Mitogen-activated Protein Kinase Gene Research Articles

Genome-Wide Analysis of the MAPKKK Gene Family Under Abiotic Stresses in Moso Bamboo (Phyllostachys edulis)

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are the upstream components of MAPK cascades and are involved in mediating stress responses and developmental processes. Although MAPKKK genes have been investigated in many plants, the identification and characterization of MAPKKKs in moso bamboo were still limited. Here, 134 MAPKKKs were identified as unevenly distributed on 23 chromosomes (except for chromosome 1) of moso bamboo and divided into three subfamilies by phylogenetic analysis. The gene structure and conserved motif of PeMAPKKKs were investigated. The expansions of PeMAPKKKs were driven by whole-genome duplication (WGD) or segmental duplication events. The duplicated gene pairs were under purifying selection based on the Ka/Ks ratios, suggesting they underwent functional conservation. Most PeMAPKKKs contained cis-elements related to development, hormones, and stress responses. Tissue expression patterns showed that PeMAPKKKs had multiple expression patterns. The qPCR analysis showed distinct expression patterns of PeMAPKKKs under drought, salt, and cold stress conditions. Taken together, this study provides a solid foundation for future functional characterizations of MAPKKKs and identifies candidate stress-responsive genes for further study in moso bamboo.

Identification of growth-related genes based on BSA in Pacific white shrimp Litopenaeus vannamei

The development of SNP genotyping techniques has facilitated the application of genome-wide association studies (GWAS) and genomic selection (GS) in plants and animals. However, the cost of high-throughput SNP genotyping is particularly high in the case of aquaculture animals which can produce a large number of offspring. Consequently, a cost effective method for trait-associated SNP identification and application was urgently needed in aquaculture breeding. In this study, a Bulk Segregation Analysis sequencing (BSA-seq) method was employed to identify growth-related single nucleotide polymorphisms (SNPs) and genes in the Pacific white shrimp Litopenaeus vannamei. Two independent families were used for this analysis to reduce the false positive. The DNA of individuals with the highest and lowest body weights was pooled separately. Two methods including the ED (Euclidean distance) method and the chi-square test were applied to calculate the allele differences between high and low growth rate individuals. A total of 24,325,437 SNPs were identified and consequently 1,299 SNPs and 163 genes were identified to be associated with growth traits. KEGG enrichment analysis showed that these genes were significantly enriched in phosphatidylinositol signaling system and phospholipase D signaling pathway. The diacylglycerol kinase genes, phosphatidylinositol 3-kinase genes, inositol polyphosphate 5-phosphatase genes, mitogen-activated protein kinase genes, myotubularin-related protein genes and epidermal growth factor receptor genes in these pathways were considered as candidate growth genes. This study demonstrated that the BSA-seq based on whole genome resequencing is cost-effective and useful in aquaculture. The results not only provide useful information for understanding the genetic basis of growth traits, but also offer a source of SNPs for marker-assisted selection (MAS) and genomic selection (GS) in shrimp.

Functional allocation of Mitogen-activated protein kinases (MAPKs) unveils thermotolerance in scallop Argopecten irradians irradians

Global warming has significantly impacted agriculture, particularly in animal husbandry and aquaculture industry. Rising ocean temperatures due to global warming are severely affecting shellfish production, necessitating an understanding of how shellfish cope with thermal stress. The mitogen-activated protein kinases (MAPK) signaling pathway plays a crucial role in cell growth, differentiation, adaptation to environmental stress, inflammatory response, and managing high temperature stress. To investigate the function of MAPKs in bay scallops, a comparative genomics and bioinformatics approach identified three MAPK genes: AiERK, Aip38, and AiJNK. Structural and phylogenetic analyses of these proteins were conducted to determine their evolutionary relationships. Spatiotemporal expression patterns were examined at different developmental stages and in various tissues of healthy adult scallops. Additionally, the expression regulation of these genes was studied in selected tissues (hemocyte, gill, heart, mantle) following exposure to high temperatures (32 °C) for different durations (0 h, 6 h, 12 h, 24 h, 3 d, 6 d, 10 d). The spatiotemporal expressions of AiMAPKs were ubiquitous, with significant increases in AiERK expression observed at the umbo larval stage (3.09-fold), while Aip38 and AiJNK were identified as potential maternal effect genes. In adult scallops, different gene expression patterns of AiMAPKs were observed across eight tissues, with high expressions in the foot and gill, and lower expressions in the striated muscle. Following high temperature stress, AiMAPKs expressions in the gill and mantle were mainly up-regulated, while in the hemocyte, they were primarily down-regulated. These findings indicate time- and tissue-dependent expression patterns with functional allocation in response to different thermal durations. This study enhances our understanding of the function and evolution of AiMAPKs genes in shellfish and provides a theoretical basis for elucidating the energy regulation mechanism of bay scallops in response to high temperature stress.

