Effects of abamectin nanocapsules on bees through host physiology, immune function, and gut microbiome.

Pesticide usage is a common practice to increase crop yields. Nevertheless, the existence of pesticide residues in the surrounding environment presents a significant hazard to pollinators, specifically the potential undisclosed dangers related to emerging nanopesticides. This study examines the impact of abamectin nanocapsules (AbaNCs), created through electrostatic self-assembly, as an insecticide on honey bees. It was determined that AbaNCs upregulated detoxification genes, including CYP450, as well as antioxidant and immune genes in honey bees. Furthermore, AbaNCs affected the activity of crucial enzymes such as superoxide dismutase (SOD). Although no apparent damage was observed in bee gut tissue, AbaNCs significantly decreased digestive enzyme activity. Microbiome sequencing revealed that AbaNCs disrupted gut microbiome, resulting in a reduction of beneficial bacteria such as Bifidobacterium and Lactobacillus. Additionally, these changes in the gut microbiome were associated with decreased activity of digestive enzymes, including lipase. This study enhances our understanding of the impact of nanopesticides on pollinating insects. Through the revelation of the consequences arising from the utilization of abamectin nanocapsules, we have identified potential stress factors faced by these pollinators, enabling the implementation of improved protective measures.

Isolation of the AccCDK8 gene of Apis cerana cerana and its functional analysis under pesticide and heavy metal stress.

Environmental pollution has gained negative attention in recent years. The pesticides and heavy metals are top list of environmental toxicants directly endangering the survival and development of Apis cerana cerana. Cyclin-dependent kinases (CDKs) are heteromeric serine/threonine kinases that participate in cell cycle regulation and have a vital role in pesticide and heavy metal stress in Apis cerana cerana. In this experiment, we filtered out CDK8 gene from Apis cerana cerana (AccCDK8) and investigated its functions of pesticide and heavy metals resistance. Sequence analysis indicated that AccCDK8 is highly homologous to multiple CDK8s and contains a highly conserved CDK active site sequence. Phylogenetic analysis showed that AmCDK8 and AccCDK8 were closely related evolutionarily in Apis mellifera. Transcriptome analysis revealed that AccCDK8 expression was differentially affected after exposure to pesticide and heavy metal stresses. This indicates that AccCDK8 has a significant role in the resistance of Apis cerana cerana to pesticide and heavy metal stresses. It has implications for studying the function of CDK in other insects in response to stress.

The gene AccCyclin H mitigates oxidative stress by influencing trehalose metabolism in Apis cerana cerana.

BACKGROUND: Environmental stress can induce oxidative stress in Apis cerana cerana, leading to cellular oxidative damage, reduced vitality, and even death. Currently, owing to an incomplete understanding of the molecular mechanisms by which A. cerana cerana resists oxidative damage, there is no available method to mitigate the risk of this type of damage. Cyclin plays an important role in cell stress resistance. The aim of this study was to explore the in vivo protection of cyclin H against oxidative damage induced by abiotic stress in A. cerana cerana and clarify the mechanism of action. We isolated and identified the AccCyclin H gene in A. cerana cerana and analysed its responses to different exogenous stresses. RESULTS: The results showed that different oxidative stressors can induce or inhibit the expression of AccCyclin H. After RNA-interference-mediated AccCyclin H silencing, the activity of antioxidant-related genes and related enzymes was inhibited, and trehalose metabolism was reduced. AccCyclin H gene silencing reduced A. cerana cerana high-temperature tolerance. Exogenous trehalose supplementation enhanced the total antioxidant capacity of A. cerana cerana, reduced the accumulation of oxidants, and improved the viability of A. cerana cerana under high-temperature stress. CONCLUSION: Our findings suggest that trehalose can alleviate adverse stress and that AccCyclin H may participate in oxidative stress reactions by regulating trehalose metabolism. © 2023 Society of Chemical Industry.

Toxic effects of the heavy metal Cd on Apis cerana cerana (Hymenoptera: Apidae): Oxidative stress, immune disorders and disturbance of gut microbiota.

