MAPKKK Genes Research Articles

Fungal plant pathogen "mutagenomics" reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor.

"Mutagenomics" is the combination of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated "random" T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have a distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the predicted protein's N-terminus, while the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains' wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines.

A MAPKKK gene from rice, RBG1res, confers resistance to Burkholderia glumae through negative regulation of ABA

Burkholderia glumae causes bacterial seedling rot (BSR) of rice and is a threat to a consistent food supply. When previously screening for resistance against B. glumae in the resistant cultivar Nona Bokra (NB) versus the susceptible cultivar Koshihikari (KO), we detected a gene, Resistance to Burkholderia glumae 1 (RBG1), at a quantitative trait locus (QTL). Here, we found that RBG1 encodes a MAPKKK gene whose product phosphorylates OsMKK3. We also found that the kinase encoded by the RBG1 resistant (RBG1res) allele in NB presented higher activity than did that encoded by the RBG1 susceptible (RBG1sus) allele in KO. RBG1res and RBG1sus differ by three single-nucleotide polymorphisms (SNPs), and the G390T substitution is essential for kinase activity. Abscisic acid (ABA) treatment of inoculated seedlings of RBG1res-NIL (a near-isogenic line (NIL) expressing RBG1res in the KO genetic background) decreased BSR resistance, indicating that RBG1res conferred resistance to B. glumae through negative regulation of ABA. The results of further inoculation assays showed that RBG1res-NIL was also resistant to Burkholderia plantarii. Our findings suggest that RBG1res contributes to resistance to these bacterial pathogens at the seed germination stage via a unique mechanism.

Comprehensive analysis of MAPK gene family in Populus trichocarpa and physiological characterization of PtMAPK3-1 in response to MeJA induction.

Mitogen-activated protein kinases (MAPK) play important roles in plant growth and development, as well as hormone and stress responses by signaling, through MAPK cascade, to eukaryotic cells the presence of various cues; thereby, regulating various responses. The MAPK cascade consists mainly of three gene families, MAPK, MAPKK and MAPKKK, which activate downstream signaling pathways through sequential phosphorylation. Although the MAPK cascade gene family has been reported in several species, there is a lack of comprehensive analysis in poplar. We identified 21 MAPK genes, 11 MAPKK genes, and 104 MAPKKK genes in Populus trichocarpa. The phylogenetic classification was supported by conservative motif, gene structure and motif analysis. Whole-genome duplication has an important role in the expansion of MAPK cascade genes. Analysis of promoter cis-elements and expression profiles indicates that MAPK cascade genes have important roles in plant growth and development, abiotic and biotic stresses, and phytohormone response. Expression profiling revealed a significant up-regulation of PtMAPK3-1 expression in response to drought, salt and disease stresses. Poplar transiently overexpressing PtMAPK3-1 and treated with MeJA had higher CAT and POD levels than non-overexpressing poplar. This work represents the first complete inventory of the MAPK cascade in P. trichocarpa, which reveals that PtMAPK3-1 is induced by the MeJA hormone and participates in the MeJA-induced enhancement of the antioxidant enzyme system. This article is protected by copyright. All rights reserved.

Genome-Wide Identification of MAPKK and MAPKKK Gene Family Members and Transcriptional Profiling Analysis during Bud Dormancy in Pear (Pyrus x bretschneideri).

