JCS039354 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Table S3 -

• Supplemental Figure S1 -

  Fig. S1. Mitochondrial morphologies in Δdnm1 mutants. (A) Strain FB2/pKS1. (B-D) Strain FB2Δdnm/pKS1. Representative examples of mitochondrial patterns after 16 hours of growth in CM/Ara are shown. Arrowheads mark a branching site (C) and expanded blebs (D). (A1-D1) GFP fluorescence, (A2-D2) DIC (differential interference contrast) light microscopy. Scale bar (10 µm) refers to all panels.

• Supplemental Figure S2 -

  Fig. S2. Microscopy of mitochondrial structures in a dikaryotic hyphae from the FB1/pKS2 × FB2Δlga2/pKS1/pCLN1 combination. Example of partially fused mitochondria. Note the accumulation of smaller fragments derived from the a2 partner and the faint GFP fluorescence coincident with RFP in the regions of detectable fusion (brackets). Scale bar: 5 µm.

• Supplemental Figure S3 -

  Fig. S3. Mitochondrial morphologies in response to dnm1 overexpression. (A) Northern blot analysis of strains FB2/pMB2-2/pCud1 (independent transformants #1 and #2) and FB2/pMB2-2 (wt) before (CM) and 4h after transfer to CM/Ara (starting OD₆₀₀: 0.35). Filters were hybridised with the ³²P-labelled dnm1 probe. Radioactive signals were quantified (value in #2 CM/Ara set to 100). Staining with methylene blue (28S) reflects the amounts of RNA (9 µg) loaded. All lanes are from the same blot. The small differences in transcript sizes might reflect different transcription start sites. (B) Comparison of mitochondrial morphologies in response to dnm1 overexpression in strains FB2/pMB2-2 (wt), FB2/pMB2-2/pCud1#1 and #2 immediately (T₀), 14h (T₁₄), and 24h (T₂₄) after transfer to CM/Ara (starting OD₆₀₀: 0.15). >100 cells were analysed per strain and time point. See legend of Fig. 2C for classification as long (white bars) and short tubules (grey bars); fragmented mitochondria (black bars) show a punctuate pattern (see Fig. 4C). (C) Part of the Northern blot shown in Fig. 6A (lanes 3,5) showing the additional hybridisation with ³²P-labelled dnm1. Note the comparable dnm1 transcript levels irrespective of lga2 overexpression in CM/Ara.

• Supplemental Figure S4 -

  Fig. S4. Requirement of the mitochondrial localisation of Mrb1 for pathogenicity. (A) Localisation of Mrb1 derivatives. Proteins of mitochondrial (M) and cytosolic (C) fractions (each 20 µg) from U. maydis Δmrb1 strains CV2 (Mrb1ΔN-myc; see Table S1) and CV4 (Mrb-myc) were subjected to SDS-PAGE for immunoblot analysis. Anti-CCHL, anti-C-Myc or anti-α-tubulin antibodies were used. Note the presence of Mrb1-myc in both cytosolic and mitochondrial pools, whereas the localisation of Mrb1ΔN-myc in strain CV2 is confined to the cytosolic pool due to the removed N-terminal mitochondrial signal sequence. (B) Pathogenicity in dependence on the mitochondrial localization of Mrb1. Plant infection experiments were performed using strain FB1Δmrb1 combined with each two independent CV2 and CV4 strains. The combination FB1Δmrb1/FB2 was used for comparison. Symptoms of infected plants (tumours or stunting) were assessed six days after inoculation. n, number of inspected plants. Note the only mild pathogenicity symptoms triggered with the mixture including strain CV2. The reduced pathogenicity symptoms in the presence of strain CV4 compared to the control infection may be due to the Myc tag fusion at the C-terminus of Mrb1, which is most conserved among p32 proteins and may have a function in oligomerization (Jiang et al., 1999; Bortfeld et al., 2004).