Genome-wide identification and analysis of abiotic stress responsiveness of the mitogen-activated protein kinase gene family in Medicago sativa L.

BackgroundThe mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa.ResultsIn this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses.ConclusionThis study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance.

Characterization of MKK family genes and their responses to temperature stress and Vibrio parahaemolyticus infection in noble scallop Chlamys nobilis

Mitogen-activated protein kinase kinase (MKK), the key element of the Mitogen-activated protein kinase (MAPK) signaling pathway, is crucial for the immune response to adverse environments in aquatic animals. Nevertheless, there is limited information regarding the role of the MKK gene family in mollusks. In our study, genome data and transcriptome were used to identify four MKK genes (CnMKK4, CnMKK5, CnMKK6, and CnMKK7) in the noble scallop. The result of the gene structure, motif analysis, and phylogenetic tree revealed that MKK genes are relatively conserved in bivalves. Moreover, four CnMKK genes were significantly highly expressed in immune-related tissues, suggesting that CnMKKs may related to bivalve immunity. Furthermore, CnMKK6 and CgMKK4 were significantly differentially expressed (P < 0.05) under 24 h of temperature stress, and all CnMKKs were significantly differentially expressed (P < 0.05) under 24 h of Vibrio parahaemolyticus infection. These results showed that the CnMKKs may have a significant impact under biotic and abiotic stresses. In conclusion, the result of the CnMKKs provides valuable insights into comprehending the function of MKK genes in mollusks.

Re-routing MAP kinase signaling for penetration peg formation in predator yeasts.

Saccharomycopsis yeasts are natural organic sulfur auxotrophs due to lack of genes required for the uptake and assimilation of sulfate/sulfite. Starvation for methionine induces a shift to a predatory, mycoparasitic life strategy that is unique amongst ascomycetous yeasts. Similar to fungal plant pathogens that separated from Saccharomycopsis more than 400 million years ago, a specialized infection structure called penetration peg is used for prey cell invasion. Penetration pegs are highly enriched with chitin. Here we demonstrate that an ancient and conserved MAP kinase signaling pathway regulates penetration peg formation and successful predation in the predator yeast S. schoenii. Deletion of the MAP kinase gene SsKIL1, a homolog of the Saccharomyces cerevisiae ScKSS1/ScFUS3 and the rice blast Magnaporthe oryzae MoPMK1 genes, as well as deletion of the transcription factor SsSTE12 generate non-pathogenic mutants that fail to form penetration pegs. Comparative global transcriptome analyses using RNAseq indicate loss of the SsKil1-SsSte12-dependent predation response in the mutant strains, while a methionine starvation response is still executed. Within the promoter sequences of genes upregulated during predation we identified a cis-regulatory element similar to the ScSte12 pheromone response element. Our results indicate that, re-routing MAP-kinase signaling by re-wiring Ste12 transcriptional control towards predation specific genes contributed to the parallel evolution of this predacious behaviour in predator yeasts. Consequently, we found that SsSTE12 is dispensable for mating.

