Heterokaryon Incompatibility and Programmed Cell Death

Fig. 1 Hyphal fusion between colonies that are identical in specificity at het loci results in colonies that are wild type in phenotype, while hyphal fusion between colonies that differ in specificity at het loci results in hyphal compartmentation and death.	Fig. 2 Confocal image of hyphal fusion between Neurospora crassa strains containing histone H1::GFP strain (green nuclei) or stained with FM4-64 (membrane selective dye; red). The two strains differ in het specificity. Note the movement of green nuclei into the fusion cell at time 0'. Soon thereafter, the fusion cell is compartmentalized and dies. Note loss of H1::GFP signal (1 hr).

Self/nonself discrimination is a ubiquitous and essential function of both multicellular and microbial species, and is an aspect of biology that has long fascinated scientists. In filamentous fungi, nonself recognition is important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and programmed cell death of the fusion compartment, an event analogous to nonself recognition following fusion in colonial marine invertebrates such as Hydractinia and Botryllus. Nonself recognition during heterokaryon formation in filamentous fungi is regulated by genetic loci, termed het (for heterokaryon incompatibility) loci. Heterokaryon incompatibility in filamentous fungi has been shown to reduce the risk of transmission of pathogenic elements, such as infectious virus-like dsRNAs. Among filamentous fungal species, Neurospora crassa is the best model to study nonself recognition via heterokaryon incompatibility. Three het loci, mat, het-c, and het-6, have been cloned and characterized at the molecular level and have been shown to encode genes with highly polymorphic allelic specificity domains.

Fig. 3 Phenotype of a heterokaryon between two strains of identical het-c specificity, which is identical to a wild-type strain.	Fig. 4 Phentoype of a heterokaryon between two strains that differ in het -c specificity, which shows highly inhibited growth and PCD.

Molecular mechanism of nonself recognition

At the het-c locus, nonself recognition is dependent on the het-c haplotype. Nonself recognition requires both allelic (het-c) and non-allelic interactions (between het-c and pin-c). Individuals carrying het-c1 pin-c1 specificity are incompatible with individuals carrying het-c2 pin-c2 or het-c3 pin-c3 specificity. The specificity domain of het-c (shown as a red, blue box in het-c) and pin-c (shown in yellow) are highly polymorphic (i.e. pin-c alleles are ~50% identical and DNA and protein level). However, strains of alternative het-c haplotype are completely inter-fertile, indicating that heterokaryon incompatibility is suppressed during sexual development. We are currently assessing protein-protein interactions between HET-C and PIN-C and regions of the proteins required for this interaction.

Fig. 5 The het-c haplotype region of N. crassa.

 

Molecular mechanism of signal transduction and death

A number of mutants have been identified that affect nonself recognition and PCD in Neurospora. These include a putative transcription factor, vib-1. Our current research efforts are aimed at understanding how nonself recognition triggers programmed cell death via activation of VIB-1. We are currently using microarray technology and chromatin immunoprecipitation to identify targets of VIB-1 to gain an understanding of how the hyphal compartments are compartmentalized and killed and the molecular and physiological mechanism of growth inhibition.

Fig. 6 Model for non-self recognition and PCD in Neurospora. Mutations at vib-1 suppress heterokaryon incompatibility mediated by het-c/pin-c and mat A-1/mat a-1. The vib-1 locus encodes a putative transcription factor.

Evolution of nonself recognition

In vertebrate species, self/non-self recognition relies on the major histocompatibility complex (MHC), which is an array of genetic loci that generate proteins important in pathogen recognition and activation of defense mechanisms.Alleles at MHC loci show long term persistence, such that an allele from one species is often more closely related to an allele in a different species, a pattern that is termed trans-species polymorphisms. Self/non-self recognition during sexual reproduction in many plant species is mediated by the gametophytic or sporophytic self-incompatibility locus, S, which elicits recognition and rejection of self-pollen. Alleles at the S locus are also extremely polymorphic and show trans-species polymorphisms. Allelic polymorphisms at some het loci in Neurospora show evidence of balancing selection, i.e. alleles are highly polymorphic, alleles of alternative specificity are equally frequent in populations and show trans-species polymorphisms. We are currently taking a genomic approach to identify all of the nonself recognition loci in the genome of N. crassa and to compare evolutionary trajectories of these loci in filamentous fungi.

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