The mitogen-activated protein kinase (MAPK) cascade consisting of three types of reversibly major signal transduction module (MAPKKK, MAPKK, and MAPK) is distributed in eukaryotes. MAPK cascades participate in various aspects of plant development, including hormone responses, cell division and plant dormancy. Pear is one of the most economically important species worldwide, and its yield is directly affected by dormancy. In this study, genome-wide identification of MAPKK and MAPKKK gene family members in Pyrus x bretschneideri and transcriptional expression analysis of MAPK cascades during pear dormancy were performed. We identified 8 MAPKKs (PbrMKKs) and 100 MAPKKKs (PbrMAPKKKs) in Pyrus using recent genomic information. PbrMAPKKs were classified into four subgroups based on phylogenetic analysis, whereas PbrMAPKKKs were grouped into 3 subfamilies (MEKK, Raf, and ZIK). Most PbrMAPKKKs and PbrMAPKKs in the same subfamily had similar gene structures and conserved motifs. The genes were found on all 17 chromosomes. The comprehensive transcriptome analysis and quantitative real-time polymerase chain reaction (qRT–PCR) results showed that numerous MAPK cascade genes participated in pear bud dormancy. The interaction network and co-expression analyses indicated the crucial roles of the MAPK member-mediated network in pear bud dormancy. Overall, this study advances our understanding of the intricate transcriptional control of MAPKKK-MAPKK-MAPK genes and provides useful information on the functions of dormancy in perennial fruit trees.

Genome-Wide Identification of MAPK, MAPKK, and MAPKKK Gene Families in Fagopyrum tataricum and Analysis of Their Expression Patterns Under Abiotic Stress

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signal transduction pathway, ubiquitous in eukaryotes, such as animals and plants. The MAPK cascade has a dominant role in regulating plant adaptation to the environment, such as through stress responses, osmotic adjustment, and processes that modulate pathogenicity. In the present study, the MAPK cascade gene family was identified in Fagopyrum tataricum (Tartary buckwheat), based on complete genome sequence data. Using phylogenetic tree, conservative motif, and chromosome location analyses, a total of 65 FtMAPK cascade genes, distributed on five chromosomes, were classified into three families: MAPK (n = 8), MAPKK (n = 1), and MAPKKK (n = 56). Transcriptome data from Tartary buckwheat seedlings grown under different light conditions demonstrated that, under blue and red light, the expression levels of 18 and 36 FtMAPK cascade genes were up-regulated and down-regulated, respectively. Through qRT-PCR experiments, it was observed that FtMAPK5, FtMAPKK1, FtMAPKKK8, FtMAPKKK10, and FtMAPKKK24 gene expression levels in the Tartary buckwheat seedlings increased under three types of abiotic stress: drought, salt, and high temperature. A co-expression network of FtMAPK cascade genes was constructed, based on gene expression levels under different light conditions, and co-expressed genes annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, which identified numerous transcription factors related to plant abiotic stress. The authors conclude that FtMAPK cascade genes have important roles in the growth and development of Tartary buckwheat, as well as its responses to abiotic stress.

Comprehensive analysis of MAPK cascade genes in sorghum (Sorghum bicolor L.) reveals SbMPK14 as a potential target for drought sensitivity regulation.

The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in regulating many important biological processes in plants. Here, we identified and characterized eight MAPKK and 49 MAPKKK genes in sorghum and analyzed their differential expression under drought treatment; we also characterized 16 sorghum MAPK genes. RNA-seq analysis revealed that 10 MAPK cascade genes were involved in drought stress response at the transcriptome level in sorghum. Overexpression of SbMPK14 in Arabidopsis and maize resulted in hypersensitivity to drought by promoting water loss, indicating that SbMPK14 functions as a negative regulator of the drought response. Subsequent transcriptome analysis and qRT-PCR verification of maize SbMPK14 overexpression lines revealed that SbMPK14 likely increases plant drought sensitivity by suppressing the activity of specific ERF and WRKY transcription factors. This comprehensive study provides valuable insight into the mechanistic basis of MAPK cascade gene function and their responses to drought in sorghum.