The regulatory effects of arginine on ovary development in the Chinese mitten crab Eriocheir sinensis

This study aimed to investigate the effects of arginine (Arg) on the vitellogenesis and ovarian maturation of Chinese mitten crab. Six isonitrogenous and isolipidic diets (1.83, 2.52, 3.26, 3.96, 4.81 and 5.52 % Arg) were fed to adult female Chinese mitten crab (60.05 ± 4.33 g). The feeding period lasted for eleven weeks. The results showed that gonadosomatic index of crabs increased with increasing dietary Arg concentrations from 1.83 % to 2.52 % and significantly decreased from 2.52 % to 5.52 %. The quadratic regression analysis based on gonadosomatic index estimated that the optimal Arg requirement for adult female crabs was 2.60 %. Dietary supplementation with 2.52 % Arg significantly increased the crude protein concentration in the ovary. A dose of 2.52 % Arg significantly decreased the vitellogenesis-inhibiting hormone (VIH) content in the hemolymph. Arg (2.52 %) significantly upregulated the Vg and protein kinase A (PKA) gene expressions and increased the cyclic adenosine monophosphate (cAMP) concentration in the hepatopancreas. Moreover, dietary supplementation with 2.52 % Arg significantly decreased the cyclic guanosine monophosphate (cGMP) concentration and downregulated protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) gene expression. Arg at 2.52 % significantly increased hemolymph Vg content and the gene expression of the vitellogenin receptor (VgR). In conclusion, Arg at 2.52 % improved vitellogenesis and ovarian maturation by reducing the VIH content and regulating Vg expression via the cGMP/cAMP pathway.

Companion basil plants prime the tomato wound response through volatile signaling in a mixed planting system

Key messageVolatile compounds released from basil prime the tomato wound response by promoting jasmonic acid, mitogen-activated protein kinase, and reactive oxygen species signaling.Within mixed planting systems, companion plants can promote growth or enhance stress responses in target plants. However, the mechanisms underlying these effects remain poorly understood. To gain insight into the molecular nature of the effects of companion plants, we investigated the effects of basil plants (Ocimum basilicum var. minimum) on the wound response in tomato plants (Solanum lycopersicum cv. ‘Micro-Tom’) within a mixed planting system under environmentally controlled chamber. The results showed that the expression of Pin2, which specifically responds to mechanical wounding, was induced more rapidly and more strongly in the leaves of tomato plants cultivated with companion basil plants. This wound response priming effect was replicated through the exposure of tomato plants to an essential oil (EO) prepared from basil leaves. Tomato leaves pre-exposed to basil EO showed enhanced expression of genes related to jasmonic acid, mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling after wounding stress. Basil EO also enhanced ROS accumulation in wounded tomato leaves. The wound response priming effect of basil EO was confirmed in wounded Arabidopsis plants. Loss-of-function analysis of target genes revealed that MAPK genes play pivotal roles in controlling the observed priming effects. Spodoptera litura larvae-fed tomato leaves pre-exposed to basil EO showed reduced growth compared with larvae-fed control leaves. Thus, mixed planting with basil may enhance defense priming in both tomato and Arabidopsis plants through the activation of volatile signaling.

Comprehensive analysis of MAPK genes in the prognosis, immune characteristics, and drug treatment of renal clear cell carcinoma using bioinformatic analysis and Mendelian randomization

Mitogen-activated protein kinase (MAPK) signalling is vitally important in tumour development and progression. This study is the first to comprehensively analyse the role of MAPK-family genes in the progression, prognosis, immune-cell infiltration, methylation, and potential therapeutic value drug candidates in ccRCC. We identified a novel prognostic panel of six MAPK-signature genes (MAP3K12, MAP3K1, MAP3K5, MAPK1, MAPK8, MAPK9), and introduced a robust MAPK-signature risk model for predicting ccRCC prognosis. Model construction, evaluation, and external validation using datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database demonstrated its stability, as well as high sensitivity and specificity. Enrichment analysis suggested the participation of immune-mediated mechanism in MAPK dysregulation in ccRCC. Immune-infiltration analysis confirmed the relationship and revealed that the MAPK-signature risk model might stratify immunotherapy response in ccRCC, which was verified in drug sensitivity analysis and validated in external ccRCC immunotherapy dataset (GSE67501). Potential therapeutic drug predictions for key MAPKs using DSigDB, Network Analyst, CTD, and DGIdb were subsequently verified by molecular docking with AutoDock Vina and PyMol. Mendelian randomization further demonstrated the possibilities of the MAPK-signature genes as targets for therapeutic drugs in ccRCC. Methylation analysis using UALCAN and MethSurv revealed the participation of epigenetic modifications in dysregulation and survival difference of MAPK pathway in ccRCC. Among the key MAPKs, MAP3K12 exhibited the highest significance, indicating its independent prognostic value as single gene in ccRCC. Knockout and overexpression validation experiments in vitro and in vivo found that MAP3K12 acted as a promoter of tumour progression in RCC, suggesting a pivotal role for MAP3K12 in the proliferation, migration, and invasion of RCC cells. Our findings proposed the potential of MAPK-signature genes as biomarkers for prognosis and therapy response, as well as targets for therapeutic drugs in ccRCC.

