Reconstructing NOD-like receptor alleles with high internal conservation in <i>Podospora anserina</i> using long-read sequencing

NOD-like receptors (NLRs) are intracellular immune receptors that detect pathogen-associated cues and trigger defence mechanisms, including regulated cell death. In filamentous fungi, some NLRs mediate heterokaryon incompatibility, a self-/non-self-recognition process that prevents the vegetative fusion of genetically distinct individuals, reducing the risk of parasitism. The <i>het-d</i> and <i>het-e</i> NLRs in <i>Podospora anserina</i> are highly polymorphic incompatibility genes (<i>het</i> genes) whose products recognize different allelic variants of the HET-C protein via a sensor domain composed of WD40 repeats. These repeats display unusually high sequence identity maintained by concerted evolution. However, some sites within individual repeats are hypervariable and under diversifying selection. Despite extensive genetic studies, inconsistencies in the reported WD40 domain sequence have hindered functional and evolutionary analyses. Here, we confirm that the WD40 domain can be accurately reconstructed from long-read sequencing (Oxford Nanopore and PacBio) data, but not from Illumina-based assemblies. Functional alleles are usually formed by 11 highly conserved repeats, with different repeat combinations underlying the same phenotypic <i>het-d</i> and <i>het-e</i> incompatibility reactions. AlphaFold 3 structure models suggest that their WD40 domain folds into two 7-blade <i>β</i>-propellers composed of the highly conserved repeats, as well as three cryptic divergent repeats at the C-terminus. We additionally show that one particular <i>het-e</i> allele does not have an incompatibility reaction with common <i>het-c</i> alleles, despite being 11-repeats long. Finally, we present evidence that the recognition phenotypes of <i>het-e</i> and <i>het-d</i> arose through convergent evolution. Our findings provide a robust foundation for future research into the molecular mechanisms and evolutionary dynamics of <i>het</i> NLRs, while also highlighting both the fragility and the flexibility of <i>β</i>-propellers as immune sensor domains.