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.
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.
- Tyler C Detomasi
- Adriana M Rico Ramírez
- Richard I Sayler
- Michael A Marletta
- N Louise Glass
- Tyler C Detomasi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Antonis Rokas, Vanderbilt University, United States
© 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.
Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. 13C-tracing revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate in these cells. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at a consistent doubling time of 3.2 days. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.
Posterior urethral valves (PUV) are the commonest cause of end-stage renal disease in children, but the genetic architecture of this rare disorder remains unknown. We performed a sequencing-based genome-wide association study (seqGWAS) in 132 unrelated male PUV cases and 23,727 controls of diverse ancestry, identifying statistically significant associations with common variants at 12q24.21 (p=7.8 × 10−12; OR 0.4) and rare variants at 6p21.1 (p=2.0 × 10-8; OR 7.2), that were replicated in an independent European cohort of 395 cases and 4151 controls. Fine mapping and functional genomic data mapped these loci to the transcription factor TBX5 and planar cell polarity gene PTK7, respectively, the encoded proteins of which were detected in the developing urinary tract of human embryos. We also observed enrichment of rare structural variation intersecting with candidate cis-regulatory elements, particularly inversions predicted to affect chromatin looping (p=3.1 × 10-5). These findings represent the first robust genetic associations of PUV, providing novel insights into the underlying biology of this poorly understood disorder and demonstrate how a diverse ancestry seqGWAS can be used for disease locus discovery in a rare disease.