Identification of Allorecognition Loci in Neurospora crassa by Genomics and Evolutionary Approaches.

Understanding the genetic and molecular bases of the ability to distinguish self from nonself (allorecognition) and mechanisms underlying evolution of allorecognition systems is an important endeavor for understanding cases where it becomes dysfunctional, such as in autoimmune disorders. In filamentous fungi, allorecognition can result in vegetative or heterokaryon incompatibility, which is a type of programmed cell death that occurs following fusion of genetically different cells. Allorecognition is genetically controlled by het loci, with coexpression of any combination of incompatible alleles triggering vegetative incompatibility. Herein, we identified, characterized, and inferred the evolutionary history of candidate het loci in the filamentous fungus Neurospora crassa. As characterized het loci encode proteins carrying an HET domain, we annotated HET domain genes in 25 isolates from a natural population along with the N. crassa reference genome using resequencing data. Because allorecognition systems can be affected by frequency-dependent selection favoring rare alleles (i.e., balancing selection), we mined resequencing data for HET domain loci whose alleles displayed elevated levels of variability, excess of intermediate frequency alleles, and deep gene genealogies. From these analyses, 34 HET domain loci were identified as likely to be under balancing selection. Using transformation, incompatibility assays and genetic analyses, we determined that one of these candidates functioned as a het locus (het-e). The het-e locus has three divergent allelic groups that showed signatures of positive selection, intra- and intergroup recombination, and trans-species polymorphism. Our findings represent a compelling case of balancing selection functioning on multiple alleles across multiple loci potentially involved in allorecognition.

Diversification of a protein kinase cascade: IME-2 is involved in nonself recognition and programmed cell death in Neurospora crassa.

Kinase cascades and the modification of proteins by phosphorylation are major mechanisms for cell signaling and communication, and evolution of these signaling pathways can contribute to new developmental or environmental response pathways. The Saccharomyces cerevisiae kinase Ime2 has been well characterized for its role in meiosis. However, recent studies have revealed alternative functions for Ime2 in both S. cerevisiae and other fungi. In the filamentous fungus Neurospora crassa, the IME2 homolog (ime-2) is not required for meiosis. Here we determine that ime-2 interacts genetically with a transcription factor vib-1 during nonself recognition and programmed cell death (PCD). Mutations in vib-1 (Δvib-1) suppress PCD due to nonself recognition events; however, a Δvib-1 Δime-2 mutant restored wild-type levels of cell death. A role for ime-2 in the post-translational processing and localization of a mitochondrial matrix protein was identified, which may implicate mitochondria in N. crassa nonself recognition and PCD. Further, Δvib-1 strains do not produce extracellular proteases, but protease secretion reverted to near wild-type levels in a Δvib-1 Δime-2 strain. Mass spectrometry analysis revealed that the VIB-1 protein is phosphorylated at several sites, including a site that matches the IME-2 consensus. The genetic and biochemical data for ime-2 and vib-1 indicate that IME-2 is a negative regulator of VIB-1 and suggest parallel negative regulation by IME-2 of a cell death pathway in N. crassa that functions in concert with the VIB-1 cell death pathway. Thus, IME2 kinase function has evolved following the divergence of S. cerevisiae and N. crassa and provides insight into the evolution of kinases and their regulatory targets.