Cadmium (Cd) is a toxic non-essential metal element that can enter the honey bee body through air, water and soil. Currently, there is a lack of sufficient research on the effects of Cd on A. cerana cerana, especially the potential risks of long-term exposure to sublethal concentrations. In order to ascertain the toxicological effects of the heavy metal Cd on bees, we performed laboratory-based toxicity experiments on worker bees and conducted analyses from three distinctive facets: antioxidative, immunological, and gut microbiota. The results showed that exposure of bees to high concentrations of Cd resulted in acute mortality, and the increase in mortality was concentration dependent. In long-term exposure to sublethal concentrations, Cd reduced the number of transcripts of antioxidant genes (AccSOD1, AccTPx3 and AccTPx4) and superoxide dismutase activity, causing an increase in malondialdehyde content. Simultaneously, the transcription of immune-related genes (AccAbaecin and AccApidaecin) and acetylcholinesterase activities was inhibited. Furthermore, Cd changes the structural characteristics of bacterial and fungal communities in the gut, disrupting the balance of microbial communities. In conclusion, the health and survival of honey bees are affected by Cd. This study provides a scientific basis for investigating the toxicological mechanisms and control strategies of the heavy metal Cd on honey bees, while facilitating a better understanding and protection of these valuable honey bees.

Functional analysis of AccCDK2-like and AccCINP-like genes in Apis cerana cerana under pesticide and heavy metal stress.

Heavy metals and pesticides represent prominent sources of pollution in the natural habitat of Apis cerana cerana, potentially endangering their health through the induction of oxidative stress reactions. This study aimed to address this issue by isolating AccCDK2-like and AccCINP-like proteins from Apis cerana cerana and investigating their functional roles in honey bee resistance against pesticide and heavy metal stresses. Bioinformatics analysis revealed significant homology of these proteins with those found in other species. Functional studies confirmed their participation in interaction with each other, alongside demonstrating distinct patterns of expression and localization. Specifically, AccCDK2-like exhibited higher expression levels in prepupae and muscle tissues, while AccCINP-like showed maximal expression in brown pupae and abdomen. Furthermore, the expression levels of these proteins were found to be modulated in response to pesticide and heavy metal stresses. Notably, overexpression of AccCDK2-like and AccCINP-like led to a noticeable alteration in E. coli's ability to withstand external stresses. Additionally, silencing of the AccCDK2-like and AccCINP-like genes resulted in a significant reduction in antioxidant enzyme activity and the expression levels of genes related to antioxidant function. Consequently, the mortality rate of Apis cerana cerana under pesticide and heavy metal stresses conspicuously increased. Hence, our findings suggest that AccCDK2-like and AccCINP-like proteins potentially play a crucial role in the response of Apis cerana cerana to pesticide and heavy metal stress, likely by modulating the antioxidant pathway.

Identification of the AccCDK7 and AccCDK9 genes and their involvement in the response to resist external stress in Apis cerana cerana.

Previous studies examining the functions of cyclin-dependent kinases (CDKs) have mainly focused on the regulation of the cell cycle. Recent studies have found that cyclin-dependent kinase 7 (CDK7) and cyclin-dependent kinase 9 (CDK9) play important roles in cell stress, metabolism of toxic substances and maintaining the stability of the internal environment. Here, we found that under stress conditions, the transcription and protein expression of AccCDK7 and AccCDK9 were induced to varying degrees. Meanwhile, the silencing of AccCDK7 and AccCDK9 also affected the expression of antioxidant genes and the activity of antioxidant enzymes, and reduced the survival rate of bees under high temperature stress. Furthermore, the exogenous overexpression of AccCDK7 and AccCDK9 improved the viability of yeast under stress conditions. Therefore, AccCDK7 and AccCDK9 may play roles in A.cerana cerana resistance to oxidative stress caused by external stimuli, potentially revealing a new mechanism of the honeybee response to oxidative stress.

The role of AccCDK20 and AccCDKN1 from Apis cerana cerana in development and response to pesticide and heavy metal toxicity.

Apis cerana cerana is a native bee species in China and plays a key role in agricultural production and ecological balance. However, the growth and development of Apis cerana cerana has not been smooth, and pesticide and heavy metal stress are key factors that have forced a dramatic decline in population size. This study was performed with the objective of investigating the role of AccCDK20 and AccCDKN1 in honey bee resistance to pesticide and heavy metal stress. RT-qPCR analysis revealed that AccCDK20 transcript levels were highest in brown-eyed pupae and AccCDKN1 transcript levels were highest in 1-day-old worker bees. In different tissues and body parts of adult bees, AccCDK20 transcript levels were highest in the head, and AccCDKN1 transcript levels were highest in the thorax. It was further observed that environmental stress can affect the transcript levels of the AccCDK20 and AccCDKN1 genes. Silencing of the AccCDK20 and AccCDKN1 genes resulted in altered activities of antioxidant-related genes and antioxidant-related enzymes. AccCDK20 and AccCDKN1 transcript levels were upregulated under glyphosate stress, and silencing of the genes resulted in reduced resistance to glyphosate and greatly increased mortality in Apis cerana cerana. In addition, gene function was verified by in vitro repression assays. Overexpression of the AccCDK20 and AccCDKN1 proteins in E. coli cells increased the resistance to ROS damage induced by CHP. In conclusion, AccCDK20 and AccCDKN1 play an indispensable role in honey bee resistance to pesticide and heavy metal stress.

