WD-repeat instability and diversification of the Podospora anserina hnwd non-self recognition gene family

Abstract

Background: Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus Podospora anserina, three genes controlling this allorecognition process het-d, het-e and het-r are paralogs belonging to the same hnwd gene family. HNWD proteins are STAND proteins (signal transduction NTPase with multiple domains) that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different P. anserina isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members.

Results: Herein, we directly analyzed the genetic instability and diversification of this allorecognition gene family. We have constituted a collection of 143 spontaneous mutants of the het-R (HNWD2) and het-E (hnwd5) genes with altered recognition specificities. The vast majority of the mutants present rearrangements in the repeat arrays with deletions, duplications and other modifications as well as creation of novel repeat unit variants.

Conclusions: We investigate the extreme genetic instability of these genes and provide a direct illustration of the diversification strategy of this eukaryotic allorecognition gene family.

Figure 1

Constitution of the RV collection of mutants. A/Schematic representation of het-e and het-r alleles at the hnwd loci under study in the het-R het-V and the het-C het-E self incompatible strains. Plain arrowheads indicate non allelic incompatible interactions. B/Genesis and selection of new alleles at hnwd loci. During vegetative growth, the WD40 repeat instability promotes the rapid genesis of allelic variants at the hnwd loci for selection to act on. Our experimental set up selects for loss of function mutations while in natural conditions in the wild, selection will promote the maintenance of new recognition specificities. C/Selection of loss of function mutants in our experimental set up. Basically, SI strains were grown for 24 h in permissive condition before incubation for five days in non permissive conditions. Arrows indicate mutant sectors escaping from cell death from a single culture, as opposed to a culture where no mutant sector appeared. A single mutant per subculture was picked up for further analysis.

Figure 2

Classes of mutants identified by the number of WD40 repeat units. A/The WD domain of the het-r gene of a selection of mutants from the RV collection were PCR amplified and separated on an 0.8% agarose gel (molecular marker: 1 kb ladder). The sizes of the amplicons are compared to the natural inactive (het-r) and active (het-R) alleles. B/Three classes of mutants are defined according to the size of the WD repeat domain of the het-r or the het-e gene: the deletion mutants (dWD), the gain of WD40 repeat mutants (gWD) and the mutants with the same number of WD40 repeats as the initial allele (snWD). Subclasses of mutants are defined according to the number of WD40 repeat units deleted (-) or gained (+) compared to the initial allele. The percentage of the total number of mutants comprised in each subclass is represented.

Figure 3

Mapping of the WD40 repeat deletions. A/On this schematic representation of the het-R WD repeat domain dashed lines delineate the WD40 repeat sequences and BglII restriction sites are labelled B1 to B6. At the top are indicated the size of the expected BglII generated restriction fragments and below are indicated the size of the non repeat fragments from the end of the amplicon to the beginning of the repeated sequences. B/For the three most represented subclasses of mutants, the intervals in which deletion occur are represented. Top labels refer to the intervals defined (additional file 2). Numbers below each interval represent the number of observed vs the number of expected mutants is generated at random. C/Representative agarose gel presenting the BglII restriction profiles of the wild type het-R allele along with selected mutants. F4, N5 and E4 belong to the -1 subclass, L16 to the -2 subclass and M14 to the -3 subclass.

Figure 4

Schematic representation of the gWD and snWD mutants. A/Parental active het-R allele and mutants from the RV collection. B/Parental active het-E allele and mutants from the CE collection. Nucleic sequence WD40 repeat units sharing the same colour are identical. Repeat units represented in white differ by a single nucleotide from their parental repeat unit (indicated by a number). Repeat units represented in black differ by more than one nucleotide from any parental units. Stop codons are indicated by arrowheads. Incomplete boxes of the e2 mutant delineate a 21 bp in frame deletion.