Identification and characterization of genes required for cell-to-cell fusion in Neurospora crassa

Abstract

A screening procedure was used to identify cell fusion (hyphal anastomosis) mutants in the Neurospora crassa single gene deletion library. Mutants with alterations in 24 cell fusion genes required for cell fusion between conidial anastomosis tubes (CATs) were identified and characterized. The cell fusion genes identified included 14 genes that are likely to function in signal transduction pathways needed for cell fusion to occur (mik-1, mek-1, mak-1, nrc-1, mek-2, mak-2, rac-1, pp2A, so/ham-1, ham-2, ham-3, ham-5, ham-9, and mob3). The screening experiments also identified four transcription factors that are required for cell fusion (adv-1, ada-3, rco-1, and snf5). Three genes encoding proteins likely to be involved in the process of vesicular trafficking were also identified as needed for cell fusion during the screening (amph-1, ham-10, pkr1). Three of the genes identified by the screening procedure, ham-6, ham-7, and ham-8, encode proteins that might function in mediating the plasma membrane fusion event. Three of the putative signal transduction proteins, three of the transcription factors, the three putative vesicular trafficking proteins, and the three proteins that might function in mediating cell fusion had not been identified previously as required for cell fusion.

Fig. 1.

Slants showing the conidiation pattern phenotypes of cell fusion mutants. Images of the wild-type strain and the ham-6, ham-7, ham-8, and amph-1 deletion mutants grown on Vogel's sucrose medium slants for 72 h are shown.

Fig. 2.

Photograph of the wild-type strain showing fusion hyphae. The wild-type strain was grown between two sheets of cellophane, and a region behind the growing edge of the colony containing fusion hyphae was photographed. The thicker arrows show locations where the fusion hyphae have completed the process of cell fusion. The thinner arrow points to two fusion hyphae that are growing toward each other but have not yet fused.

Fig. 3.

Complementation of cell fusion gene deletion mutations with wild-type copies of cell fusion genes. (Top) Conidiation patterns of the ham-6, ham-7, ham-8, and amph-1 mutants grown for 72 h on Vogel's sucrose medium. (Bottom) Transformants of these mutants (ham-6T, ham-7T, ham-8T, and amph-1T) in which a wild-type copy of the cell fusion gene complements the mutant conidiation pattern phenotype.

Fig. 4.

Protocol used to identify cell fusion genes. The four steps in the protocol used to identify genes required for cell fusion are shown. The first step involved screening each of the 10,000 mutants in the single gene deletion library to identify those with a cell fusion phenotype. The second step involved mating each of the putative mutants with a wild-type isolate to demonstrate that the mutant phenotype cosegregated with the deletion mutation. The third step involved assessing whether the mutants were able to generate CATs. In the final step, complementation experiments were carried out to demonstrate that the deletion mutation was responsible for the mutant phenotype.

Fig. 5.

Mutations in the ham-10 gene give rise to a cell fusion phenotype. To demonstrate that ham-10 is a cell fusion gene, RIP mutants with mutations of ham-10 were generated. Shown are photographs of slants containing the ham-10 deletion mutant, a transformant containing a copy of the ham-10 gene at the his-3 locus (his-3::ham-10), and a ham-10 RIP mutant (ham-10^RIP) that was obtained as progeny from the transformant.

Fig. 6.

Schematic representation of the functions proposed for cell fusion proteins. The transcription factor PP-1 is a target for phosphorylation by MAK-2. PP-1, along with the other transcription factors identified (ADV-1, ADA-3, SNF5, and the dimeric RCO-1/RCM-1), is proposed to reside in the nucleus (N) and to direct the transcription of the cell fusion genes. HAM-5 is proposed to function in directing MAK-2 into the nucleus, where it phosphorylates PP-1 and perhaps some of the other transcription factors. In addition to regulating transcriptional activity, the two MAP kinase signaling pathways (NRC-1/MEK-2/MAK-2 and MIK-1/MEK-1/MAK-1) also regulate CAT formation and growth by controlling vesicular trafficking to and from the hyphal tip. The two MAP kinase pathways are activated by cell-to-cell signaling, and the plasma membrane-associated HAM-6, HAM-7, and HAM-8 proteins might function as receptors for the signaling factor(s) or might be directly involved in fusion between the plasma membranes. As part of the cell-to-cell signaling process, the HAM-1/SO protein and the RAC-1/NRC-1/MEK-2/MAK-2 complex transiently associate with the plasma membrane in an oscillatory “ping pong” manner. The PP2A phosphatase might function in dephosphorylating the MAP kinase pathway proteins. The HAM-2/HAM-3/MOB3 complex is located on an intracellular vesicle (V) and is proposed to regulate vesicular trafficking to the hyphal tip. We propose that the HAM-2/HAM-3/MOB3 complex could receive input from the MAP kinase pathways and that the HAM-9 protein could function to connect the HAM-2/HAM-3/MOB3 complex to the MAP kinase pathways. AMPH-1 is proposed to play roles in mediating the formation of endosomes and to be necessary to maintain a balance between exocytosis and endocytosis of vesicles during CAT growth. The HAM-10 protein may function in regulating the movement of vesicles at the hyphal tip. The PKR1 protein is found in the ER and is necessary for efficient vesicular trafficking in the secretory pathway.