Group IIc WRKY transcription factors regulate cotton resistance to Fusarium oxysporum by promoting GhMKK2-mediated flavonoid biosynthesis.

WRKY transcription factors (TFs) are crucial regulators in response to pathogen infection. However, the regulatory mechanisms of WRKY TFs in response to Fusarium oxysporum f. sp. vasinfectum (Fov), the most devastating pathogen of cotton, remain unclear. Here, transcriptome sequencing indicated that the group IIc WRKY TF subfamily was the most important TF subfamily in response to Fov. Gain-of-function and loss-of-function analyses showed that group IIc WRKY TFs positively regulated cotton resistance to Fov. A series of chromatin immunoprecipitation sequencing, yeast one-hybrid assay and electrophoresis mobility shift assay experiments indicated that group IIc WRKY TFs directly bound to the promoter of GhMKK2 and regulated its expression. Importantly, a novel mitogen-activated protein kinase (MAPK) cascade composed of GhMKK2, GhNTF6 and GhMYC2 was identified. The functional analysis indicated that group IIc WRKY TFs induced the GhMKK2-GhNTF6 pathway to increase resistance to Fov by upregulating the GhMYC2-mediated expression of several flavonoid biosynthesis-related genes, which led to flavonoid accumulation. In conclusion, our study demonstrated a novel disease defense mechanism by which the WRKY-MAPK pathway promotes flavonoid biosynthesis to defend against pathogen infection. This pathway improves our understanding of the interaction mode between WRKY TFs and MAPK cascades in plant immunity and the vital role of plant flavonoids in pathogen defense.

Identification of the AccCDK1 gene in Apis cerana cerana and its relationship with the oxidative stress response.

The cyclin-dependent kinase (CDK) protein family plays an important role in regulating life functions, such as the cell cycle and metabolism. This study reports the first cloning and functional analysis of A. cerana cerana CDK1 (AccCDK1). The distribution profile of AccCDK1 in different developmental periods and different tissues was determined. The experimental results showed that the distribution of AccCDK1 was tissue-specific. AccCDK1 distribution at the transcriptional and translational levels was affected by stress conditions induced by H2O2, UV, HgCl2, CdCl2, extreme temperatures (4 °C, 44 °C) and pesticides (avermectin, lambda-cyhalothrin, haloxyfop-R-methyl, and glyphosate), which resulted in changes in the expression levels. These results suggest that AccCDK1 may have an important part to play in honey bee resistance to stress. The expression of a recombinant AccCDK1 protein in vitro enhanced the antistress capacities of E. coli and yeast, which suggests that AccCDK1 is related to the stress response. When AccCDK1 was silenced, the expression of some antioxidant genes was downregulated, and the enzymatic potencies of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were reduced, which suggests that AccCDK1 takes part in the body's resistance to oxidative stress upon external stimulation by influencing relevant antioxidants. Notably, the survival rate of A. cerana cerana under high-temperature-induced stress decreased after AccCDK1 silencing, which verifies our results. In conclusion, we found that AccCDK1 played an indispensable function in resisting oxidative stress and maintaining normal cellular functions.

The Protein Phosphatase GhAP2C1 Interacts Together with GhMPK4 to Synergistically Regulate the Immune Response to Fusarium oxysporum in Cotton.

