Rapid screening for Neurospora crassa strains lacking CuZn superoxide dismutase (sod-1)

We have developed a simple system for determining if strains of N. crassa produce functional CuZn superoxide dismutase. We previously isolated N. crassa mutants with null alleles of the sod-1 gene, which encodes this enzyme (Chary et al. submitted)

GENOMICS BASED APPROACHES TO FUNGAL EVOLUTION

Advances in DNA sequencing and data analysis make it possible to address questions in population genetics and evolution at the genomic level. Fungi are excellent subjects for such studies, because they are found in diverse environments, have short generation times, can be maintained in culture and have relatively small genomes. My research employed genetic approaches using a variety of sequencing technologies and methods of analysis to explore questions in fungal evolution. In one study, I explored the genetics behind differences in thermotolerance between isolates of Neurospora discreta from Alaska and New Mexico. Isolates from the two states exhibited differences in maximal growth temperature, with New Mexico isolates being substantially more thermotolerant than isolates from Alaska. Genomic scale comparisons of progeny from crosses between isolates from New Mexico and Alaska indicated that two regions, one on chromosome III and another on chromosome I, are responsible for differences in thermotolerance. Examination of these regions revealed numerous differences between the New Mexico and Alaska isolates at nucleotide and amino-acid levels; and it identified candidate genes for being important for differences in maximal growth temperatures. In a second study, I explored the genomic differences between pathogenic and endophytic isolates in the genus Monosporascus. Culture and sequence-based surveys of root associating fungi at the Sevilleta National Wildlife Refuge (SNWR) revealed the ubiquitous presence of members of this genus. Although M. cannonballus is known as a severe pathogen of melon roots in agricultural settings, all of the host plants associating with Monosporascus species in natural settings appeared to be disease free. Complete genome sequences were obtained from three M. cannonballus isolates, an M. ibericus isolate and six SNWR isolates. Comparative genome analyses revealed that 1) isolates of Monosporascus possess genomes that are more than twice the size of those typical for members of the Sordariomycetes, while having typical numbers of protein-coding genes; 2) isolates from diverse grasses, tree and forbs include lineages closely-related to previously described species including M. cannonballus, in addition to novel lineages; and 3) species of Monosporascus and other Xylariales lack mating-type gene regions typical of other members of the Pezizomycotina

Neurospora proteome 2000

The filamentous fungus, Neurospora crassa, has an eminent history as a central organism in the elucidation of the tenets of classical and biochemical genetics. Of particular significance are the experiments of George Beadle and Edward Tatum in the 1940s with N. crassa that led to the one gene-one enzyme hypothesis (Beadle and Tatum 1941 Proc. Natl. Acad. Sci. USA 27:499 506). In six decades, over 1,000 genes have been mapped and characterized (Perkins, Radford and Sachs 2000 The Neurospora Compendium: Chromosomal Loci. Academic Press; Perkins 2000 Fungal Genet. Newsl., this volume), but that leaves perhaps 10,000 or more genes not yet identified by classical genetics. High-throughput, automated partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) allows for the rapid identification and characterization of preferentially expressed genes in different tissues, as well as the discovery of novel genes (Adams et al. 1991 Science252:1651-1656; Okubo et al. 1992 Nature Genet. 1:173-179)

A provisional UniGene clone set based on ESTs from Neurospora crassa

We have constructed a list of N. crassa cDNA clones for which partial sequences exist, toward the goal of maximizing the number of genes represented while avoiding redundancy. This effort employed GenBank sequences from the combined N. crassa EST projects at the University of New Mexico, the University of Oklahoma and Dartmouth College (27,557 ESTs; Nelson et al. 1997 Fungal Genet. Biol.21:348-363; Zhu et al. 2001 Genetics 157: 1057-1065). The current list, subject to ongoing revision, includes 2842 clones and is available at the web site of the Neurospora Genome Project (NGP) at the University of New Mexico (http://www.unm.edu/~ngp/), along with details of its construction. Each cDNA clone in the list represents a unique gene. We have also assembled a UniGene set of cDNA clones for that portion of the UniGene set that is represented in libraries constructed by the NGP at UNM. This UniGene library is comprised of 1786 clones distributed in 20 96-well dishes, and it is available through the Fungal Genetics Stock Center

Massive Changes in Genome Architecture Accompany the Transition to Self-Fertility in the Filamentous Fungus Neurospora tetrasperma

A large region of suppressed recombination surrounds the sex-determining locus of the self-fertile fungus Neurospora tetrasperma. This region encompasses nearly one-fifth of the N. tetrasperma genome and suppression of recombination is necessary for self-fertility. The similarity of the N. tetrasperma mating chromosome to plant and animal sex chromosomes and its recent origin (<5 MYA), combined with a long history of genetic and cytological research, make this fungus an ideal model for studying the evolutionary consequences of suppressed recombination. Here we compare genome sequences from two N. tetrasperma strains of opposite mating type to determine whether structural rearrangements are associated with the nonrecombining region and to examine the effect of suppressed recombination for the evolution of the genes within it. We find a series of three inversions encompassing the majority of the region of suppressed recombination and provide evidence for two different types of rearrangement mechanisms: the recently proposed mechanism of inversion via staggered single-strand breaks as well as ectopic recombination between transposable elements. In addition, we show that the N. tetrasperma mat a mating-type region appears to be accumulating deleterious substitutions at a faster rate than the other mating type (mat A) and thus may be in the early stages of degeneration

Genomic Analysis of Diverse Members of the Fungal Genus Monosporascus Reveals Novel Lineages, Unique Genome Content and a Potential Bacterial Associate

The genus Monosporascus represents an enigmatic group of fungi important in agriculture and widely distributed in natural arid ecosystems. Of the nine described species, two (M. cannonballus and M. eutypoides) are important pathogens on the roots of members of Cucurbitaceae in agricultural settings. The remaining seven species are capable of colonizing roots from a diverse host range without causing obvious disease symptoms. Recent molecular and culture studies have shown that members of the genus are nearly ubiquitous as root endophytes in arid environments of the Southwestern United States. Isolates have been obtained from apparently healthy roots of grasses, shrubs and herbaceous plants located in central New Mexico and other regions of the Southwest. Phylogenetic and genomic analyses reveal substantial diversity in these isolates. The New Mexico isolates include close relatives of M. cannonballus and M. ibericus, as well as isolates that represent previously unrecognized lineages. To explore evolutionary relationships within the genus and gain insights into potential ecological functions, we sequenced and assembled the genomes of three M. cannonballus isolates, one M. ibericus isolate, and six diverse New Mexico isolates. The assembled genomes were significantly larger than what is typical for the Sordariomycetes despite having predicted gene numbers similar to other members of the class. Differences in predicted genome content and organization were observed between endophytic and pathogenic lineages of Monosporascus. Several Monosporascus isolates appear to form associations with members of the bacterial genus Ralstonia (Burkholdariaceae)