Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.

Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans.

Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding ß-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both ß-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB. IMPORTANCE Branched-chain amino acid (BCAA) biosynthesis is important for pathogenic fungi to successfully cause disease in human and plant hosts. The enzymes for their production are absent from humans and, therefore, provide potential antifungal targets. While BCAA biosynthesis is well characterized in yeasts, it is poorly understood in filamentous fungal pathogens. Developing a thorough understanding of both the genes encoding the metabolic enzymes for BCAA biosynthesis and how their expression is regulated will inform target selection for antifungal drug development.

Unbiased K-mer Analysis Reveals Changes in Copy Number of Highly Repetitive Sequences During Maize Domestication and Improvement.

The major component of complex genomes is repetitive elements, which remain recalcitrant to characterization. Using maize as a model system, we analyzed whole genome shotgun (WGS) sequences for the two maize inbred lines B73 and Mo17 using k-mer analysis to quantify the differences between the two genomes. Significant differences were identified in highly repetitive sequences, including centromere, 45S ribosomal DNA (rDNA), knob, and telomere repeats. Genotype specific 45S rDNA sequences were discovered. The B73 and Mo17 polymorphic k-mers were used to examine allele-specific expression of 45S rDNA in the hybrids. Although Mo17 contains higher copy number than B73, equivalent levels of overall 45S rDNA expression indicates that transcriptional or post-transcriptional regulation mechanisms operate for the 45S rDNA in the hybrids. Using WGS sequences of B73xMo17 doubled haploids, genomic locations showing differential repetitive contents were genetically mapped, which displayed different organization of highly repetitive sequences in the two genomes. In an analysis of WGS sequences of HapMap2 lines, including maize wild progenitor, landraces, and improved lines, decreases and increases in abundance of additional sets of k-mers associated with centromere, 45S rDNA, knob, and retrotransposons were found among groups, revealing global evolutionary trends of genomic repeats during maize domestication and improvement.

Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat.

Wheat leaf rust, caused by the basidiomycete Puccinia triticina, can cause yield losses of up to 20% in wheat producing regions. During infection, the fungus forms haustoria that secrete proteins into the plant cell and effect changes in plant transcription, metabolism, and defense. It is hypothesized that new races emerge as a result of overcoming plant resistance via changes in the secreted effector proteins. To understand gene expression during infection and find genetic differences associated with races, RNA from wheat leaves infected with six different rust races, at 6 days post inoculation, was sequenced using Illumina. As P. triticina is an obligate biotroph, RNA from both the host and fungi were present and separated by alignment to the P. triticina genome and a wheat EST reference. A total of 222,571 rust contigs were assembled from 165 million reads. An examination of the resulting contigs revealed 532 predicted secreted proteins among the transcripts. Of these, 456 were found in all races. Fifteen genes were found with amino acid changes, corresponding to putative avirulence effectors potentially recognized by 11 different leaf rust resistance (Lr) genes. Twelve of the potential avirulence effectors have no homology to known genes. One gene had significant similarity to cerato-platanin, a known fungal elicitor, and another showed similarity to fungal tyrosinase, an enzyme involved in melanin synthesis. Temporal expression profiles were developed for these genes by qRT-PCR and show that the genes expression patterns were consistent between races from infection initiation to just prior to spore eruption.