The plant mitogen-activated protein kinase (MAPK) cascade plays an important role in mediating responses to biotic and abiotic stresses and is the main pathway through which extracellular stimuli are transduced intracellularly as signals. Our previous research showed that the GhMKK6-GhMPK4 cascade signaling pathway plays an important role in cotton immunity. To further analyze the role and regulatory mechanism of the GhMKK6-GhMPK4 cascade signaling pathway in cotton resistance to Fusarium wilt, we functionally analyzed GhMPK4. Our results show that silencing GhMPK4 reduces cotton tolerance to Fusarium wilt and reduces the expression of several resistance genes. Further experiments revealed that GhMPK4 is similar to GhMKK6, both of whose overexpression cause unfavorable cotton immune response characteristics. By using a yeast two-hybrid screening library and performing a bioinformatics analysis, we screened and identified a negative regulator of the MAPK kinase-protein phosphatase AP2C1. Through the functional analysis of AP2C1, it was found that, after being silenced, GhAP2C1 increased resistance to Fusarium wilt, but GhAP2C1 overexpression caused sensitivity to Fusarium wilt. These findings show that GhAP2C1 interacts together with GhMPK4 to regulate the immune response of cotton to Fusarium oxysporum, which provides important data for functionally analyzing and studying the feedback regulatory mechanism of the MAPK cascade and helps to clarify the regulatory mechanism through which the MAPK cascade acts in response to pathogens.

Distinct molecular impact patterns of abamectin on Apis mellifera ligustica and Apis cerana cerana.

The effects of insecticides on bee health are a topic of intensive research. Although abamectin is toxic to bees, the molecular impact of abamectin needs to be clarified. Here, we found that Apis cerana cerana exhibited a higher mortality rate when exposed to abamectin than Apis mellifera ligustica. In addition, A. cerana cerana had markedly higher numbers of differentially expressed genes (DEGs), differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs) than A. mellifera ligustica during exposure to abamectin. These results indicate that abamectin exposure exerts stronger effects on A. cerana cerana than on A. mellifera ligustica. In addition, six DEGs, two DEPs and two DEMs overlapped between the two bee species under abamectin exposure; however, some genes or proteins from the zinc finger protein, superoxide dismutase and peroxiredoxin families and the energy metabolism pathway were only unregulated in A. cerana cerana, which indicates a significant difference in the impact of abamectin on the two bee species. Despite these differences, several of the same gene families, such as heat shock proteins, cytochrome P450, odorant-binding proteins and cuticle proteins, and pathways, including the carbohydrate metabolism, immune system, lipid metabolism, amino acid metabolism, sensory system, locomotion and development pathways, were influenced by abamectin exposure in both A. cerana cerana and A. mellifera ligustica. Together, our results indicate that abamectin causes adverse effects on bees and thus poses a risk to bee populations and that abamectin exposure affects A. cerana cerana more strongly than A. mellifera ligustica. These findings improve our understanding of the behavioural and physiological effects of abamectin on bees.

Identification and characterization of an Apis cerana cerana nucleoside diphosphate kinase (AccNDPK) associated with oxidative stress.

Nucleoside diphosphate kinases (NDPKs) are widespread nucleotide-metabolizing enzymes that are involved in a variety of biological processes, including responses to oxidative stress. Although studies have been conducted on NDPKs in mammals and some plants, there is scant research on insect NDPKs, especially in honey bees. In the present study, we isolated AccNDPK from Apis cerana cerana. Sequence analysis showed that AccNDPK has high homology with many NDPKs and contains a highly conserved NDPK active site motif. Based on phylogenetic analysis, AccNDPK has a relatively recent evolutionary relationship with NDPKs in other hymenopteran insects. AccNDPK was found to be highly expressed in newly emerged honey bees and muscle tissues, and RT-qPCR analysis and bacteriostatic assays showed that the expression level of AccNDPK is affected by abnormal temperature, UV light, H2O2, heavy metals, and various pesticides. After AccNDPK knockdown, antioxidant-related genes, including AccCAT, AccCYP4G11, AccGSTS4, AccTpx1 and AccMsrA, were upregulated, whereas AccGSTD, AccGST1, AccHSP22.6 and AccTrx1 were downregulated. Furthermore, catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities were significantly increased, and the tolerance of bees to oxidative stress caused by cyhalothrin was reduced by silencing of AccNDPK. Given these findings, we speculate that AccNDPK plays an important role in the oxidative stress response of A. cerana cerana.

Identification of an Apis cerana zinc finger protein 41 gene and its involvement in the oxidative stress response.

