Characterization of fsd-1 Mutant Alleles in Neurospora crassaMark Soto and Elizabeth Hutchison

Biology Department, SUNY Geneseo, Geneseo, NY 14454

Introduction:• NDT80 is a key meiotic transcription factor in Saccharomyces

cerevisiae, and NDT80 homologs are present in filamentous fungi [6].

• fsd-1, a homolog of NDT80, has been identified as an integral regulator of sexual development in Neurospora crassa [4].

• N. crassa, a well-studied model organism, is a filamentous ascomycete fungus that can undergo asexual or sexual reproduction [1].

• To further understand the fsd-1 gene and its role in N. crassa, we will construct mutant alleles of fsd-1 and assess whether these mutations affect fsd-1 function.

• In S. cerevisiae, several Ndt80 loss-of-function mutations have been shown to induce a decrease in both DNA binding and sporulation [2][5].

• Our study focuses on mutating amino acid residues located in the DNA binding domain and the carboxyl terminus.

• To accomplish this, primers will be designed and used in double-joint polymerase chain reaction, a method used to construct alleles that target to the native locus without the need for subcloning [3].

• Along with the mutation is the green fluorescent protein tag for protein localization study, and hygromycin resistance for selection purposes.

Stay happy, you have incredible value- Barnabas Gikonyo

Procedure:• Identified several conserved amino acid residues in various

fungal strains using the Biomatters program Geneious.• Targeted these residues for mutation and constructed forward

primers that were used to amplify the final fragment in Double Jointed-PCR (DJ-PCR).

• Used DJ-PCR to construct mutant alleles (Figure 1) [3].

• Transformed into N. crassa strain P8-43 (rid his-3; Dmus-52::bar A), and grown on Vogel’s minimal media and histidine.

• Isolated transformants and grew them on minimal medium.

Acknowledgements:The authors gracefully acknowledge financial support from the McNairScholars Program, materials and logistical support from the BiologyDepartment, and Dr. Patrick Shiu for strain P8-43.

References:1. Davis, R. H. and D. D. Perkins. 2002. Neurospora: a Model of Model

Microbes. Nat Rev Genet. 3:397.

2. Fingerman, I. M., Sutphen, K., & Montano, S. P. 2004. Characterization of

Critical Interactions between Ndt80 and MSE DNA Defining a Novel Family of Ig-

fold Transcription Factors. Nucleic Acids Research 32(9): 2947-2956.

3. Hammond, T. M., Xiao, H., & Rehard, D. G. 2011. Fluorescent and

Biomolecular-Fluorescent Protein Tagging of Genes at their Native Loci in

Neurospora crassa Using Specialized Double-Joint PCR Plasmids. Fungal

Genetics and Biology 48: 866-873.

4. Hutchison E. A., & Glass N. L. 2010. Meiotic Regulators Ndt80 and Ime2

Have Different Roles in Saccharomyces and Neurospora. Genetics 185(4):

1271-1282.

5. Montano, S. P., Coté M. L., & Fingerman, I. 2002. Crystal structure of the

DNA-binding Domain from Ndt80, a Transcriptional Activator Required for

Meiosis in Yeast. PNAS 99(22): 14041-14046.

6. Winter, E. 2012. The Sum1/Ndt80 Transcriptional Switch and Commitment to

Meiosis in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 76:1-15.

Figure 3. Sequence alignment of fsd-1 homologs. The purple arrow indicates the DNA binding domain inthe yeast, Saccharomyces cerevisiae. The region preceding this domain represents the amino terminus, andfollowing this domain is the carboxyl terminus. Similarity of color in specific columns indicate highly conservedamino acid residues. Black boxes indicate choice of mutation. Primer design referenced these sequences.

Figure 4. DJ-PCR method to obtain mutant alleles. Wild-type N. crassa genomic DNA was the template for all

PCR reactions. (A) DJ-PCR left fragments were amplified using the Invitrogen Platinum High Fidelity Supermix. From each50 μl reaction product, approximately 20 μl were examined by gel-electrophoresis on a 1% agarose/TBE gel. The singlepredominant band in each lane matched the expected band size listed in Table 1. Note: Step 2 of DJ-PCR was performedand products were amplified by the Roche Expand Long Range dNTPack. Nested primers were used to amplify full-lengthfragments (Step 3). (B) Full-fragment DJ-PCR products were examined under the same conditions. Multiple non-specificproducts were observed, however, bands of interest are indicated by red asterisks and match the predicted sizes.

Table 1

Figure 1. A three step process of DJ-PCR. Three fragments are required for DJ-PCR. The left flank which contains the incorporated mutation, the center which contains a GFP tag and Hygromycin resistance marker, and a right flank. Step 1. Amplify and extract the separate fragments by gel-purifying. Step 2. Perform a PCR reaction using the left flank, center, and right flank, but lacking conventional primers. Due to partial complementarity between the end of the flank fragments and center, these will serve as primers for each other and fuse properly (joint 1 and joint 2). Step 3. Perform another PCR reaction using product from Step 2 and incorporate mutated, nested primers. Nested primers (np1 and np2) will be used to amplify the final fragment.

Figure 2. Localization of GFP Labeled FSD-1. GFP fluorescence shown in green, and DNA shown in blue

(DAPI staining). The corresponding phase contrast image is shown on the right. FSD-1-GFP localizes to nuclei. Seven days post-cross, with red arrows indicating a crozier. No FSD-1-GFP expression observed in croziers, but it is expressed within paraphyses tissue. (Data from Thomas Hurysz)

Preliminary Findings:

Results:

Future Directions:• Transformants will be screened for the correct genotype

(1) Using PCR to amplify the region surrounding the introduced mutation(2) Send PCR products for sequencing or use restriction enzyme digest to test for the presence of the mutant allele.

• Strains with correct mutant allele genotype will be crossed to obtain homokaryotic strains (strains with a single genotype).

• Strains with mutant alleles will be assessed for complementation and their phenotype will be analyzed

(1) Their ability to be crossed will be determined(2) Visualize FSD-1-GFP localization during development(3) Whether mutant strains have an ascosporic maturation defect will also be determined.

Figure 5. Transformation of mutants. (C) Flasks of strain P8-43, with genotype rid his-3; mus-52Δ::bar A, were inoculated with N. crassa conidia. Strain P8-43 was grown in a 500 ml flask containing 100 ml of Vogel’s minimal media at 30⁰C for a week, and another week in a lighted environment at room temperature. (D) Post-plating, transformants were picked using syringe needles and inoculated onto new minimal medium slant tubes. Approximately 30 transformants were isolated for each mutant allele.

R125A H182A K188A K260A S483A

base pairsbase pairs

R125A H182A K188A K260A S483Aladder ladderA B

603

234194

125

72

872

1,078

310

603

2,0272,322

4,3616,5579,416

2,0272,322

* * * * *

Results: