Identification of AnSte7 [MkkB], and AnSte50 [SteD] associated proteins and role of MAPK pathway on secondary metabolism.

Abstract

<p>(A) A silver stained 5–14% gradient SDS polyacrylamide gel of AnSte7 [MkkB]::cTAP from the wild type and <i>steC</i>Δ [lacking AnSte11] background grown for vegetatively at 30°C for 20 hours. (B) Identified peptides of the proteins from the excised lanes of the wild type and <i>steC</i>Δ strains (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002816#pgen.1002816.s013" target="_blank">Table S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002816#pgen.1002816.s014" target="_blank">S5</a>). AnSte11 [SteC] (AN2269) and AnSte50 [SteD] (AN7252) were found as interactors of AnSte7 [MkkB]. (C) Interaction partners of the AnSte50::cTAP fusion from vegetatively, asexually and sexually grown cultures at 30°C for 20 hours (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002816#pgen.1002816.s015" target="_blank">Table S6</a>) (M2B; MkkB, MB; MpkB, SD; SteD, SC; SteC). (D) Identified polypeptides from the bands. (E) Monitoring of the phosphorylation status of the MpkB by phospho-p44/42 MAPK (Thr182/Tyr184) antibody in the wild type as well as pheromone pathway mutants grown for 24, 48 and 72 hours vegetatively. VeA protein levels served as loading control. 80 µg protein extract was loaded on each lane. (F) Production of secondary metabolite sterigmatocystin (ST) in the mutants of pheromone pathway, <i>Anste11</i> [<i>steC</i>Δ], <i>Anste50</i> [<i>steD</i>Δ], <i>Anste7</i> [<i>mkkB</i>Δ], <i>Anfus3</i> [<i>mpkB</i>Δ], <i>Anste12</i> [<i>steA</i>Δ], respectively. Developed TLC plates show sterigmatocystin production. Sts; Sterigmatocystin standard. (G) Quantification of the ST production from the TLC plates. Wild type ST levels served as 100% standard. (H) Expression of the developmental, secondary metabolite genes in the pheromone pathway mutants. <i>laeA, aflR, stcU</i> for ST production, <i>tdiA</i> and <i>tdiB</i> for terrequinone (TQ) production and <i>veA, mkkB, steD, mpkB, steA</i> for developmental purposes were monitored. Strains were grown in the liquid medium for 24, 48 and 72 hours and total RNA was isolated and blotted (20 µg). Glycolytic gene <i>gpdA</i> expression and ethidium bromide stained rRNA was used as loading control.</p

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