Zinc finger proteins (ZFPs) are a class of transcription factors that contain zinc finger domains and play important roles in growth, aging, and responses to abiotic and biotic stresses. These proteins activate or inhibit gene transcription by binding to single-stranded DNA or RNA and through RNA/DNA bidirectional binding and protein-protein interactions. However, few studies have focused on the oxidation resistance functions of ZFPs in insects, particularly Apis cerana. In the current study, we identified a ZFP41 gene from A. cerana, AcZFP41, and verified its function in oxidative stress responses. Real-time quantitative polymerase chain reaction showed that the transcription level of AcZFP41 was upregulated to different degrees during exposure to oxidative stress, including that induced by extreme temperature, UV radiation, or pesticides. In addition, the silencing of AcZFP41 led to changes in the expression patterns of some known antioxidant genes. Moreover, the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and glutathione S-transferase (GST) in AcZFP41-silenced honeybees were higher than those in a control group. In summary, the data indicate that AcZFP41 is involved in the oxidative stress response. The results provide a theoretical basis for further studies of zinc finger proteins and improve our understanding of the antioxidant mechanisms of honeybees.

Functions of RPM1-interacting protein 4 in plant immunity.

KEY MESSAGE: We reviewed recent advances related to RIN4, including its involvement in the immune process through posttranslational modifications, PM H+-ATPase activity regulation, interaction with EXO70 and identification of RIN4-associated NLR proteins. RPM1-interacting protein 4 (RIN4) is a conserved plant immunity regulator that has been extensively studied and can be modified by pathogenic effector proteins. RIN4 plays an important role in both PTI and ETI. In this article, we review the functions of the two conserved NOI domains of RIN4, the C-terminal cysteine residues required for membrane localization and the sites targeted and modified by effector proteins during plant immunity. In addition, we discuss the effect of RIN4 on the stomatal virulence of pathogens via the regulation of PM H+-ATPase activity, which is involved in the immune process through interactions with the exocyst subunit EXO70, and progress in the identification of RIN4-related R proteins in multiple species. This review provides new insights enhancing the current understanding of the immune function of RIN4.

Cloning and expression studies on glutathione S-transferase like-gene in honey bee for its role in oxidative stress.

Glutathione S-transferases (GSTs) constitute an important multifunctional enzyme family that plays vital roles in cellular detoxification and protecting organisms against oxidative stress caused by reactive oxygen species (ROS). In this study, we isolated a GST-like gene from Apis cerana cerana (AccGSTL) and investigated its antioxidant functions under stress conditions. We found that AccGSTL belongs to the Sigma class of GSTs. Real-time quantitative PCR and western blotting analyses showed that the mRNA and protein levels of AccGSTL were altered in response to oxidative stress caused by various external stimuli. In addition, a heterologous expression analysis showed that AccGSTL overexpression in Escherichia coli (E. coli) cells enhanced resistance to oxidative stress. After AccGSTL silencing with RNA interference (RNAi) technology, the expression of some antioxidant genes was inhibited, and the enzymatic activities of POD, CAT, and SOD were decreased. In conclusion, these data suggest that AccGSTL may be involved in antioxidant defense under adverse conditions in A. cerana cerana.

Molecular and functional characaterization of the novel odorant-binding protein gene AccOBP10 from Apis cerana cerana.

Odorant-binding proteins (OBPs) play an important role in odour perception and transport in insects. However, little is known about whether OBPs perform other functions in insects, particularly in Apis cerana cerana. Within this study, an OBP gene (AccOBP10) was isolated and identified from A. c. cerana. Both homology and phylogenetic relationship analyses indicated that the amino acid sequence of AccOBP10 had a high degree of sequence identity with other members of the gene family. Analysis of quantitative real-time PCR (qRT-PCR) showed that AccOBP10 mRNA was expressed at higher levels in the venom gland than in other tissues. The mRNA transcript expression of AccOBP10 was upregulated by low temperature (4°C), hydrogen peroxide (H2O2), pyridaben, methomyl and imidacloprid but downregulated by heat (42°C), ultraviolet light, vitamin C, mercuric chloride, cadmium chloride, paraquat and phoxim. Expression of AccOBP10 under abiotic stress was analysed by western blotting, and the results were consistent with those of qRT-PCR. And as a further study of AccOBP10 function, we demonstrated that knockdown of AccOBP10 by RNA interference could slightly increase the expression levels of some stress-related genes. Collectively, these results suggest that AccOBP10 is mainly involved in the response to stress conditions.

Transcriptomic and metabolomic landscape of the molecular effects of glyphosate commercial formulation on Apis mellifera ligustica and Apis cerana cerana.

