The actin cytoskeleton is involved in toxisome formation.

(A) The interaction of Actin-RFP and Tri1-GFP was verified by the Co-IP assay. Total proteins (input) extracted from the strain bearing Actin-RFP and Tri1-GFP constructs or a single construct (Actin-RFP or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-GFP and anti-RFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-GFP agarose and further detected with anti-RFP antibody (GFP pull-down panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (B) Co-IP analysis for verification of the interaction between FgMyo1-GFP and Actin-RFP. Total proteins (input) extracted from the strain bearing Actin-RFP and FgMyo1-GFP constructs or a single construct (Actin-RFP or FgMyo1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-GFP agarose and further detected with anti-RFP antibody (GFP pull-down panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (C) The actin polymerization inhibitor latrunculin A inhibited toxisome formation. After growth in TBI for 24 h, ΔTri1::Tri1-GFP was treated with 0.1 μg/ml latrunculin A for another 24 h before examination (left panel). The solvent DMSO was used as a control. Bar = 10 μm. The accumulation of Tri1-GFP protein was further verified by western blotting assay using the anti-GFP antibody (right panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) DON was extracted from mycelia of PH-1 grown in TBI supplemented with 0.1 μg/ml latrunculin A. The solvent DMSO was used as a control. Values on the bars followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05.