Identification and analysis of MAPK cascade gene families of Camellia oleifera and their roles in response to cold stress.

Low-temperature severely limits the growth and development of Camellia oleifera (C. oleifera). The mitogen-activated protein kinase (MAPK) cascade plays a key role in the response to cold stress. Our study aims to identify MAPK cascade genes in C. oleifera and reveal their roles in response to cold stress. In our study, we systematically identified and analyzed the MAPK cascade gene families of C. oleifera, including their physical and chemical properties, conserved motifs, and multiple sequence alignments. In addition, we characterized the interacting networks of MAPKK kinase (MAPKKK)-MAPK kinase (MAPKK)-MAPK in C. oleifera. The molecular mechanism of cold stress resistance of MAPK cascade genes in wild C. oleifera was analyzed by differential gene expression and real-time quantitative reverse transcription-PCR (qRT-PCR). In this study, 21 MAPKs, 4 MAPKKs and 55 MAPKKKs genes were identified in the leaf transcriptome of C. oleifera. According to the phylogenetic results, MAPKs were divided into 4 groups (A, B, C and D), MAPKKs were divided into 3 groups (A, B and D), and MAPKKKs were divided into 2 groups (MEKK and Raf). Motif analysis showed that the motifs in each subfamily were conserved, and most of the motifs in the same subfamily were basically the same. The protein interaction network based on Arabidopsis thaliana (A. thaliana) homologs revealed that MAPK, MAPKK, and MAPKKK genes were widely involved in C. oleifera growth and development and in responses to biotic and abiotic stresses. Gene expression analysis revealed that the CoMAPKKK5/CoMAPKKK43/CoMAPKKK49-CoMAPKK4-CoMAPK8 module may play a key role in the cold stress resistance of wild C. oleifera at a high-elevation site in Lu Mountain (LSG). This study can facilitate the mining and utilization of genetic resources of C. oleifera with low-temperature tolerance.

FsCGBP, a Cutinase G-Box Binding Protein, Regulates the Growth, Development, and Virulence of Fusarium sacchari, the Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari is a causal agent of sugarcane Pokkah boeng, an important fungal disease that causes a considerable reduction in yield and sugar content in susceptible varieties of sugarcane worldwide. Despite its importance, the fungal factors that regulate the virulence of this pathogen remain largely unknown. In our previous study, mapping of an insertional mutant defect in virulence resulted in the identification of a cutinase G-box binding protein gene, designated FsCGBP, that encodes a C2H2-type transcription factor (TF). FsCGBP was shown to localize in the nuclei, and the transcript level of FsCGBP was significantly upregulated during the infection process or in response to abiotic stresses. Deletion or silencing of FsCGBP resulted in a reduction in mycelial growth, conidial production, and virulence and a delay in conidial germination in the F. sacchari. Cutinase genes FsCUT2, FsCUT3, and FsCUT4 and the mitogen-activated protein kinase (MAPK) genes FsHOG1, FsMGV1, and FsGPMK1, which were significantly downregulated in ΔFsCGBP. Except for FsHOG1, all of these genes were found to be transcriptionally activated by FsCGBP using the yeast one-hybrid system in vitro. The deletion of individual cutinase genes did not result in any of the phenotypes exhibited in the ΔFsCGBP mutant, except for cutinase activity. However, disruption of the MAPK pathway upon deletion of FsMGV1 or FsGPMK1 resulted in phenotypes similar to those of the ΔFsCGBP mutant. The above results suggest that FsCGBP functions by regulating the MAPK pathway and cutinase genes, providing new insights into the mechanism of virulence regulation in F. sacchari.

