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

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 (cell  wall remodeling), 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 catalytic (PMO) domain of CWR-1 was sufficient for checkpoint  function and cell fusion blockage; however, through analysis of active-site, histidine29 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 CWR-1 PMO domain.