CRV::GhMORG1 up, increased phosphorylated proteins from CRV::GhMORG1 cotton infected with F. oxysporum. (c) Venn diagram illustrating the overlap of increased‐ or decreased‐regulated phosphopeptides identified in CRV::00 and CRV::GhMORG1 cotton after F. oxysporum infection. (a, b) GO‐based enrichment analysis of the increased‐ and decreased‐regulated phosphorylated proteins from CRV::00 and CRV::GhMORG1 cotton infected with F. oxysporum. Screening of putative substrates of the GhMORG1‐dependent GhMKK6‐GhMPK4 cascade. Different letters indicate significant differences ( P < 0.01) based on Tukey’s HSD test. Data in (f) are means ± SE of three independent experiments ( n = 15). Asterisks above the lines indicate significant differences (* P < 0.05, ** P < 0.01) based on Tukey’s HSD test. The error bars in (b, c and e) indicate the mean ± SE of three independent experiments ( n = 15). (f) Expression levels of SA‐mediated defence pathway genes in GhMORG1‐overexpressing tobacco. (e) Pathogen disease index in GhMORG1‐overexpressing tobacco after F. oxysporum infection. (d) Representative phenotypes of GhMORG1‐overexpressing tobacco after F. oxysporum infection. (c) Expression levels of SA‐mediated defence pathway genes in CRV::00 and CRV::GhMORG1 cotton. (b) Pathogen disease index in GhMORG1‐silenced cotton after five days of F. oxysporum infection. (a) Representative phenotypes of GhMORG1‐silenced cotton plants after five days of F. oxysporum infection. GhMORG1 positively regulated the resistance of cotton to F. oxysporum. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. Gossypium hirsutum MAPK scaffold protein disease resistance phosphoproteomics. Taken together, these findings reveal a new plant MAPK scaffold protein and provide insights into the mechanism of plant resistance to pathogens. These results suggest that GhMORG1 regulates several different disease resistance processes by facilitating the phosphorylation of GhMKK6-GhMPK4 cascade substrates. oxysporum was decreased in the substrate-silenced cottons. For functional analysis, nine putative substrates were silenced in cotton, respectively. The kinase assays verified that most of the putative substrates were phosphorylated by the 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. 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. GhMORG1 interacted with GhMKK6 and GhMPK4, and the overexpression of GhMORG1 in cotton protoplasts dramatically increased the activity of the GhMKK6-GhMPK4 cascade. Here, we identified GhMORG1, a GhMKK6-GhMPK4 cascade scaffold protein that positively regulates the resistance of cotton to Fusarium oxysporum. 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. In eukaryotes, MAPK scaffold proteins are crucial for regulating the function of MAPK cascades.
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