A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in allorecognition in Neurospora crassa

Abstract

Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr, encodes two linked genes, cwr-1 and cwr-2. Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the PMO domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein-protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR1 PMO domain.

Evolutionary genetics of nonself recognition regarding social and sexual interactions of fungi

“…The interaction between doc alleles seems to involve cell wall receptors or ligands, and differences at the doc locus downregulates fusion (Heller et al, 2016). A second pathway seems to involve the cwr genes, a chitin lytic polysaccharide monooxygenase, which are also highly polymorphic and where allelic differences also downregulate cell fusion (Detomasi et al, 2022). While these two pathways have similar outcomes, limiting sustained fusion between nonself, the mechanisms are likely very different.…”