Figure 1. Isolation and characterization of N. crassa strains with reduced levels of Tob37 or Tob38.

(a) Sheltered heterokaryons with deletions of either tob37 or tob38 in one nucleus of the heterokaryon were constructed using a split marker approach. Boxes symbolize heterokaryons while circles within the boxes represent the different component nuclei of the heterokaryon. The Figure shows an example for tob37, but the process was identical for tob38. The starting heterokaryon (HP1) contained nuclei with different genetic markers: either his-3 and mtr (provides resistance to fpa) or pan-2 and Bml (provides resistance to benomyl). Strains chosen for further work carried the knockouts in the his-3 mtr nucleus (see Methods). (b) Serial dilutions of conidiaspores (actual numbers spotted shown at top of panel) from the strains indicated on the left were spotted onto plates containing either minimal medium (min), which maintains both nuclei of the heterokaryon approximately equally; minimal medium containing pantothenate and benomyl (pan ben), which forces the nucleus carrying benomyl resistance (Bml, Figure 1A) to predominate the culture; or minimal medium containing histidine plus fpa (his fpa), which forces the nucleus carrying fpa resistance (mtr, Figure 1A) to predominate the culture. The control was strain HP1. (c) Cells from the indicated strains (top of panel) were grown in the presence of histidine (His) and fpa to force the predominance of the nucleus bearing the deletion of either tob37 or tob38. This results in reduction of the levels of Tob37 or Tob38, respectively. Mitochondria were isolated and subjected to SDS-PAGE followed by transfer to nitrocellulose, and immunodecoration with the anitbodies indicated on the left. The control strain was HP1. Multiple bands in the Tob55 lane correspond to different isoforms of the protein [27]. (d) Mitochondria isolated from the indicated strains were either untreated (Mitos) or incubated in the presence of proteinase K (Mitos + pK) for 15 min. Mitochondrial proteins were then subjected to SDS-PAGE and western blotting. The blot was examined for the presence of Tom70 and the intermembrane space protein CCHL. (e) As in panel C, except strains were grown in minimal medium which maintains the numbers of both types of nuclei in the culture approximately equally. (f) Conidia produced from the sheltered heterokaryons (ΔTob37 and ΔTob38) were streaked onto medium containing histidine and pantothenate. Individual colonies were isolated and tested for nutritional requirements to determine if they were histidine-requiring homokaryons (His-req), pantothenate requiring homokaryons (Pan-req), or heterokaryons (Het).