The basic leucine zipper transcription factor MeaB is critical for biofilm formation, cell wall integrity, and virulence in Aspergillus fumigatus.

The regulation of fungal cell wall biosynthesis is crucial for cell wall integrity maintenance and directly impacts fungal pathogen virulence. Although numerous genes are involved in fungal cell wall polysaccharide biosynthesis through multiple pathways, the underlying regulatory mechanism is still not fully understood. In this study, we identified and functionally characterized a direct downstream target of SomA, the basic-region leucine zipper transcription factor MeaB, playing a certain role in Aspergillus fumigatus cell wall integrity. Loss of meaB reduces hyphal growth, causes severe defects in galactosaminogalactan-mediated biofilm formation, and attenuates virulence in a Galleria mellonella infection model. Furthermore, the meaB null mutant strain exhibited hypersensitivity to cell wall-perturbing agents and significantly alters the cell wall structure. Transcriptional profile analysis revealed that MeaB positively regulates the expression of the galactosaminogalactan biosynthesis and β-1,3-glucanosyltransferase genes uge3, agd3, and sph3 and gel1, gel5, and gel7, respectively, as well as genes involved in amino sugar and nucleotide sugar metabolism. Further study demonstrated that MeaB could respond to cell wall stress and contribute to the proper expression of mitogen-activated protein kinase genes mpkA and mpkC in the presence of different concentrations of congo red. In conclusion, A. fumigatus MeaB plays a critical role in cell wall integrity by governing the expression of genes encoding cell wall-related proteins, thus impacting the virulence of this fungus.IMPORTANCEAspergillus fumigatus is a common opportunistic mold that causes life-threatening infections in immunosuppressed patients. The fungal cell wall is a complex and dynamic organelle essential for the development of pathogenic fungi. Genes involved in cell wall polysaccharide biosynthesis and remodeling are crucial for fungal pathogen virulence. However, the potential regulatory mechanism for cell wall integrity remains to be fully defined in A. fumigatus. In the present study, we identify basic-region leucine zipper transcription factor MeaB as an important regulator of cell wall galactosaminogalactan biosynthesis and β-1,3-glucan remodeling that consequently impacts stress response and virulence of fungal pathogens. Thus, we illuminate a mechanism of transcriptional control fungal cell wall polysaccharide biosynthesis and stress response. As these cell wall components are promising therapeutic targets for fungal infections, understanding the regulatory mechanism of such polysaccharides will provide new therapeutic opportunities.

Mechanism of IL-17 Signaling Pathway in Spleen Inflammatory Response Induced by Altitude Hypoxia in Mice