Cloning, characterization, and expression analysis of two MAPKKK genes in Chrysanthemum

Cloning, characterization, and expression analysis of two MAPKKK genes in Chrysanthemum

Molecular characterization, expression and interaction of MAPK, MAPKK and MAPKKK genes in upland cotton

Mitogen-activated protein kinase (MAPK) signaling cascades, consisting of three types of sequentially phosphorylated kinases (MAPKKK, MAPKK, and MAPK), play vital roles in various processes including plant development and stress response. In this study, 52 GhMAPKs, 23 GhMAPKKs, and 166 GhMAPKKKs were identified in upland cotton. Chromosomal locations, gene duplication and structure, motifs, cis-regulatory elements, and protein subcellular localization were further analyzed. With the identified MAPK cascade genes in G. arboretum and G. raimondii, a syntenic diagram of three cotton species was constructed. The interactions of seven GhMAPK cascade genes were investigated. Two complete signaling modules were defined: The GhMEKK24/GhMEKK31-GhMAPKK9-GhMAPK10 and GhMEKK3/GhMEKK24/GhMEKK31-GhMAPKK16-GhMAPK10/GhMAPK11 cascades. Moreover, interaction networks and the interaction pairs were combined with their expression patterns and demonstrated that the network mediated by the MAPK signaling cascade participates in abiotic stress signaling. Our research provides a foundation for studying the molecular mechanism of the MAPK signaling pathway under abiotic stress

Genome-wide identification and functional characterization of cotton (Gossypium hirsutum) MAPKKK gene family in response to drought stress

BackgroundMitogen-activated protein kinase kinase kinases (MAPKKKs) are significant components in the MAPK signal pathway and play essential roles in regulating plants against drought stress. To explore MAPKKK gene family functioning in cotton response and resistance to drought stress, we conducted a systematic analysis of GhMAPKKKs.ResultsIn this study, 157 nonredundant GhMAPKKKs (including 87 RAFs, 46 MEKKs and 24 ZIKs) were identified in cotton (Gossypium hirsutum). These GhMAPKKK genes are unevenly distributed on 26 chromosomes, and segmental duplication is the major way for the enlargement of MAPKKK family. Furthermore, members within the same subfamily share a similar gene structure and motif composition. A lot of cis-elements relevant to plant growth and response to stresses are distributed in promoter regions of GhMAPKKKs. Additionally, these GhMAPKKKs show differential expression patterns in cotton tissues. The transcription levels of most genes were markedly altered in cotton under heat, cold and PEG treatments, while the expressions of some GhMAPKKKs were induced in cotton under drought stress. Among these drought-induced genes, we selected GhRAF4 and GhMEKK12 for further functional characterization by virus-induced gene silencing (VIGS) method. The experimental results indicated that the gene-silenced cotton displayed decreased tolerance to drought stress. Malondialdehyde (MDA) content was higher, but proline accumulation, relative leaf water content and activities of superoxide dismutase (SOD) and peroxidase (POD) were lower in the gene-silenced cotton, compared with those in the controls, under drought stress.ConclusionCollectively, a systematic survey of gene structure, chromosomal location, motif composition and evolutionary relationship of MAPKKKs were performed in upland cotton (Gossypium hirsutum). The following expression and functional study showed that some of them take important parts in cotton drought tolerance. Thus, the data presented here may provide a foundation for further investigating the roles of GhMAPKKKs in cotton response and resistance to drought stress.

Genome-wide identification of MAPKKK genes and their responses to phytoplasma infection in Chinese jujube (Ziziphus jujuba Mill.)

