Creating and screening Cochliobolus heterostrophus non-ribosomal peptide synthetase mutants

An exhaustive characterization of the set of non-ribosomal peptide synthetase (NRPS) genes of the corn pathogen, Cochliobolus heterostrophus, and the small molecule peptides produced by the enzymes they encode, has been undertaken to ascertain the role of the peptide metabolites in the fungal cell. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. In addition to structural diversity, non-ribosomal peptides have a broad spectrum of biological activities, many are useful in medicine, agriculture, industry, and biological research. However, to suggest that inter-organismal activities is their primary function is likely incorrect; in fact, the physiological significance of these peptides to the producing fungi is largely unknown. We document that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches and that some are conserved, but most are not.     

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat–, and Saccharomyces cerevisiae MAT1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.The first three authors contributed equally to the work     

A mitochondrial plasmid from the plant pathogenic fungus Cochliobolus heterostrophus

Summary Twenty-three strains of Cochliobolus heterostrophus were examined for the presence of plasmid DNA. One isolate, T40, contained a 1.9 kb sequence which occurred as a series of circular head-to-tail multimers with from 1 to 17 or more monomers per plasmid molecule. The plasmid was cloned in pBR322 to facilitate analysis. It was homologous to the mitochondrial chromosome of isolate T40 as well as to the mitochondrial DNAs of C. heterostrophus isolates that did not contain the plasmid; each isolate, including T40, had only one copy of the plasmid sequence integrated into the mitochondrial chromosome and the sequence mapped at the same location in all isolates tested. In the T40 isolate there were about 30 excised copies per chromosome in addition to the single integrated sequence. Presence of the plasmid had no apparent effect on the structural integrity of the mitochondrial chromosome. There was no detectable homology between the plasmid and either C. heterostrophus nuclear DNA or plasmids that have been isolated from mitochondria of Neurospora or Podospora. A circular map was constructed which has 6 sites for hexan-ucleotide-recognizing enzymes and the region of the splice site; no sites were detected in the plasmid for an additional 17 restriction enzymes. The plasmid functioned as an ARS (autonomously replicating sequence) in yeast, although it was highly unstable compared to other ARSs.     

Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene

Summary Cochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.     

Chromosomal heteromorphism linked to the mating type locus of the oomycetePhytophthora infestans

The mating type locus of the oomycete,Phytophthora infestans, is embedded in a region of DNA that displays distorted and non-Mendelian segregation. By using DNA probes linked to the mating type locus to genetically and physically characterize that region, a large zone of chromosomal heteromorphism was detected. LocusS1 was shown to represent a tandemly repeated array of DNA that was typically present in a hemizygous state in A1 isolates while being absent from A2 isolates. The analysis of the parents and progeny of seven crosses indicated that the tandem array was linked in cis to the A1-determining allele of the mating type locus. A worldwide survey of genotypically diverse field isolates ofP. infestans indicated thatS1 was present in each of 48 isolates of the A1 mating type that were tested, but was absent in 46 of 47 A2 strains. Physical analysis ofS1 indicated that the tandemly repeated DNA sequence spanned about 300 kb and had evolved from a 1.35-kb monomer. Internal deletions occurred withinS1 during sexual propagation. This and other mutations apparently contributed to a high degree of polymorphism within theS1 array.     

Novel mating type-dependent transcripts at the mating type locus in Magnaporthe oryzae

The mating type locus (MAT1) of Magnaporthe oryzae has similar structural organization to MAT in other ascomycetes and encodes the mating type genes MAT1-1-1 with an alpha-box motif and MAT1-2-1 with an HMG-box motif in the MAT1-1 and MAT1-2 idiomorphs, respectively. Sequence and expression analyses of the MAT1 locus indicated a second open reading frame (ORF), MAT1-1-2, in the MAT1-1 idiomorph, and novel mating-type dependent ORFs (MAT1-1-3 and MAT1-2-2) at the locus. The MAT1-1-3 ORF initiated within the MAT1-1 idiomorph while the MAT1-2-2 ORF initiated at the border of the MAT1-2 idiomorph with both ORFs sharing most of their reading frames in the MAT1 flanking region. This suggests that the encoded proteins (MAT1-1-3 and MAT1-2-2) should be similar in their primary structures but can be distinguished by distinct N-termini with amino acids of 1 and 32, respectively, in each mating type. A CT dinucleotide repeat, (CT)n, present in the upstream region of MAT1-1-3, was polymorphic among the isolates.     

