Model for the interplay between Lga2 and Rga2 to control uniparental mtDNA inheritance in U. maydis. (A) Wild-type case. Pheromone stimulation confers low-level expression of lga2 and rga2 in the a2 partner (Urban et al. 1996b). Rga2 (gray dots) protects the mt nucleoid (m2) from Lga2 (black ellipse) by a currently unknown mechanism. After fusion of the cells (gray lines), lga2 is strongly upregulated in response to the bE/bW complex (Romeis et al. 2000; Brachmann et al. 2001). High amounts of Lga2 enter unprotected m1 mitochondria and cause loss of m1 mtDNA. The outcome is the m2 mitotype. (B) In the absence of Lga2, m1 and m2 mitotypes are maintained and mtDNA recombination occurs after mitochondrial fusion. Rga2 has no effect. The outcome is parental and X mitotypes. (C) Expression of rga2 in the a1 cell protects the m1 mitotype from elimination. Since this leads to a very similar outcome as in the absence of lga2 (B), mitochondrial fusion must also occur here, conceivably due to downregulation of lga2 during sexual progression in the host (Bortfeld et al. 2004). The outcome is parental and X mitotypes. (D) In the absence of protective Rga2 in the a2 cell, the m2 mtDNA is eliminated in response to Lga2. In this combination, the m1 mitotype is maintained.

Discrimination among U. maydis mitotypes. (A) The scheme shows the polymorphic region of parental mitotypes (F, W, and B) and of recombinant mitotypes (X1, X2, and X3) in the region of the LSU rRNA gene (drawn to scale). Open boxes (top) refer to deduced exon sequences of the LSU rRNA gene (the three major ones are numerated from I to III). All numbers refer to the complete mitochondrial genome sequence of U. maydis strain 521 (accession no. DQ157700). Thick solid arrows mark the positions of predicted HEGs within intronic regions (named LRI1, -2, -3 and LRII1). The W- and B/X-type exon sequences are indicated by dotted and hatched boxes, respectively. Stippled lines connect homologous exon sequences. HindIII restriction sites are marked (H). The position of the probe (solid bar) used for RFLP analysis and the sizes of the detected HindIII fragments are indicated (asterisks).

Influence of the a2 locus genes lga2 and rga2 on mtDNA inheritance. (A) Combinations with Δlga2 strains (left: FB1 with GF8 or GF8Δlga2; right: GF25 with FB2 or FB2Δlga2). (B) Combinations with Δrga2 strains (left: FB1 with GF8Δrga2, FB1 with GF5 or GF5Δrga2; right: GF25 with FB2Δrga2, MF34 with FB2 or FB2Δrga2). (A and B) Maize plants were inoculated with the combinations indicated above the panels for RFLP analysis. The a1 partner (m1) is on the left, and the a2 partner (m2) is on the right. Each lane represents the total DNA isolated from one tumor and hybridized with the mtDNA-specific probe (see Figure 1). Each tumor was isolated from a different plant. Lanes m1 and m2 are controls loaded with DNA from the corresponding parental strains. All lanes in each panel are from the same blot. Mitochondrial bands indicative for the W, X1, X2, X3, and F types are marked (arrowheads). Lanes clearly showing X2 are denoted by open triangles. (C) Quantitative analysis of the influence of the Δlga2, Δrga2, and Δlga2Δrga2 deletions on uniparental mtDNA inheritance in combinations with three different a1 partners. Bands corresponding to the W, X1, and F types were quantified from Southern blot analysis. For each strain combination (written as a1/a2 below the graph; Δl = Δlga2, Δr = Δrga2), average values and standard deviations of the W (solid), X1 (shaded), and F types (open bars) are indicated. Values of each bar express relative percentages, with a sum of 100% for all bars within a bracket. Quantification of the W band may include signals of the X2 band due to close proximity of the W and X2 bands. The number of individual tumors evaluated for each combination is indicated in parentheses.

Effect of rga2 expression in the a1 mating partner on uniparental mtDNA inheritance. (A) RNA gel blot analysis to verify rga2 expression in the individual strains transformed with pR-rga2 or pot-rga2 (bottom). The individual strains are marked at the top of each lane. Strains FB1, FB1/pR-rga2#1, and FB1/pR-rga2#2 (lanes 1–3) were cultivated in CM and stimulated with Mfa2 pheromone for 6 hr. The remaining strains (lanes 4–9) were cultivated in YEPSl medium for 12 hr. Two identical filters were hybridized with [³²P]-labeled rga2 and ppi fragments, respectively. Radioactive signals (both for rga2 in lanes 2 and 3; bracket) were quantified and the ratios of rga2/ppi signals were calculated, with the ratio in lane 9 set to 100. Scheme of pR-rga2 and pot-rga2: pR-rga2 expresses rga2 under its own regulatory sequences. Nontranslated sequences (5′ and 3′) (shaded boxes) flank the rga2 ORF. pot-rga2 expresses rga2 under the constitutive otef promoter (solid arrow). The thick lines represent the plasmid backbone. (B) Combination of either FB1/pR-rga2#1 or FB1/pR-rga2#2 with GF5. (C) Combinations of MF18 or MF18/pot-rga2 with FB2 (top panels) and MF34 or MF34/pot-rga2 with FB2 (bottom panels). #1 and #2 refer to the two MF18/pot-rga2 or MF34/pot-rga2 strains (A). (B and C) Note the additional appearance of the X and/or W bands in dependence on rga2 expression in the a1 partner. Lanes clearly showing X2 are denoted by open triangles. Maize plants were inoculated with the combinations indicated at the top of the panels for RFLP analysis. See legend of Figure 2 for further details. All lanes in all panels are from the same blot.

Influence of lga2 and rga2 on mitochondrial fusion in F/W combinations. (A) FB1/pKS2 combined with GF5/pKS1. (B) FB1/pKS2 combined with GF5Δlga2/pKS1. (C) FB1/pKS2 combined with GF5Δrga2/pKS1. Mating was performed with cells of opposite mating types preloaded with either mtGFP or mtRFP. Hyphae of fused cells were observed by DIC light microscopy and mitochondrial structures were assayed by fluorescence microscopy to detect GFP and RFP. The yellow color in merged images (Me) indicates coincidence. Representative examples are shown. Bars, 5 μm (A and B) and 10 μm (C).