Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype

Abstract

Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa, and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so/ham-1, and show that it is the absence of this gene-and not of ada-3-that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures. IMPORTANCE This study emphasizes the need for careful and detailed characterization of strains from mutant collections. Specifically, we found a common deletion in various strains from the Neurospora crassa gene knockout collection that results in a cell fusion-defective phenotype. This is noteworthy because this collection is known to contain background mutations-of a largely unclear nature-that produce cell fusion-defective phenotypes. Our results describe an example of such mutations, and highlight how this common genetic defect could have impacted previous studies that have used the affected strains. Furthermore, they provide a cautionary note about the use of Neurospora strains with similar phenotypes. Lastly, these findings offer additional details relevant to our understanding of the origin and spread of cell fusion-defective cheater variants in N. crassa cultures.

Keywords: Neurospora crassa; cell fusion; gene knockout; secondary mutations.

Fig 1

The absence of ada-3 is not responsible for the ADA phenotype of FGSC 11070. (A) (left) Strains were imaged after culture on slants of VMM for 7 days at 25°C under constant light. Clones #52 and #71 (Δada-3, a) were obtained by crossing 11070 with a WT strain (a his-3+ derivative of FGSC 9720, as female), after which germinated ascospores were individually transferred to slants of VMM + 200 µg/mL hygromycin, as described in Materials and Methods. (Right) Genomic DNA (gDNA) was extracted from the strains listed, and PCR was used to evaluate the presence of either ada-3 or the hph cassette at the ada-3 locus. Note that even though both reactions were tested per homokaryotic strain, each strain can only be positive for either the WT gene or the KO cassette, but not both. The arrows denote the position of the primers used (see Table 1). (B) For crosses, strains used as females were first grown on synthetic crossing medium for 7 days at 25°C under constant light, after which they were fertilized with conidia from different strains with the opposite mating type (i.e., males) and incubated under the same conditions for an additional 7 days before imaging. Reciprocal crosses were set up for each strain pair, so that every strain could be tested for both female and male fertility. For every cross, the strain used as female is listed first (i.e., female x male).

Fig 2

The ADA phenotype of FGSC 11070 is due to the absence of so/ham-1. (A) Integrative Genomics Viewer image of a portion of linkage group I (LG I), showing coverage tracks (blue, green) for the eight N. crassa strains sequenced and the corresponding location of annotated genes (orange). Four independent WT-like Δada-3 strains (blue, WT) and four independent ADA-like Δada-3 strains (green, 11070) were sequenced. (B) Phenotypic analysis of candidate strains. The strains shown were imaged after culture on slants of VMM for 7 days at 25°C under constant light. FGSC 11292, 18264, and 21640 correspond to full deletion strains of NCU02794, NCU02793, and NCU10987, respectively. (C) Complementation assay. WT, 11070, and a 11070 his-3 strain transformed with Pccg-1-so-GFP (564tss05), Pso-so-GFP (565tss05), or pBM61 (empty vector) (see Materials and Methods), were grown on slants of VMM for 7 days at 25°C under constant light before imaging. (D) The same strains as in (C) were tested for female fertility. To do that, the strains were first grown on synthetic crossing medium for 7 days at 25°C under constant light, after which they were fertilized with conidia from WT of the opposite mating type (i.e., males). The crosses were incubated under the same conditions for an additional 7 days before imaging. For every cross, the strain used as female is listed first (i.e., female × male).

Fig 3

Strains FGSC 12957 and 12958 also harbor the ~30-Kb deletion. (A) PCR analysis to evaluate the presence of so/ham-1 or the ~30-Kb gap in the genome of the strains listed. To detect so/ham-1, primers within its coding region were used. To evaluate the presence of the deletion, primers flanking the ~30-Kb gap were used such that under normal conditions (i.e., no deletion), the size of the DNA fragment between the primers (~30 Kb) would be too large to be amplified with the PCR settings used. Conversely, if the deletion is present, a PCR product could be obtained under the conditions used. We used a nested PCR strategy to detect the gap (Table 1). Note that a homokaryotic strain can either be positive for so/ham-1 or harbor the deletion, but not both. As such, even though all strains were tested for both targets, only one can be detected. (B) (Left) Phenotypic analysis. WT, FGSC 12957, FGSC 12958, and two WT-like Δacw-4 progeny derived from crossing WT with 12957 (xc1478, clones 21 and 22) were imaged after culture on slants of VMM for 7 days at 25°C under constant light. (Right) Schematic diagram and genotyping of the strains on the left, to confirm the Δacw-4 status (and concomitant replacement of acw-4 with the hph cassette) of the 12957, 12958, and xc1478 strains. Note that even though both reactions were tested per homokaryotic strain, each strain can only be positive for either the WT gene or the KO cassette, but not both. (C) PCR analysis to evaluate the presence of so/ham-1 or the ~30-Kb gap in the genome of the strains shown in (B), with 11070 as reference for the deletion. Note that a homokaryotic strain can either be positive for so/ham-1 or harbor the deletion, but not both. All primer sequences are shown in Table 1.

Fig 4

FGSC 11372, an rco-1 KO strain, harbors the ~30-Kb deletion. (A) (Left) Phenotypic analysis of WT and the two rco-1 KO strains generated as part of Neurospora Functional Genomics Project, FGSC 11371 and 11372. The strains were imaged after culture on slants of VMM for 7 days at 25°C under constant light. (Right) Schematic diagram and genotyping of the strains on the left, to confirm the Δrco-1 status (and concomitant replacement of rco-1 with the hph cassette) of the 11371 and 11372 strains. Note that even though both reactions were tested per homokaryotic strain, each strain can only be positive for either the WT gene or the KO cassette, but not both. (B) Growth rate assay. Conidia from the strains listed were inoculated on race tubes containing VMM and were then placed under constant light conditions at 25°C. The growth front was marked on the tubes every 24 h, and the distance between the marks was then used to calculate the linear growth rate per day. Dots represent data from four to six independent biological replicates. Shown are the mean ± 95% confidence intervals. Analysis of variance, F (3, 18) = 257.5, P < 0.0001. Different letters indicate statistically significant differences between groups (Tukey's Honest Significant Difference , P < 0.05). All statistical analyses were performed using GraphPad Prism v. 9.5.1 (733). (C) PCR analysis to evaluate the presence of so/ham-1 or the ~30-Kb gap in the genome of the strains shown in (B). Note that a homokaryotic strain can either be positive for so/ham-1 or harbor the deletion, but not both. All primer sequences are shown in Table 1.