A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition
- University of California, Berkeley, United States
- National Cheng Kung University, Taiwan
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, 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 CWR-1 PMO domain.
Data availability
Materials AvailabilityAll strains and plasmids listed in Supplementary file 1a, b and d are available upon request or from the Fungal Genetics Stock Center (https://www.fgsc.net). Primers used in this study are listed in Supplementary file 1c. P value data for Figures 1, 3, 5, 6 and Figure 1-figure supplement 3B are provided in the Figure 1-source data 4, Figure 3-source data 1, Figure 5-source data 5, Figure 6-source data 1, Figure 6-source data 2 and Figure 1-figure supplement 3-source data 1. Data for biochemical analyses of CWR-1 are provided in Figure 1-source data 3; Figure 4-source data 4; Figure 5-source data 4 (HRP oxygen reduction assays); Figure 1-source data 2; Figure 4-source data 3; Figure 5-source data 3 (ICP); Figure 1-source data 1; Figure 4-source data 1; Figure 4-source data 2; Figure 4-source data 5; Figure 4-source data 6; Figure 4-source data 7; Figure 5-source data 1, Figure 5-source data 2, Figure-1-figure supplement 5-source data 1; Figure 1-figure supplement 5-source data 1; Figure 4-figure supplement 1-source data 1 (HPAEC-PAD traces). Data for construction of the SSN is provided in Supplementary file 1g and the raw data as Figure 1-figure supplement 2-source data 1. Original data for Figure 1-supplement 4A are provided as Figure-1-figure supplement 4-source data 1. Whole protein MS data are provided in Figure 1-figure supplement 4-source data 2, Figure 5-figure supplement 1-source data 1. EPR source data are provided in Figure 5-figure supplement 2-source data 1. Tandem MS data are provided in Figure 1-figure supplement 6-source data 1.
Article and author information
Author details
Adriana M Rico Ramírez
Michael A Marletta
Funding
National Science Foundation (MCB 1818283)
- Tyler C Detomasi
- Adriana M Rico Ramírez
- Richard I Sayler
- Michael A Marletta
- N Louise Glass
National Science Foundation (CHE-1904540)
- Tyler C Detomasi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Detomasi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition
eLife 11:e80459.
https://doi.org/10.7554/eLife.80459
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