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 GhMORG1dependent 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.
Summary
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.
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