To explore the mechanism of spleen tissue inflammatory response induced by altitude hypoxia in mice. C57BL/6 mice were randomly assigned to a plain, i.e., low-altitude, normoxia group and an altitude hypoxia group, with 5 mice in each group. In the plain normoxia group, the mice were kept in a normoxic environment at the altitude of 400 m above sea level (with an oxygen concentration of 19.88%). The mice in the altitude hypoxia group were kept in an environment at the altitude of 4200 m above sea level (with an oxygen concentration of 14.23%) to establish the animal model of altitude hypoxia. On day 30, spleen tissues were collected to determine the splenic index. HE staining was performed to observe the histopathological changes in the spleen tissues of the mice. Real time fluorogenic quantitative PCR (RT-qPCR) and Western blot were conducted to determine the mRNA and protein expressions of interleukin (IL)-6, IL-12, and IL-1β in the spleen tissue of the mice. High-throughput transcriptome sequencing was performed with RNA sequencing (RNA-seq). KEGG enrichment analysis was performed for the differentially expressed genes (DEGs). The DEGs in the key pathways were verified by RT-qPCR. Compared with the plain normoxia group, the mice exposed to high-altitude hypoxic environment had decreased spleen index (P<0.05) and exhibited such pathological changes as decreased white pulp, enlarged germinal center, blurred edge, and venous congestion. The mRNA and protein expression levels of IL-6, IL-12, and IL-1β in the spleen tissue of mice in the altitude hypoxia group were up-regulated (P<0.05). According to the results of transcriptome sequencing and KEGG pathway enrichment analysis, 4218 DEGs were enriched in 178 enrichment pathways (P<0.05). DEGs were significantly enriched in multiple pathways associated with immunity and inflammation, such as T cell receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway (P<0.05) in the spleen of mice exposed to high-altitude hypoxic environment. Among them, IL-17 signaling pathway and the downstream inflammatory factors were highly up-regulated (P<0.05). Compared with the plain normoxia group, the mRNA expression levels of key genes in the IL-17 signaling pathway, including IL-17, IL-17R, and mitogen-activated protein kinase genes (MAPKs), and the downstream inflammatory factors, including matrix metallopeptidase 9 (MMP9), S100 calcium binding protein A8 gene (S100A8), S100 calcium binding protein A9 gene (S100A9), and tumor necrosis factor α (TNF-α), were up-regulated or down-regulated (P<0.05) in the altitude hypoxia group. According to the validation of RT-qPCR results, the mRNA expression levels of DEGs were consistent with the RNA-seq results. Altitude hypoxia can induce inflammatory response in the mouse spleen tissue by activating IL-17 signaling pathway and promoting the release of downstream inflammatory factors.

OsMKK1 is a novel element that positively regulates the Xa21-mediated resistance response to Xanthomonas oryzae pv. oryzae in rice.

OsMKK1, a MAPK gene, positively regulates rice Xa21-mediated resistance response and also plays roles in normal growth and development process of rice. The mitogen-activated protein kinase (MAPK) cascade was highly conserved among eukaryotes, which played crucial roles in plant responses to pathogen infection. Bacterial blight is the most devastating bacterial disease. Xa21 confers broad-spectrum resistance to Xanthomonas oryzae pv. Oryzae (Xoo). This study identified that the transcription level of OsMKK1 was up-regulated in resistant response against Xoo, thus overexpression (OsMKK1-OX) and RNA interference (OsMKK1-RNAi) transgenic rice lines under the background of Xa21 was constructed. Compared with recipient control plants 4021, the OsMKK1-OX lines significantly enhanced disease resistance to Xoo, on the contrary, the resistance of OsMKK1-RNAi lines was weakened, demonstrated that OsMKK1 played a positive role in Xa21-mediated disease resistance pathway. A number of pathogenesis-related proteins, including PR1A, PR2 and PR10A showed enhanced expression in OsMKK1-OX lines, supported that these PR genes may be regulated by OsMKK1 to participate in the defense responses. In addition, the agronomic traits of OsMKK1 transgenic plants were affected. Overall, these results revealed the role of OsMKK1 in Xa21-mediated resistance against Xoo and in the normal growth and development process in rice.

Genome-wide identification of Glycyrrhiza uralensis Fisch. MAPK gene family and expression analysis under salt stress relieved by Bacillus subtilis.