Understanding the causes of the decline in bee population has attracted intensive attention worldwide. The indiscriminate use of agrochemicals is a persistent problem due to their physiological and behavioural damage to bees. Glyphosate and its commercial formulation stand out due to their wide use in agricultural areas and non-crop areas, such as parks, railroads, roadsides, industrial sites, and recreational and residential areas, but the mode of action of glyphosate on bees at the molecular level remains largely unelucidated. Here, we found that the numbers of differentially expressed genes and metabolites under glyphosate commercial formulation (GCF) stress were significantly higher in Apis cerana cerana than in Apis mellifera ligustica. Despite these differences, the number of differentially expressed transcripts increased following an increase in the GCF treatment time in both A. cerana cerana and A. mellifera ligustica. GCF exerted adverse impacts on the immune system, digestive system, nervous system, amino acid metabolism, carbohydrate metabolism, growth and development of both bee species by influencing their key genes and metabolites to some extent. The expression of many genes involved in immunity, agrochemical detoxification and resistance, such as antimicrobial peptides, cuticle proteins and cytochrome P450 families, was upregulated by GCF in both bee species. Collectively, our results indicate that both A. cerana cerana and A. mellifera ligustica strive to mitigate the pernicious effects caused by GCF by regulating detoxification and immune systems. Moreover, A. cerana cerana might be better able to withstand the toxic effects of GCF with lower fitness costs than A. mellifera ligustica. Our work will contribute to elucidating the deleterious physiological and behavioural impacts of GCF on bees.

Interfacial Engineering of MoO2 -FeP Heterojunction for Highly Efficient Hydrogen Evolution Coupled with Biomass Electrooxidation.

Simultaneous highly efficient production of hydrogen and conversion of biomass into value-added products is meaningful but challenging. Herein, a porous nanospindle composed of carbon-encapsulated MoO2 -FeP heterojunction (MoO2 -FeP@C) is proposed as a robust bifunctional electrocatalyst for hydrogen evolution reaction (HER) and biomass electrooxidation reaction (BEOR). X-ray photoelectron spectroscopy analysis and theoretical calculations confirm the electron transfer from MoO2 to FeP at the interfaces, where electron accumulation on FeP favors the optimization of H2 O and H* absorption energies for HER, whereas hole accumulation on MoO2 is responsible for improving the BEOR activity. Thanks to its interfacial electronic structure, MoO2 -FeP@C exhibits excellent HER activity with an overpotential of 103 mV at 10 mA cm-2 and a Tafel slope of 48 mV dec-1 . Meanwhile, when 5-hydroxymethylfurfural is chosen as the biomass for BEOR, the conversion is almost 100%, and 2,5-furandicarboxylic acid (FDCA) is obtained with the selectivity of 98.6%. The electrolyzer employing MoO2 -FeP@C for cathodic H2 and anodic FDCA production requires only a low voltage of 1.486 V at 10 mA cm-2 and can be powered by a solar cell (output voltage: 1.45 V). Additionally, other BEORs coupled with HER catalyzed by MoO2 -FeP@C also have excellent catalytic performance, implying their good versatility.

Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by facilitating the MKK6-MPK4 cascade.

In eukaryotes, MAPK scaffold proteins are crucial for regulating the function of MAPK cascades. However, only a few MAPK scaffold proteins have been reported in plants, and the molecular mechanism through which scaffold proteins regulate the function of the MAPK cascade remains poorly understood. Here, we identified GhMORG1, a GhMKK6-GhMPK4 cascade scaffold protein that positively regulates the resistance of cotton to Fusarium oxysporum. GhMORG1 interacted with GhMKK6 and GhMPK4, and the overexpression of GhMORG1 in cotton protoplasts dramatically increased the activity of the GhMKK6-GhMPK4 cascade. Quantitative phosphoproteomics was used to clarify the mechanism of GhMORG1 in regulating disease resistance, and thirty-two proteins were considered as the putative substrates of the GhMORG1-dependent GhMKK6-GhMPK4 cascade. These putative substrates were involved in multiple disease resistance processes, such as cellular amino acid metabolic processes, calcium ion binding and RNA binding. The kinase assays verified that most of the putative substrates were phosphorylated by the GhMKK6-GhMPK4 cascade. For functional analysis, nine putative substrates were silenced in cotton, respectively. The resistance of cotton to F. oxysporum was decreased in the substrate-silenced cottons. These results suggest that GhMORG1 regulates several different disease resistance processes by facilitating the phosphorylation of GhMKK6-GhMPK4 cascade substrates. Taken together, these findings reveal a new plant MAPK scaffold protein and provide insights into the mechanism of plant resistance to pathogens.