BackgroundMitogen-activated protein kinase (MAPK) cascades play vital roles in signal transduction in response to a wide range of biotic and abiotic stresses. In a previous study, we identified ten ZjMAPKs and five ZjMAPKKs in the Chinese jujube genome. We found that some members of ZjMAPKs and ZjMAPKKs may play key roles in the plant’s response to phytoplasma infection. However, how these ZjMAPKKs are modulated by ZjMAPKKKs during the response process has not been elucidated. Little information is available regarding MAPKKKs in Chinese jujube.ResultsA total of 56 ZjMAPKKKs were identified in the jujube genome. All of these kinases contain the key S-TKc (serine/threonine protein kinase) domain, which is distributed among all 12 chromosomes. Phylogenetic analyses show that these ZjMAPKKKs can be classified into two subfamilies. Specifically, 41 ZjMAPKKKs belong to the Raf subfamily, and 15 belong to the MEKK subfamily. In addition, the ZjMAPKKKs in each subfamily share the same conserved motifs and gene structures. Only one pair of ZjMAPKKKs (15/16, on chromosome 5) was found to be tandemly duplicated. Using qPCR, the expression profiles of these MAPKKKs were investigated in response to infection with phytoplasma. In the three main infected tissues (witches’ broom leaves, phyllody leaves, and apparently normal leaves), ZjMAPKKK26 and − 45 were significantly upregulated, and ZjMAPKKK3, − 43 and − 50 were significantly downregulated. ZjMAPKKK4, − 10, − 25 and − 44 were significantly and highly induced in sterile cultivated tissues infected by phytoplasma, while ZjMAPKKK6, − 7, − 17, − 18, − 30, − 34, − 35, − 37, − 40, − 41, − 43, − 46, − 52 and − 53 were significantly downregulated.ConclusionsFor the first time, we present an identification and classification analysis of ZjMAPKKKs. Some ZjMAPKKK genes may play key roles in the response to phytoplasma infection. This study provides an initial understanding of the mechanisms through which ZjMAPKKKs are involved in the response of Chinese jujube to phytoplasma infection.

Mutations in the Ras Pathway in Pre-Treatment Chronic Lymphocytic Leukemia Are Associated with VH1-69: Linking B-Cell Receptor Stereotypy to Downstream Signaling Events

Introduction: Hairy cell leukemia and some other low-grade B-LPDs are characterized by a high incidence of oncogenic mutations in the Ras-MAPK pathway, most commonly in BRAF and MAP2K1 genes. In contrast, chronic lymphocytic leukemia (CLL) has a low frequency of such mutations. To determine the patterns of emergence of Ras pathway mutations in CLL, we assessed the frequency and timing of mutations in KRAS, NRAS, BRAF and MAP2K1 and their association with other CLL-associated mutations and B-cell receptor (BCR) type. We also compared expression profiles for signaling molecules for CLL with or without these mutations.Methods: A custom-designed 50-gene, Ampliseq/Ion-S5 next-generation sequencing (NGS) panel was performed on DNA from purified B cells for 221 CLL cases presenting for therapy at our institution. Panel included mutation hotspots for the genes above and all other common CLL-associated pathogenic mutations, with an analytic sensitivity of 1-2% VAF. For post-therapy samples demonstrating a Ras pathway mutation, prior/pre-therapy samples were tested to determine whether mutations were present at baseline or acquired with therapy (or likely so based on findings in off-therapy