Allen D. Budde

Characterization of the Ustilago maydis sid2 Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster

Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation of L-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodes L-ornithine N(5)-oxygenase, which catalyzes hydroxylation of L-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulated sid2 transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1 of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.

Genome organization of Magnaporthe grisea: genetic map, electrophoretic karyotype, and occurrence of repeated DNAs

A genetic map of Magnaporthe grisea (anamorph=Pyricularia oryzae and P. grisea), the causal agent of rice blast disease, was generated from segregation data utilizing 97 RFLP markers, two isoenzyme loci and the mating type locus among progeny of a cross between parental strains Guy 11 and 2539. Of the seven chromosomes of M. Grisea, three were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis, while the remaining four migrated as two doublet bands. By utilizing differences between CHEF mobilities of unresolved chromosomes from the parental strains, Southern analysis with selected markers allowed the chromosomal assignment of all linkage groups. A small translocation involving 1 marker was found in the parental strains used to produce the segregating population from which the map was constructed. Nine classes of repetitive DNA elements were found in the genome of a fungal isolate pathogenic to rice. These occurred only a few times or not at all in the genomes of isolates showing reduced virulence on rice. One repetitive DNA was shown to have structural similarity to the Alu sequences found in primates, a sequence similarity to the copia-like elements of Drosophila, and peptide similarity to transposable elements found in Drosophila, other fungi, and higher plants.

New cosmid vectors for library construction, chromosome walking and restriction mapping in filamentous fungi

New cosmid vectors were constructed for the ascomycete fungus, Magnaporthe grisea and the basidiomycete fungus, Ustilago maydis. These vectors are capable of transforming M. grisea at frequencies of up to 5 transformants/micrograms linear DNA and U. maydis at up to 25 transformants/microgram circular DNA for integrative transformation. In addition, 2800 transformants/microgram DNA are possible when using an autonomously replicating vector. Since the promoters used in these vectors function in other ascomycete and basidiomycete fungi, we anticipate that these vectors will be widely applicable.

Structural and functional characterization of a winter malting barley

The development of winter malting barley (Hordeum vulgare L.) varieties is emerging as a worldwide priority due to the numerous advantages of these varieties over spring types. However, the complexity of both malting quality and winter hardiness phenotypes makes simultaneous improvement a challenge. To obtain an understanding of the relationship between loci controlling winter hardiness and malt quality and to assess the potential for breeding winter malting barley varieties, we structurally and functionally characterized the six-row accession “88Ab536”, a cold-tolerant line with superior malting quality characteristics that derives from the cross of NE76129/Morex//Morex. We used 4,596 SNPs to construct the haplotype structure of 88Ab536 on which malting quality and winter hardiness loci reported in the literature were aligned. The genomic regions determining malting quality and winter hardiness traits have been defined in this founder germplasm, which will assist breeders in targeting regions for marker-assisted selection. The Barley1 GeneChip array was used to functionally characterize 88Ab536 during malting. Its gene expression profile was similar to that of the archetypical malting variety Morex, which is consistent with their similar malting quality characteristics. The characterization of 88Ab536 has increased our understanding of the genetic relationships of malting quality and winter hardiness, and will provide a genetic foundation for further development of more cold-tolerant varieties that have malt quality characteristics that meet or exceed current benchmarks.

Calcofluor Fluorescence Assay for Wort β-Glucan in a Microplate Format

The level of β-glucan in worts produced from malted barley is a critical malting quality parameter. Measurement of wort β-glucan levels allows maltsters to determine whether production malts meet commercial specifications, helps brewers avoid production problems due to slow lautering, and enables barley breeders to develop lines with the potential to meet commercial needs. Many laboratories performing routine wort β-glucan analyses use flow injection analysis (FIA) methods or related segmented flow analysis (SFA) methods that measure the increase in fluorescence with Calcofluor binding to β-glucan. Such automated systems are attractive due to low per-sample reagent costs, relatively high sample throughput, and a minimal number of sample processing steps prior to automated analysis, limiting the need for manual operations. However, FIA or SFA methodology requires significant capital investment for instrument acquisition, discouraging its use outside dedicated quality assurance laboratories. Laboratories that routinely analyze relatively few samples often find kits for enzymatic or colorimetric analysis of β-glucans more attractive. Such kits require relatively simple laboratory instrumentation, reducing capital costs, but may involve multiple sample manipulations, increasing the level of technical support necessary to perform the analyses. Per-analysis reagent costs may also be greater for the kits. In this work, we have adapted the Calcofluor fluorescence method to a microplate reader to achieve a simple and cost-effective assay for wort β-glucan that avoids the acquisition costs of FIA or SFA instrumentation.

Quantitative trait loci of barley malting quality trait components in the Stellar/01Ab8219 mapping population

Malting barley is of high economic and scientific importance. Determining barley grains that are suitable for malting involves measuring malting quality, which is an expensive and complex process. In order to decrease the cost of phenotyping and accelerate the process of developing superior malting barley cultivars, markers for marker-assisted breeding are needed. In this study, we identified quantitative trait loci (QTLs) for malting traits in a Stellar/01Ab8219 F6:8 recombinant inbred line population grown at Aberdeen and Tetonia, Idaho, USA in 2009 and 2010. We identified QTLs associated with malt extract (ME), wort protein, soluble/total protein (S/T), diastatic power (DP), alpha-amylase, beta-glucan (BG) and free amino nitrogen (FAN) at a logarithm of odds score ≥2.5 using a high-density genetic map produced by merging Diversity Arrays Technology markers with the current single nucleotide polymorphism map. Novel QTLs were identified for DP and FAN on chromosome 5H, S/T on 6H, and BG and ME on 7H. Dissection of the genetic regions associated with malting traits suggests the involvement of multiple molecular pathways. The resulting molecular markers may prove useful for barley improvement.

Molecular Analysis of Pathogenesis in Ustilago maydis

We are isolating and studying genes required for pathogenicity of Ustilago maydis, the causative agent of corn smut disease (Christensen, 1963). This phytopathogenic basidiomycete offers an attractive system in which to gain a molecular understanding of host-parasite interactions. The organism grows as a haploid yeast on defined laboratory media, mutants are readily generated by UV or chemical mutagenesis, stable diploids can be constructed for mitotic recombination and complementation analysis, and the fungus is amenable to Mendelian genetic analysis (Holliday, 1974). These attributes are not found in combination in any other phytopathogenic fungus. Although this pathogen is no longer a production constraint in North America, where resistant hybrid corn is grown, it continues to be a problem in third world countries where susceptible varieties are still cultivated. Moreover, we hope that an understanding of mechanisms of pathogenesis in this host-parasite interaction will have application to more economically important and difficult to study fungal diseases such as the bunts and rusts. We have initiated a molecular genetic analysis of two gene systems thought to control pathogenic growth of U. maydis in maize. These include genes that program sexual development and genes involved in the high affinity, siderophore-mediated iron uptake system of the fungus. In order to conduct these analyses, one of our first goals has been to develop tools which enable us to clone, study, and transfer genes in U. maydis.