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Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of amil, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that amil is involved in nuclear distribution during the sexual cycle and that alteration of amil delays the fruiting-body development. We also demonstrate that amil alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an amil mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the amil mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.

The heterothallic fungus Podospora anserina has two mating-type alleles termed mat+ and mat-. The mat+ sequence contains one gene, FPR1, while mat- contains three genes: FMR1, SMR1, and SMR2. FPR1 and FMR1 are required for fertilization, which is followed by mitotic divisions of the two parental nuclei inside the female organ. This leads to the formation of plurinucleate cells containing a mixture of parental mat+ and mat- nuclei. Further development requires a recognition between mat+ and mat- nuclei before migration of the mat+ / mat- pairs into specialized hyphae in which karyogamy, meiosis, and ascospore formation take place. FPR1, FMR1, and SMR2 control this internuclear recognition step. Initial development of the dikaryotic stage is supposed to require SMR1; disruption of SMR1 results in barren perithecia. In a systematic search for suppressors restoring fertility, we isolated 15 suppressors--all of them mutations in the mating-type genes. These fmr1, smr2, and fpr1 mutants, as well as the strains disrupted for FMR1, SMR2, and FPR1, are weakly self-fertile. They are able to act as the male partner on a strain of the same mating type and give a mixture of biparental and uniparental progeny when crossed with a wild-type strain of opposite mating type. These observations lead us to propose that SMR2, FMR1, and FPR1 act as activators and repressors of fertilization and internuclear recognition functions.

The mating types and their role in the sexual development of the filamentous ascomycetes are summarized. Bipolar heterothallism is a distinguishing feature of filamentous ascomycetes. The mating types of Neurospora crassa, Podospora anserina and Cochliobolus heterostrophus comprise unconnected and unique sequences. The presence of putative DNA binding domains in the proteins encoded by the mating-type genes indicates that there are transcriptional fators.

Mating type behavior in Podospora anserina was examined to determine mat+ and mat- nuclei compartmentalization factors and the impact of mating type locus. In vitro mutagenesis and transformation experiments yielded mat gene strains with interaction that indicated dikaryotic state development based on mat information. Other ascus information generated are selfishness for nucleus with mating-type mutation and karyogamy, meiosis and sporulation processes with absent opposite mating types.