baseline samples). We performed transcript analysis on total RNA from highly pure CLL samples in a subset of baseline samples using the 770-gene Pan-cancer Immune Panel, which included many signaling and downstream AP1, MAPK, MAPKK and MAPKKK genes (nCounter System, Nanostring). Transcript normalization and clustering was performed in nSolver software with log-2 transcript levels compared for significance following Benjamini-Hochberg FDR adjustment. Pathway analysis was performed using R scripts with Bioconductor and Pathview visualization.Results: Mutations in KRAS, NRAS, BRAF and MAP2K1 were identified in 32/221 CLL (14.4%), including pre-therapy/baseline in 17 cases. Ras pathway-mutated cases were IGVH-unmutated (U) in 29/32 cases (90.6%). Multiple (31.3%) and subclonal mutations (75%) were common with atypical sites of BRAF mutation predominating (Table). These BRAF mutations were mostly predicted to have minimally increased or decreased kinase activity. Particularly among cases where the mutations were likely or definitively present pretherapy, IGH variable (IGVH) gene sequencing revealed a striking predominance of cases expressing VH1-69/51p1 type (14/17, 82.4%, p<0.0001, Fisher exact). In contrast, cases likely or definitely arising post-therapy did not show any association with BCR stereotype. These most frequently arose following BTK inhibitor therapy (Table).The case percentage of other co-occurring CLL-associated mutations included: BIRC3 6.3%, BCOR 21.9%, BCORL1 6.3%, NOTCH1 28.1%, SF3B1 25% and TP53 12.5% and 3.1% for FBXW7, MYD88, POT1, RPS15 and XPO1. There was a trend (not reaching significance) for lower TP53 and higher SF3B1 and NOTCH1 mutations than the overall data set. Cytogenetics revealed an increased proportion of +12 by karyotype and/or FISH in the Ras pathway-mutated cases (13/32, 40.6% versus 24.6% for other cases, p = 0.001). The co-association of +12 CLL requiring treatment with NOTCH1 mutations has been previously noted.We compared RNA expression in the 770-gene panel for 11 baseline 1-69/IGVHU CLL (6 Ras pathway-mutated) and a control group of 6 baseline CLL expressing other IGVH genes. There were 50 genes that were significantly differentially expressed in the 1-69 group (adjusted p value <0.05) with 3 that distinguished Ras-mutated from unmutated 1-69/IGVHU CLL (IFNL1 and IL13 increased; TOLLIP decreased). In unsupervised clustering, Ras pathway-mutated cases clustered together except for 1 case. An analysis of differentially regulated BCR-associated genes showed coordinate shifts in expression in the components of the co-regulatory PIR-B and Leu13 complexes among Ras pathway-mutated as compared to the unmutated groupConclusion: We identify Ras pathway mutations in CLL in two different contexts; prior to therapy in the VH1-69 subgroup of IGVHU CLL and separately emerging following therapy with BTK inhibitors. In both scenarios, multiple and subclonal mutations were common implicating Ras pathway dysregulation as a progression factor. The apparent complementation of Ras pathway mutations with the putatively antigen-driven unmutated VH1-69 BCR stereotype may indicate a unique pattern of regulated signaling in that CLL subgroup. [Display omitted] DisclosuresLozanski:Beckman: Research Funding; BI: Research Funding; Stem Line: Research Funding; Genentech: Research Funding; Coulter: Research Funding; Novartis: Research Funding.

