Fig 1.
Survival of heterokaryons depends on TER export from the TER + nucleus.
A. The asexual life cycle of Aspergillus nidulans includes uni- and multi-nucleate stages. B and C. Diagrams represent heterokaryotic cells on selective media lacking UU. B. 1) If TER (blue) is exported from the TER + nucleus, 2) it can join with trtA (green) in the cytoplasm. 3) Functional telomerase enters both TER + and TERΔ::pyrGAf; pyrG89 nuclei to maintain telomeres. 4) The TERΔ::pyrGAf; pyrG89 nucleus transcribes orotidine-5’-phosphate decarboxylase (pyrG), producing uracil and uridine (purple) for the cell. The cell survives through this heterokaryotic relationship. C. 1) If TER is not exported from the TER + nucleus, 2) trtA in the cytoplasm remains incomplete and 3) can only function in the TER + nucleus. The TERΔ nucleus cannot replenish its telomeres, and ultimately the cell will die, 4) even if it produces uridine and uracil.
Fig 2.
Generation of TER knockout construct using selective marker pyrGAf.
A. Schematic of the TER knockout construct generation and subsequent gene replacement. The TER locus (red) is replaced with the Aspergillus fumigatus pyrG gene (pyrGAf, gray) through fusion PCR. This pyrGAf gene synthesizes uridine and uracil, compensating for the pyrG89 mutation in the parent SO451 A. nidulans strain. B. PCR amplification of the construct fragments. Lane 2 shows the pyrGAf-containing fragment (~1700 bp, gray), while lanes 4 and 5 display the left (~1000 bp, blue) and right (~1000 bp, purple) flanking regions, all amplified to the expected sizes. No template DNA (-) in the PCR reaction. C. The TER knockout construct formation. Lane 2 shows the complete TER knockout construct, ~ 3500 bp. Faint bands between 2,000 and 3,000 bp indicate unligated fragments.
Fig 3.
Expected results from the heterokaryon test.
Conidia from transformants are plated on A. nonselective media containing UU and B. selective media lacking UU. Bubbles in each sector represent expected growth for conidia of each genotype shown in the legend, with circles representing conidia and white lines indicating growth. 1) Heterokaryon with Essential Gene Knockout: Nonselective Media (A): Healthy growth due to the presence of the essential gene and UU supplementation. Selective Media (B): Weak, sickly brown growth, as pyrGAf conidia cannot maintain telomeres without UU. No growth of conidia containing the essential gene due to lack of UU. 2) Nonessential Gene Knockout: Nonselective Media (A) and Selective Media (B): Healthy growth of pyrGAf conidia, indicating the gene of interest is nonessential. 3) Diploid Formation with Essential Gene: Nonselective Media (A) and Selective Media (B): Healthy growth on both media types, as diploid conidia can grow regardless of UU presence. 4) Heterologous Recombination: Nonselective Media (A) and Selective Media (B): Healthy growth, as conidia contain nuclei with both genes due to pyrGAf integration elsewhere in the genome, somewhere outside of the target gene locus. This haploid conidium grows well on both media types because it contains wild-type copies of both essential genes.
Fig 4.
Heterokaryon tests of trtAΔ and TERΔ primary transformant colonies.
The heterokaryon tests demonstrate distinct growth patterns between TERΔ and trtAΔ primary transformant colonies, indicating heterokaryon formation in trtAΔ but not in TERΔ. Healthy growth is observed on the nonselective (+UU) plates for both TERΔ (sectors 1 and 2) and trtAΔ (sectors 3 and 4) samples. On the selective (-UU) plate, TERΔ colonies exhibit healthy growth, while trtAΔ colonies show severely impaired growth. This lack of growth in the trtAΔ background indicates the formation of a trtAΔ heterokaryon, but TERΔ colonies did not form heterokaryons.
Table 1.
TERΔ and trtAΔ transformant types verified by Heterokaryon (Het) Test and Integration Check (IC) PCR.
Fig 5.
Integration check PCR validates heterokaryon and diploid test results.
A. Schematic of PCR design for TERΔ transformants. The upper arrow represents the wild-type TER + amplicon (~1,600 bp), while the lower arrow corresponds to the TERΔ::pyrGAf amplicon (~2,300 bp). B. Gel electrophoresis of representative PCR products. Lane 1: Quick-Load Purple 1 kb Plus DNA Ladder. Lanes 2 and 3: The same TERΔ transformant having different amplicons in Lane 2 TER + (~1,600 bp) and Lane 3 TERΔ::pyrGAf (~2,300 bp). Lanes 4 and 5: trtA (~1,900 bp) and trtA::pyrGAf (~2,900 bp) amplicons from the trtAΔ heterokaryon. Lanes 6 and 7: Same amplicons as lanes 4 and 5 but from trtAΔ diploids. Lanes 8 to 13: Parent SO451 controls using trtAΔ and TERΔ primers on SO451 control DNA, with the pyrGAf amplicon absent as it has not been knocked in. Lane 12: Protection of Telomeres (POT1) gene as a PCR positive control. Lane 13: Negative no template control.
Fig 6.
DAPI staining reveals larger nuclei in TERΔ diploids.
A. Representative DAPI-stained conidia from a TERΔ diploid transformant colony and a parent haploid SO451 colony. The DAPI (left) and merge (right) images are shown for each conidium. B and C. Bar graphs depicting the (B) mean intensity and (C) mean volume (nucleus size) for TERΔ (shades of blue) and the haploid SO451 strain (red/orange) with standard error bars. Results show that TERΔ transformants have nuclei nearly double the size of the SO451 DAPI-stained nuclei. The DAPI stain intensity for the TERΔ background is approximately 1.5 times that of the SO451. ( * = p < 0.05, ** = p < 0.005).