Rearrangements at the mating type locus in fission yeast

Summary Crosses involving the partially defective mating type mutant B102 (functional in conjugation, defective in meiosis) have confirmed the notion that, in Schizosaccharomyces pombe, certain mating type mutations can arise by transposition. A copy of the mat2 ^P segment (specifying + mating type) is transposed and inserted into the mat1 ^M segment (usually specifying mating type). The mat1 ^M segment affected by the insertion loses its former function entirely. The function is, however, fully regained upon excision of the transposed and inserted mat2 ^P segment.At either position, the mat2 ^P segments can undergo inactivations to different states of residual activity. These events can occur about as frequent as other mutations of the mating type locus (ca. 10^–4 per cell division). In certain diploid strains, such inactivations were significantly correlated with recombination. Spontaneous reversions to full activity were also observed.     

Ceratocystidaceae exhibit high levels of recombination at the mating-type (MAT) locus

Mating is central to many fungal life cycles and is controlled by genes at the mating-type (MAT) locus. These genes determine whether the fungus will be self-sterile (heterothallic) or self-fertile (homothallic). Species in the ascomycete family Ceratocystidaceae have different mating strategies, making them interesting to consider with regards to their MAT loci. The aim of this study was to compare the composition of the MAT locus flanking regions in 11 species of Ceratocystidaceae representing four genera. Genome assemblies for each species were examined to identify the MAT locus and determine the structure of the flanking regions. Large contigs containing the MAT locus were then functionally annotated and analysed for the presence of transposable elements. Genes typically flanking the MAT locus in sordariomycetes were found to be highly conserved in the Ceratocystidaceae. The different genera in the Ceratocystidaceae displayed little synteny outside of the immediate MAT locus flanking genes. Even though species ofCeratocystis did not show much synteny outside of the immediate MAT locus flanking genes, species of Huntiella and Endoconidiophora were comparatively syntenic. Due to the high number of transposable elements present in Ceratocystis MAT flanking regions, we hypothesise that Ceratocystis species may have undergone recombination in this region.     

A dominant mutation (SAD) bypassing the requirement for the a mating type locus in yeast sporulation

Summary SAD (suppressor of a deficiencies) is a mutation that allows -mater diploids such as / or a1^-/ strains to sporulate. This mutation is unstable and reverts to wildtype (sad ⁺) even in strains homozygous for SAD. SAD is dominant to sad ⁺: / and a1^-/ sad ¹/SAD diploids are sporulation-proficient. SAD is located on chromosome III, 40 cM distal to the mating type locus, between THR4 and HMR a. The ability of SAD to support sporulation requires the presence of an mating type locus with an active 2 function. Possible models for the action of SAD are (1) SAD bypasses the need for a1 function in sporulation, and (2) SAD provides a1 function to MAT a1^- mutants by supplying a1 function itself, for example, by allowing expression of a silent copy of MAT a.     

Functional characterization of an ammonium transporter gene from Lotus japonicus

NH₄⁺ is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH₄⁺ by the plant cells.     

The biallelica mating type locus ofUstilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor

Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termeda1 anda2. We have completed the analysis of the nucleotide sequences unique toa1 anda2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of thea1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in thea2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in thea2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.     

Role of Zeste in transvection at the Bithorax locus of Drosophila

Summary We have re-examined the effect of the mutation z^a in the zeste locus on the phenotype of the bx ^34e /Ubx trans combination of bithorax mutations, in the presence and absence of rearrangements which presumably affect homologous pairing in the bithorax region. Our observations suggest the possibility of the zeste ⁺gene product being needed for transvection at the bithorax locus     

Transposable element Ds at the shrunken locus in Zea mays

Summary Maize DNAs isolated from wild type and from mutants caused by the insertion of transposable genetic element Ds at the gene encoding endosperm sucrose synthase (Sh) are compared in Southern blotting experiments by hybridization to Sh-cDNA cloned in pBR322. Differences observed between the DNAs of the wild type and the mutants indicate the presence of additional DNA at the Sh locus and/or DNA alterations that have occurred subsequent to the insertion of Ds. A double mutant exhibiting the recessive phenotype of both sh and the closely linked gene bz lacks DNA hybridizing to the probe and may be a deletion.     

Examination of the Intron in the meiosis-specific recombination gene REC114 in Saccharomyces

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes. Received: 16 October 1996 / Accepted: 10 February 1997     

Instability at the y-1 locus of Chlamydomonas reinhardtii

Summary Twenty-three spontaneous yellow mutants were isolated from two stable green strains of the unicellular green alga Chlamydomonas reinhardtii. Genetic characterization indicated that 22 of 23 mutants had a mutation at the y-1 locus, and all 22 y-1 alleles were unstable. Crosses designed to follow the inheritance of instability at the y-1 locus showed that instability is caused by a single genetic factor located at the y-1 locus or very close to it.