Divergent evolutionary patterns of the MAPK cascade genes in Brassica rapa and plant phylogenetics

Mitogen-activated protein kinase (MAPK) cascade signal transduction modules play crucial roles in regulating many biological processes in plants. These cascades are composed of three classes of hierarchically organized protein kinases, MAPKKKs, MAPKKs and MAPKs. Here, we analyzed gene retention, phylogenetic, evolution and expression patterns of MAPK cascade genes in Brassica rapa. We further found that the MAPK branches, classes III and IV, appeared after the split of bryophytes and green algae after analyzing the MAPK cascade genes in 8 species, and their rapid expansion led to the great size of the families of MAPKs. In contrast, the ancestral class I subfamily of MAPKK gene families have been highly conserved from algae to angiosperms. For the MAPKKK family, the MEKK and Raf subfamily share a common evolutionary origin, and Raf plays a major role in the expansion of the MAPKKK gene family. The cis-elements and interaction network analyses showed the important function of MAPK cascade genes in development and stress responses in B. rapa. This study provides a solid foundation for molecular evolution analyses of MAPK cascade genes.

VqMAPKKK38 is essential for stilbene accumulation in grapevine

Vitis species, including grapevine, produce a class of secondary metabolites called stilbenes that are important for plant disease resistance and can have positive effects on human health. Mitogen-activated protein kinase (MAPK) signaling cascades not only play key roles in plant defense responses but also contribute to stilbene biosynthesis in grapevine. MAPKKKs function at the upper level of the MAPK network and initiate signaling through this pathway. In this study, a Raf-like MAPKKK gene, VqMAPKKK38, was identified and functionally characterized from the Chinese wild grapevine V. quinquangularis accession ‘Danfeng-2’. We observed that VqMAPKKK38 transcript levels were elevated by powdery mildew infection, high salinity conditions and chilling stresses, as well as in response to treatments by the hormones salicylic acid (SA), methyl jasmonate (MeJA), ethylene (Eth) and abscisic acid (ABA). In addition, based on both transient overexpression and gene suppression of VqMAPKKK38 in grapevine leaves, we found that VqMAPKKK38 positively regulates stilbene synthase transcription and stilbene accumulation probably by mediating the activation of the transcription factor MYB14. In addition, both hydrogen peroxide (H2O2) and calcium influx activated VqMAPKKK38 expression and stilbene biosynthesis, which suggests that VqMAPKKK38 may be involved in the calcium signaling and ROS signaling pathways.

Mutation of the Slt2 ortholog from Cryphonectria parasitica results in abnormal cell wall integrity and sectorization with impaired pathogenicity

We assessed the biological function of CpSlt2, an ortholog of the cell wall integrity (CWI) MAPK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica. The CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, and abnormal pigmentation. In addition, the CpSlt2-null mutant exhibited CWI-related phenotypic defects including hypersensitivity to cell wall-disturbing agents and other stresses. Electron microscopy revealed the presence of abnormal hyphae such as intrahyphal hyphae. In addition, virulence assays indicated that the CpSlt2 gene plays an important role in fungal pathogenesis. As cultivation of the mutant strains progressed, the majority of the colonies showed sporadic sectorization and mycelia from the sectored area stably maintained the sectored phenotype. Although mycelial growth was partially recovered, the sectored progeny had dramatically impaired virulence, confirming the CpSlt2 gene has a role in pathogenicity. Compared to a previous mutant of the CpBck1 gene, a MAPKKK gene in CWI pathway, the CpSlt2-null mutant showed similar, although not identical, phenotypic changes and most phenotypic changes were less severe than those of the CpBck1-null mutant. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.

The MAPKKK and MAPKK gene families in banana: identification, phylogeny and expression during development, ripening and abiotic stress

The mitogen-activated protein kinase (MAPK) cascade, which is a major signal transduction pathway widely distributed in eukaryotes, has an important function in plant development and stress responses. However, less information is known regarding the MAPKKK and MAPKK gene families in the important fruit crop banana. In this study, 10 MAPKK and 77 MAPKKK genes were identified in the banana genome, and were classified into 4 and 3 subfamilies respectively based on phylogenetic analysis. Majority of MAPKKK and MAPKK genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis indicated that MAPKKK-MAPKK genes is involved in tissue development, fruit development and ripening, and response to abiotic stress of drought, cold and salt in two banana genotypes. Interaction networks and co-expression assays demonstrated that MAPK signaling cascade mediated network participates in multiple stress signaling, which was strongly activated in Fen Jiao (FJ). The findings of this study advance understanding of the intricately transcriptional control of MAPKKK-MAPKK genes and provide robust candidate genes for further genetic improvement of banana.

Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and functional characterization of MAPKKK family in Brachypodium, which will facilitate the functional analysis of BdMAPKKK genes, and also will facilitate better understanding the MAPK signal pathway in Brachypodium and beyond.

Regulation of Hippo signalling by p38 signalling.

