Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Abstract

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Figure 1

Localization of grisea on a 4.5-kb genomic SmaI fragment of P. anserina. The ORF of grisea is indicated by an arrow, the single intron is shown by the solid bar in this reading frame. The position and the nature of four sequence alterations in the mutant gene copy of grisea are indicated and correspond to the changes shown in the sequencing gels in the lower part of the figure. Arrows indicate three transitions and the dot one nucleotide addition. The numbers correspond to the positions of the corresponding nucleotides with respect to the the start codon of the grisea ORF (position +1).

Figure 2

Reverse transcription-coupled PCR analysis demonstrating a splicing defect in mutant grisea. RNA was isolated from the wild-type strain (s), from mutant grisea grown on medium with or without additional copper and from a transformant of grisea displaying wild-type characteristics due to an additional wild-type gene copy of grisea. Amplification was performed by using a specific pair of synthetic primers (un51 and un64) located upstream and downstream of the grisea intron. The four cDNA regions show the results from the PCR amplification after reverse transcription of RNA samples. Whereas the intron is almost completely spliced out of the pre-mRNA from the wild-type strain, no splicing product was detected in cDNA preparations of mutant grisea grown on medium with or without additional copper. The transformant gives rise to amplification products, 241 bp and 301 bp long, related to the spliced and unspliced RNA, respectively. The four RNA panels show the results of amplification of RNA prior to cDNA preparation (negative control).

Figure 3

Complementation assay demonstrating that a mutation in the single grisea intron leads to a loss of function. (A) A physical map of the genomic grisea locus is presented at the top. Various plasmids were constructed and used to complement long-lived mutant grisea to wild-type characteristics. In plasmids pPgr⁻6-7, pPgrP⁻6-7, and pPgrI⁻2-6, point mutations corresponding to the mutations identified in the mutant grisea allele are indicated. The right column summarizes the results of the transformation experiments. (B) Morphology of the untransformed recipient strain grisea (gr), the wild-type strain s (s), a colony regenerated from protoplasts of grisea that were not incubated with DNA of plasmid pPgrP⁻6-7 (R), and independent hygromycin B-resistant transformants of grisea. Sixty-three percent of the transformants (dark colonies) show the typical wild-type morphology.

Figure 4

Southern blot analysis of the plDNA region of mtDNA from different P. anserina cultures. Total DNA digested with BglII was fractionated on an agarose gel and hybidized with a plDNA-specific probe. The localization of the probe (pl) in the mtDNA region of the cytochrome oxidase subunit I gene (COI) and a BglII restriction map is shown in the scheme. DNA was isolated from independent cultures of the wild-type strain race s (s1, s2), long-lived mutant grisea (gr1, gr2, gr3), and grisea transformants (T1, T2) that, due to the transformation with the grisea wild-type gene copy, are complemented to wild-type characteristics. DNA was derived from juvenile (juv), middle aged (m.a.), and senescent (sen) cultures, respectively.

Figure 5

Southern blot analysis of the P. anserina mtDNA regions that do not overlap the plDNA region. (Upper) Locations of the cloned DNA probes used in the hybridization experiments are shaded. (Lower) Hybridization experiments. Fragment numbers are according to ref. . Total DNA was derived from juvenile (juv), middle aged (m.a.), and senescent (sen) cultures of the wild-type and mutant grisea. DNA was digested with BglII and fractionated on an agarose gel.