Meiotic Silencing in Dothideomycetous Bipolaris maydis

Abstract

The filamentous ascomycete Bipolaris maydis is a plant pathogen that causes corn leaf blight and has been used in cytological studies of sexual reproduction. In this fungus, when null mutants of each septin are crossed with the wild-type strain, all ascospores derived from the same asci show abnormal morphology. The phenomenon was remarkably similar to the event known as "ascus dominance" in Neurospora crassa, which is known to be caused by MSUD (meiotic silencing by unpaired DNA). However, it is not clear whether B. maydis possesses functional MSUD. The object of this study is to elucidate whether this fungus carries a functional MSUD system that causes ascus dominance in the crosses of septin mutants and the wild-type strain. The results of homozygous and heterozygous crossing tests with mutants, having the insertional CDC10-septin gene sequence into the genome, suggested that the ascus dominance in B. maydis is triggered by the unpaired DNA as in N. crassa. To investigate whether MSUD is caused by the same mechanism as in N. crassa, an RNA-dependent RNA polymerase, one of the essential factors in MSUD, was identified and disrupted (Δrdr1) in B. maydis. When the Δrdr1 strain was crossed with each mutant of the septins, ascus dominance did not occur in all crosses. These results suggest that this ascus dominance is caused by RNA silencing triggered by an unpaired gene, as in N. crassa, and septin genes were affected by this silencing. To date, although MSUD has been found only in Fusarium graminearum and N. crassa, which are classified as Sordariomycetes, this study showed that MSUD is also functional in B. maydis, which is classified as a Dothideomycete. These results showed the possibility that this posttranscriptional regulation is extensively conserved among filamentous ascomycetes.

Keywords: Bipolaris maydis; Cochliobolus heterostrophus; ascospore formation; meiotic silencing; meiotic silencing by unpaired DNA; septin.

Figure 1

Asci and ascospores from crosses between the null mutants of core septin genes and the wild-type strain. The null mutants of core septin genes were crossed with the wild-type strain. After 4 weeks, formed pseudothecia were harvested and dissected to observe asci and ascospores. Bars: 50 µm.

Figure 2

Generation and phenotype of mutants by the insertion of a CDC10 sequence. (A) Schematic illustration of the introduction of linearized pZGCDC10c and the location of primers. The CDC10^comp and the cdc10^fs were generated by the insertion of each cassette into the Δcdc10 allele of the Δcdc10 strain. (B) PCR results for confirmation of the introduction of each CDC10 cassette. Products were amplified by the primer set f6/r6 (W and WE: 1104 bp; F and C: 2321 bp). M, λ/StyI digest; W, wild type; D, null mutant; F, cdc10^fs ; C, CDC10^comp ; EC, CDC10^ect ; FE, CDC10^fsect ; N, negative control (TE buffer). (C) Colonial growth and conidial morphology of the wild-type strain, the Δcdc10 strain, the cdc10^fs strain, the CDC10^comp strain, the CDC10^ect strain, and the CDC10^fsect strain. Each strain was cultured on CV8A for 7 days. Conidia of each strain were harvested from a colony of each strain on CV8A and stained with CFW. Bars: 50 µm. (D) Morphology of ascospores. Each strain was crossed with the wild-type strain on Sachs media. Bars: 50 µm.

Figure 3

Identification of the MSUD-related RdRP and generation of the RDR1 disruption mutant in B. maydis. (A) Phylogenetic analysis of the RdRP protein sequence. A neighbor-joining tree was constructed based on the conserved domain of the RdRP. The locus tags of proteins used in this analysis are listed in Supplementary Table S2. The main three groups—quelling, MSUD, and unknown—are divided into different color zones. (B) Colonial morphology of the Δrdr1 strain. The wild-type strain and the Δrdr1 strain were cultured on CV8A for 7 days. (C) Asci and ascospores from the cross between the Δrdr1 strain and the wild-type strain. The Δrdr1 strain was crossed with the wild-type strain. After 4 weeks, resulting pseudothecia were harvested and dissected to observe the asci and ascospores. Bars: 50 µm.

Figure 4

Asci and ascospores from the cross between the Δrdr1 strain and the Δcdc10 strain. The Δrdr1 strain was crossed with the Δcdc10 strain. After 4 weeks, resulting pseudothecia were harvested and dissected to observe the asci and ascospores. Bars: 50 µm.

Figure 5

Asci and ascospores from crosses between the Δrdr1 strain and null mutants of the CDC3, CDC11, and CDC12 genes. The Δrdr1 strain was crossed with the null mutants of septins. After 4 weeks, resulting pseudothecia were harvested and dissected to observe the asci and ascospores. Bars: 50 µm.