The Hippo signalling pathway has a crucial role in growth control during development, and its dysregulation contributes to tumorigenesis. Recent studies uncover multiple upstream regulatory inputs into Hippo signalling, which affects phosphorylation of the transcriptional coactivator Yki/YAP/TAZ by Wts/Lats. Here we identify the p38 mitogen-activated protein kinase (MAPK) pathway as a new upstream branch of the Hippo pathway. In Drosophila, overexpression of MAPKK gene licorne (lic), or MAPKKK gene Mekk1, promotes Yki activity and induces Hippo target gene expression. Loss-of-function studies show that lic regulates Hippo signalling in ovary follicle cells and in the wing disc. Epistasis analysis indicates that Mekk1 and lic affect Hippo signalling via p38b and wts. We further demonstrate that the Mekk1-Lic-p38b cascade inhibits Hippo signalling by promoting F-actin accumulation and Jub phosphorylation. In addition, p38 signalling modulates actin filaments and Hippo signalling in parallel to small GTPases Ras, Rac1, and Rho1. Lastly, we show that p38 signalling regulates Hippo signalling in mammalian cell lines. The Lic homologue MKK3 promotes nuclear localization of YAP via the actin cytoskeleton. Upregulation or downregulation of the p38 pathway regulates YAP-mediated transcription. Our work thus reveals a conserved crosstalk between the p38 MAPK pathway and the Hippo pathway in growth regulation.

GhMAPKKK49, a novel cotton (Gossypium hirsutum L.) MAPKKK gene, is involved in diverse stress responses

MAPK (mitogen-activated protein kinase) family plays diverse roles in plant signal transduction and response to abiotic stress. In this study, we isolated a novel MAPKKK gene, GhMAPKKK49 from cotton (Gossypium hirsutum L.). The full-length cDNA of GhMAPKKK49 is 1,056 bp, encoding a protein of 351 amino acids. This protein contains a kinase catalytic domain, composed of an ATP binding site, a Ser/Thr kinase active site and a conserved Raf motif. The alignment analysis revealed a high sequence similarity of GhMAPKKK49 and other plant MAPKKK proteins. Our results indicated a constitutive expression of GhMAPKKK49 in roots, stems, and leaves. The expression of GhMAPKKK49 transcripts was found to be enhanced by abiotic salt, drought and wounding stresses but not the biotic stress of Ralstonia solanacearum or Rhizoctonia solani infection. Furthermore, real-time PCR showed that GhMAPKKK49 is upregulated in response to the application of various signaling molecules, including abscisic acid (ABA), hydrogen peroxide (H2O2), gibberellins (GAs), methyl jasmonate (MeJA), salicylic acid (SA), 6-benzylaminopurine (6-BA), a-naphthylacetic acid (NAA) and ethylene (ET). Furthermore, we demonstrate that GhMAPKKK49 interacts with GhMAPKK4 and GhMAPKK9. Give all that, GhMAPKKK49 may responds to various stresses and may involved in the ABA-, JA-, ET-, SA- and H2O2-mediated signaling pathways.

Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber.

BackgroundThe mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack.ResultsA genome-wide analysis revealed the presence of at least 14 MAPKs, 6 MAPKKs, and 59 MAPKKKs in the cucumber genome. Phylogenetic analyses classified all the CsMAPK and CsMAPKK genes into four groups, whereas the CsMAPKKK genes were grouped into the MEKK, RAF, and ZIK subfamilies. The expansion of these three gene families was mainly contributed by segmental duplication events. Furthermore, the ratios of non-synonymous substitution rates (Ka) and synonymous substitution rates (Ks) implied that the duplicated gene pairs had experienced strong purifying selection. Real-time PCR analysis demonstrated that some MAPK, MAPKK and MAPKKK genes are preferentially expressed in specific organs or tissues. Moreover, the expression levels of most of these genes significantly changed under heat, cold, drought, and Pseudoperonospora cubensis treatments. Exposure to abscisic acid and jasmonic acid markedly affected the expression levels of these genes, thereby implying that they may play important roles in the plant hormone network.ConclusionA comprehensive genome-wide analysis of gene structure, chromosomal distribution, and evolutionary relationship of MAPK cascade genes in cucumber are present here. Further expression analysis revealed that these genes were involved in important signaling pathways for biotic and abiotic stress responses in cucumber, as well as the response to plant hormones. Our first systematic description of the MAPK, MAPKK, and MAPKKK families in cucumber will help to elucidate their biological roles in plant.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1621-2) contains supplementary material, which is available to authorized users.