SXI1α controls uniparental mitochondrial inheritance in Cryptococcus neoformans

In the great majority of sexual eukaryotes, mitochondria are inherited almost exclusively from a single parent [1,2]. However, the mechanisms for uniparental mitochondrial inheritance remain largely unknown. Here, we identify a gene that controls mitochondrial inheritance in the fungus Cryptococcus neoformans. Strains of the basidiomycete yeast C. neoformans are predominantly haploid and exist in one of two sexes (mating types), a and α [3]. We recently discovered that mtDNA was almost exclusively transmitted from the a-parent in sexual crosses of C. neoformans [4]. Genomic sequences for the MATa and the MATα alleles were recently obtained from two strains each [5]. Sequence comparisons indicated that the sex-determining homeodomain gene SXI1α is present only in MATα and that this is the only difference between MATa and MATα [6]. To test the role of SXI1α in mtDNA inheritance, we used a series of strains and crosses (Tables 1 and 2). For each cross, cells from two genetically marked haploid strains of opposite mating type were mixed [3] and, after 16–20 hour incubation, transferred to selective media, which allow only growth of diploid zygotes. Randomly picked zygote colonies were genotyped at two mitochondrial loci: NADH dehydrogenase subunit 2 (NAD2) and subunit 4 (NAD4), using restriction enzyme digests of the PCR products [4] (Supplemental Data). Similar to previous studies [4,7], crosses involving wild type aand α-strains produced zygotes containing mtDNA almost exclusively from the a-parent (Table 2). Neither the mitochondrial genomes nor the auxotrophic markers used for selection influenced mitochondrial inheritance. In contrast, zygotes from matings between a wild-type a strain and a sxi1α∆ mutant strain (CHY618) showed biparental mitochondrial inheritance, significant heteroplasmy, and a recombinant mtDNA genotype (Table 2; Supplemental Data). Heteroplasmic zygotes included the mitochondrial genes NAD2 and NAD4 from both parental strains. As shown in cross 4 (Table 2), the change in mtDNA inheritance was not due to the introduction of the nourseothricin (NAT) resistance gene used to disrupt the SXI1α gene (Tables 1 and 2). Reintroduction of the wild-type SXI1α gene into the sxi1α∆ strain reestablished uniparental mtDNA inheritance (Table 2). This change was not due to the introduced transforming vector DNA (Table 2, cross 6). This clearly shows that SXI1α controls mtDNA transmission during sexual crosses in C. neoformans. SXI1α is a putative homeodomain transcription factor [6]. Therefore, it is likely to control mtDNA inheritance by regulating other genes. At present, the downstream targets of SXI1α are largely unknown. However, there are two mutually non-exclusive hypotheses. The first one holds that SXI1α controls mtDNA transmission through active degradation of mtDNA in the αparent. Thus, deletion of SXI1α would abolish the degradation activity towards MATα. Such selective degradation of organelle genomes has been observed recently in the green alga Chlamydomonas reinhardtii [8] and the multi-sexual protist Physarum polycephalum [9]. In C. reinhardtii, the chloroplast DNA from the parent of the mtmating type is degraded soon after zygote formation and all progeny inherit chloroplast DNA from the mt+ parent [8]. Similarly, in P. polycephalum, rapid, selective digestion of mtDNA was observed after sexual mating, resulting in uniparental mtDNA inheritance. The selectivity of mtDNA digestion in P. polycephalum has a hierarchical pattern based on mating type alleles [9]. In both organisms, the nucleases, their relationship to the mating type loci, and the mechanisms of control are unknown. Whether a similar process operates in C. neoformans is not known. Alternatively, uniparental mtDNA inheritance in C. neoformans might be the result of