Introduction: Research on Glycyrrhiza uralensis, a nonhalophyte that thrives in saline-alkaline soil and a traditional Chinese medicinal component, is focused on improving its ability to tolerate salt stress to increase its productivity and preserve its "Dao-di" characteristics. Furthermore, the inoculation of bioagents such as Bacillus subtilis to increase plant responses to abiotic stressors is currently a mainstream strategy. Mitogen-activated protein kinase (MAPK), a highly conserved protein kinase, plays a significant role in plant responses to various abiotic stress pathways. Methods: This investigation involved the identification of 21 members of the GuMAPK family from the genome of G. uralensis, with an analysis of their protein conserved domains, gene structures, evolutionary relationships, and phosphorylation sites using bioinformatics tools. Results: Systematic evolutionary analysis of the 21 GuMAPKs classified them into four distinct subgroups, revealing significant differences in gene structure and exon numbers. Collinearity analysis highlighted the crucial role of segmental duplication in expanding the GuMAPK gene family, which is particularly evident in G. uralensis and shows a close phylogenetic relationship with Arabidopsis thaliana, tomato, and cucumber. Additionally, the identification of phosphorylation sites suggests a strong correlation between GuMAPK and various physiological processes, including hormonal responses, stress resistance, and growth and development. Protein interaction analysis further supported the role of GuMAPK proteins in regulating essential downstream genes. Through examination of transcriptome expression patterns, GuMAPK16-2 emerged as a prospective pivotal regulatory factor in the context of salt stress and B. subtilis inoculation, a finding supported by its subcellular localization within the nucleus. Discussion: These discoveries offer compelling evidence for the involvement of GuMAPK in the salt stress response and for the exploration of the mechanisms underlying B. subtilis' enhancement of salt tolerance in G. uralensis.

Genome-Wide Identification, Evolution, and Expression Analysis of the MAPK Gene Family in Rosaceae Plants

Mitogen-activated protein kinases (MAPKs) are crucial regulators in coping with abiotic and biotic stresses, including drought, salinity, fungi, and pathogens. However, little is known about the characteristics, evolution process, and functional divergence of the MAPK gene family in Rosaceae plants. A total of 97 MAPK members were identified in six Rosaceae species, including 12 genes in Fragaria vesca, 22 genes in Malus domestica, 23 genes in Pyrus bretschneideri, 12 genes in Prunus mume, 14 genes in Prunus persica, and 14 genes in Rosa chinensis. All MAPK members of six Rosaceae plants were categorized into four clusters by the phylogenetic relationship analysis. Collinearity analysis discovered that both segmental duplication and tandem duplication contributed to the expansion of MAPK family genes in Rosaceae plants. And the analysis of motifs and gene structures indicated that the evolution of the MAPK gene family was highly conserved among phylogenetic clusters in Rosaceae species. In addition, the dN/dS rates of MAPK paralogous gene pairs were below one, suggesting the MAPK gene family in Rosaceae was driven by purifying selective pressure. Furthermore, functional divergence analysis discovered that 14 amino acid residues were detected as potentially key sites for functional divergence of MAPK family genes between different cluster pairs, specifically Type I functional divergence. The analysis of functional distance indicated that cluster C retained more of the original functional features, while cluster B exhibited functional specialization. Moreover, the expression profiles revealed that PmMAPK8, PmMAPK9, and PmMAPK10 were both highly expressed under drought stress and low temperature conditions. This study aims to comprehensively analyze the evolutionary process and functional analyses of the MAPK gene family in Rosaceae plants, which will lay the foundation for future studies into MAPK genes of Rosaceae in response to drought and cold stress.

Abstract B082: A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC)

