more confident representation of the MVQ–WRKY interactions. Nevertheless, it should be emphasized that it remains only a guideline for further confirmation by independent protein–protein interaction analytical methods. Some of the differences to the previous interaction dataset include the following: No interaction detected in the current experiment compared to the previous strong interaction: WRKY11 with MVQ4–6. No interaction detected now compared to interactions previously considered as weak or ambiguous: MVQ4/WRKY1; MVQ3/WRKY75; MVQ5/WRKY60; MVQ4/WRKY29; MVQ1/WRKY35; MVQ6/WRKY30; MVQ5/WRKY63. New interactions detected now, which were not in the previous report: MVQ4/WRKY4; MVQ6/WRKY26; MVQ6/WRKY33; MVQ6/WRKY8; MVQ7/WRKY1; MVQ7/WRKY58; MVQ5/WRKY28; MVQ6/WRKY28. The revised Fig. 3 is shown below. We apologize to our readers for this mistake. Reference Pecher P, Eschen-Lippold L, Herklotz S, Kuhle K, Naumann K, Bethke G, Uhrig J, Weyhe M, Scheel D, Lee J. 2014. The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of ‘VQ-motif’-containing proteins to regulate immune responses. New Phytologist 203: 592– 606. Volume215, Issue4September 2017Pages 1623-1624 FiguresReferencesRelatedInformation

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Early perception of stink bug damage in developing seeds of field-grown soybean induces chemical defences and reduces bug attack.

Southern green stink bugs (Nezara viridula L.) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva, which causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defences in seeds. This study demonstrated that mitogen-activated protein kinases MPK3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulate a defensive response after stink bug damage. Although 10-20 min after stink bug feeding on seeds induced the expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also induced early, and at increasing levels up to 72 h after damage. Damaged seeds upregulated defensive genes typically modulated by JA/ET or SA, which in turn reduced the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. This study shows that stink bug damage induces seed defences, which is perceived early by MPKs that may activate defence metabolic pathways in developing seeds of field-grown soybean. © 2015 Society of Chemical Industry.

A Critical Role of Lyst-Interacting Protein5, a Positive Regulator of Multivesicular Body Biogenesis, in Plant Responses to Heat and Salt Stresses.

Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumen and play critical roles in many cellular processes. We have recently shown that Arabidopsis (Arabidopsis thaliana) Lyst-Interacting Protein5 (LIP5), a positive regulator of the Suppressor of K(+) Transport Growth Defect1 (SKD1) AAA ATPase in MVB biogenesis, is a critical target of the mitogen-activated protein kinases MPK3 and MPK6 and plays an important role in the plant immune system. In this study, we report that the LIP5-regulated MVB pathway also plays a critical role in plant responses to abiotic stresses. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. The critical role of LIP5 in plant tolerance to abiotic stresses is dependent on its ability to interact with Suppressor of K(+) Transport Growth Defect1. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates and NaCl under heat and salt stresses, respectively. Further analysis using fluorescent dye and MVB markers reveals that abiotic stress increases the formation of endocytic vesicles and MVBs in a largely LIP5-dependent manner. LIP5 is also required for the salt-induced increase of intracellular reactive oxygen species, which have been implicated in signaling of salt stress responses. Basal levels of LIP5 phosphorylation by MPKs and the stability of LIP5 are elevated by salt stress, and mutation of MPK phosphorylation sites in LIP5 reduces the stability and compromises the ability to complement the lip5 salt-sensitive mutant phenotype. These results collectively indicate that the MVB pathway is positively regulated by pathogen/stress-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in broad plant responses to biotic and abiotic stresses.

Methylglyoxal activates the target of rapamycin complex 2-protein kinase C signaling pathway in Saccharomyces cerevisiae.

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr(1125) and Ser(1143). Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser(1143), which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1(T1125) affected the phosphorylation of Pkc1 at Ser(1143), in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser(473). Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

MicroRNA biogenesis factor DRB1 is a phosphorylation target of mitogen activated protein kinase MPK3 in both rice and Arabidopsis.

MicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein-protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant.