Ament-Velásquez S Lorena, Furneaux Brendan, Dheur Sonia, Granger-Farbos Alexandra, Stelkens Rike, Johannesson Hanna, Saupe Sven J
Department of Zoology, Stockholm University, 106 91 Stockholm, Sweden.
Department of Biological and Environmental Science, University of Jyväskylä, 40014 Jyväskylä, Finland.
Microb Genom. 2025 Jul;11(7). doi: 10.1099/mgen.0.001442.
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 and NLRs in are highly polymorphic incompatibility genes ( 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 and incompatibility reactions. AlphaFold 3 structure models suggest that their WD40 domain folds into two 7-blade -propellers composed of the highly conserved repeats, as well as three cryptic divergent repeats at the C-terminus. We additionally show that one particular allele does not have an incompatibility reaction with common alleles, despite being 11-repeats long. Finally, we present evidence that the recognition phenotypes of and arose through convergent evolution. Our findings provide a robust foundation for future research into the molecular mechanisms and evolutionary dynamics of NLRs, while also highlighting both the fragility and the flexibility of -propellers as immune sensor domains.
NOD样受体（NLRs）是细胞内免疫受体，可检测病原体相关信号并触发防御机制，包括程序性细胞死亡。在丝状真菌中，一些NLRs介导异核体不相容性，这是一种自我/非自我识别过程，可防止遗传上不同个体的营养体融合，降低寄生风险。禾本科植物中的A和B NLRs是高度多态的不相容基因（het基因），其产物通过由WD40重复序列组成的传感结构域识别HET-C蛋白的不同等位基因变体。这些重复序列通过协同进化保持异常高的序列同一性。然而，单个重复序列中的一些位点是高度可变的，并且处于多样化选择之下。尽管进行了广泛的遗传学研究，但报道的WD40结构域序列的不一致性阻碍了功能和进化分析。在这里，我们证实WD40结构域可以从长读长测序（牛津纳米孔和PacBio）数据中准确重建，但不能从基于Illumina的组装中重建。功能等位基因通常由11个高度保守的重复序列形成，相同表型的A和B不相容反应具有不同的重复组合。AlphaFold 3结构模型表明，它们的WD40结构域折叠成两个由高度保守的重复序列组成的7叶β-螺旋桨，以及C末端的三个隐蔽的 divergent重复序列。我们还表明，一个特定的A等位基因尽管有11个重复序列长，但与常见的B等位基因没有不相容反应。最后，我们提供证据表明A和B的识别表型是通过趋同进化产生的。我们的发现为未来研究禾本科植物NLRs的分子机制和进化动力学提供了坚实的基础，同时也突出了β-螺旋桨作为免疫传感结构域的脆弱性和灵活性。