Abstract Introduction: KRASG12C inhibitors are a new therapeutic approach to treat KRAS mutant-driven cancers, but as with other oncogene directed therapies, adaptive resistance develops quickly and frequently. The predominant resistance mechanisms are 1) secondary mutations in KRAS and/or other mitogen-activated protein kinase (MAPK) signaling genes and 2) amplification of KRAS or MAPK genes. Extrachromosomal DNA (ecDNA) are the predominant site of focal high copy number oncogene or resistance gene amplifications in human cancers. Previous studies have demonstrated KRASG12C amplification on ecDNA to be an acquired resistance mechanism after treatment with approved KRASG12C inhibitors. Cancers enabled by ecDNA remain underserved by current therapeutic options, including targeted therapies, and have poor prognosis. An in silico “loss of function” screen revealed ribonucleotide reductase (RNR) as a critical target required for the survival of ecDNA amplified tumor cells. RNR is the rate-limiting enzyme in the biosynthesis of deoxyribonucleotide triphosphates (dNTP), the building blocks of DNA and ecDNA replication and repair. While non-selective drugs with RNR inhibitory activity (e.g., gemcitabine) indirectly validate RNR as a cancer target, no selective small molecules targeting RNR have been approved. Methods: We have developed BBI-825, a potent, selective, and orally bioavailable RNR inhibitor (RNRi) for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers. Results: BBI-825 inhibits RNR with an IC50 of 20 nM in vitro and reduces deoxyadenosine triphosphate (dATP) levels in ecDNA amplified cancer cells, with an EC50 of less than 100 nM. On target selectivity of BBI-825 was confirmed using SAFETYscan cellular screening assay investigating target selectivity over 78 targets spanning hormone receptors, kinases, calcium, GPCR, ion channel, and transporter signaling. Importantly, BBI-825 prevented resistance-conferring ecDNA-enabled amplification of KRASG12C in KRASG12C mutated colorectal cancer cells treated with the KRAS inhibitor adagrasib in vitro. Prevention of ecDNA amplification correlated with antiproliferation of BBI-825-treated tumor cells in long-term cultures. Treatment of KRASG12C mutated CRC tumor xenograft models with single agent adagrasib resulted in rapid acquired resistance through KRASG12C amplification on ecDNA. In concordance with in vitro observations, co-treatment with BBI-825 delayed the development of adagrasib resistance concurrent with an abrogation of ecDNA-enabled KRASG12C amplification (P=0.001). Furthermore, BBI-825 significantly inhibited the growth of pre-established adagrasib-resistant KRASG12C ecDNA-amplified tumors in vivo (tumor growth inhibition (TGI) = 92%, p&amp;lt;0.002), reinforcing clinical actionability in the KRAS inhibitor-refractory setting. Conclusions: These findings support the advancement of BBI-825 as a selective RNR inhibitor for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers, including KRASG12C mutant CRC. Citation Format: Sudhir Chowdhry, Ryan Hansen, Jacques Mauger, AnneMarie Pferdekamper, Edison Tse, Ben Norman, Jafar Moininazeri, Salvador Garcia, Yen Truong, Debbie Liao, Joshua Lange, Evan Holmes, Anthony Celeste, Dave Solis, Auzon Steffy, Joshua Plum, Julie Wiese, Julius Apuy, Ardalan Ardeshiri, Snezana Milutinovic, Deepti Wilkinson, Anthony Pinkerton, Christian Hassig, Shailaja Kasibhatla. A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B082.

In Silico Analysis of the MAPK Gene Family in Cabbage and Its Expression during Development and Stress Response

Cabbage is often affected by an adverse environment and pathogens during its growth, resulting in a decline in yield and quality. Mitogen-activated protein kinases (MAPKs) play crucial roles in the development process, hormones, abiotic and biotic stresses, and cell division. So far, MAPKs have been characterized in various species. However, no systematic analysis of the MAPK family in cabbage has been performed. A total of 24 BoMAPK genes were identified and renamed according to Arabidopsis homologs; they were unevenly distributed on nine chromosomes. According to the conserved domain and phylogenetic relationship, BoMAPKs were divided into four subgroups, of which three belonged to subgroup A, four to subgroup B, three to subgroup C, and fourteen to subgroup D, respectively. There were 13 segmental duplication events in the cabbage genome. The Ka/Ks ratios for duplicated gene pairs of the MAPK family were less than 0.2, indicating that these BoMAPK genes have undergone purification selection in evolution. There were various cis-acting elements associated with stress, hormones, and development found in the promoter regions of most BoMAPK genes. Transcriptome data showed a high expression of BoMAPK3, BoMAPK4.1, and BoMAPK6.1 genes in various tissues. BoMAPK17.2, BoMAPK20.1 and BoMAPK20.2 were significantly induced by Plasmodiophora brassicae, as evidenced in the quantitative PCR (RT-qPCR) analysis, revealing their enormous potential to regulate stress response. RT-qPCR results showed how the transcriptional levels of BoMAPK genes varied substantially under abiotic stress at different time points. These analysis results provide a reference to further explore the function and evolution of BoMAPK genes in cabbage.

Selection and validation of internal control genes for quantitative real-time RT‒qPCR normalization of Phlebopus portentosus gene expression under different conditions.

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT‒qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.