Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.

An international virtual hackathon to build tools for the analysis of structural variants within species ranging from coronaviruses to vertebrates.

In October 2020, 62 scientists from nine nations worked together remotely in the Second Baylor College of Medicine & DNAnexus hackathon, focusing on different related topics on Structural Variation, Pan-genomes, and SARS-CoV-2 related research.   The overarching focus was to assess the current status of the field and identify the remaining challenges. Furthermore, how to combine the strengths of the different interests to drive research and method development forward. Over the four days, eight groups each designed and developed new open-source methods to improve the identification and analysis of variations among species, including humans and SARS-CoV-2. These included improvements in SV calling, genotyping, annotations and filtering. Together with advancements in benchmarking existing methods. Furthermore, groups focused on the diversity of SARS-CoV-2. Daily discussion summary and methods are available publicly at  https://github.com/collaborativebioinformatics provides valuable insights for both participants and the research community.

A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing.

Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

A diploid assembly-based benchmark for variants in the major histocompatibility complex.

Most human genomes are characterized by aligning individual reads to the reference genome, but accurate long reads and linked reads now enable us to construct accurate, phased de novo assemblies. We focus on a medically important, highly variable, 5 million base-pair (bp) region where diploid assembly is particularly useful - the Major Histocompatibility Complex (MHC). Here, we develop a human genome benchmark derived from a diploid assembly for the openly-consented Genome in a Bottle sample HG002. We assemble a single contig for each haplotype, align them to the reference, call phased small and structural variants, and define a small variant benchmark for the MHC, covering 94% of the MHC and 22368 variants smaller than 50 bp, 49% more variants than a mapping-based benchmark. This benchmark reliably identifies errors in mapping-based callsets, and enables performance assessment in regions with much denser, complex variation than regions covered by previous benchmarks.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

Genome Sequence for Candida albicans Clinical Oral Isolate 529L.

The diploid heterozygous yeast Candida albicans is the most common cause of fungal infection. Here, we report the genome sequence assembly of the clinical oral isolate 529L. As this isolate grows as a commensal, this genome will serve as a reference for experimental and genetic studies of mucosal colonization.

A strategy for building and using a human reference pangenome.

In March 2019, 45 scientists and software engineers from around the world converged at the University of California, Santa Cruz for the first pangenomics codeathon. The purpose of the meeting was to propose technical specifications and standards for a usable human pangenome as well as to build relevant tools for genome graph infrastructures. During the meeting, the group held several intense and productive discussions covering a diverse set of topics, including advantages of graph genomes over a linear reference representation, design of new methods that can leverage graph-based data structures, and novel visualization and annotation approaches for pangenomes. Additionally, the participants self-organized themselves into teams that worked intensely over a three-day period to build a set of pipelines and tools for specific pangenomic applications. A summary of the questions raised and the tools developed are reported in this manuscript.

Whole-Genome Analysis Illustrates Global Clonal Population Structure of the Ubiquitous Dermatophyte Pathogen Trichophyton rubrum.

Dermatophytes include fungal species that infect humans, as well as those that also infect other animals or only grow in the environment. The dermatophyte species Trichophyton rubrum is a frequent cause of skin infection in immunocompetent individuals. While members of the T. rubrum species complex have been further categorized based on various morphologies, their population structure and ability to undergo sexual reproduction are not well understood. In this study, we analyze a large set of T. rubrum and T. interdigitale isolates to examine mating types, evidence of mating, and genetic variation. We find that nearly all isolates of T. rubrum are of a single mating type, and that incubation with T. rubrum "morphotype" megninii isolates of the other mating type failed to induce sexual development. While the region around the mating type locus is characterized by a higher frequency of SNPs compared to other genomic regions, we find that the population is remarkably clonal, with highly conserved gene content, low levels of variation, and little evidence of recombination. These results support a model of recent transition to asexual growth when this species specialized to growth on human hosts.

Evaluation of a 27-gene inherited cancer panel across 630 consecutive patients referred for testing in a clinical diagnostic laboratory.

BACKGROUND: Extensive clinical and genetic heterogeneity of inherited cancers has allowed multi-gene panel testing to become an efficient means for identification of patients with an inherited predisposition to a broad spectrum of syndromic and nonsyndromic forms of cancer. This study reports our experience with a 27-gene inherited cancer panel on a cohort of 630 consecutive individuals referred for testing at our laboratory with the following objectives: 1. Determine the rates for positive cases and those with variants of uncertain clinical significance (VUS) relative to data published in the recent literature, 2. Examine heterogeneity among the constituent genes on the panel, and 3. Review test uptake in the cohort relative to other reports describing outcomes for expanded panel testing. METHODS: Clinical and genomic data were reviewed on 630 individuals tested on a panel of 27 genes selected on the basis of high (≥ 40%) or moderate to low (≤ 40%) lifetime risk of hereditary cancer. These patients were not enriched for adherence to the National Comprehensive Cancer Network (NCCN) criteria for Hereditary Breast and Ovarian Cancer (HBOC) or Lynch Syndrome (LS) and constitute a referral laboratory cohort. RESULTS: Sixty-five individuals with variants classified as pathogenic or likely pathogenic across 14 genes were identified for an overall positive rate of 10.3%. Although a family history of cancer constituted a major reason for referral, accounting for 84% of our cohort, excluding patients with a known familial variant did not have a significant impact on the observed positive rate (9% vs 10.3%). More than half (58%) of the pathogenic or likely pathogenic variants were observed in high or moderate to low risk genes on the panel, while only 42% occurred in classic HBOC or LS-associated genes. CONCLUSION: These results provide the actual percentage of family or personal history of cancer that can be attributed to pathogenic or likely pathogenic variants in one or more of the genes on our panel and corroborate the utility of multi-gene panels over sequential testing to identify individuals with an inherited predisposition to cancer.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci.

Three members of the Puccinia genus, Pucciniatriticina (Pt), Pstriiformis f.sp. tritici (Pst), and Pgraminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

Genome Sequence of Spizellomyces punctatus.

Spizellomyces punctatus is a basally branching chytrid fungus that is found in the Chytridiomycota phylum. Spizellomyces species are common in soil and of importance in terrestrial ecosystems. Here, we report the genome sequence of S. punctatus, which will facilitate the study of this group of early diverging fungi.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.

The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia.

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.

Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii.

UNLABELLED: Cryptococcus gattii is a fungal pathogen of humans, causing pulmonary infections in otherwise healthy hosts. To characterize genomic variation among the four major lineages of C. gattii (VGI, -II, -III, and -IV), we generated, annotated, and compared 16 de novo genome assemblies, including the first for the rarely isolated lineages VGIII and VGIV. By identifying syntenic regions across assemblies, we found 15 structural rearrangements, which were almost exclusive to the VGI-III-IV lineages. Using synteny to inform orthology prediction, we identified a core set of 87% of C. gattii genes present as single copies in all four lineages. Remarkably, 737 genes are variably inherited across lineages and are overrepresented for response to oxidative stress, mitochondrial import, and metal binding and transport. Specifically, VGI has an expanded set of iron-binding genes thought to be important to the virulence of Cryptococcus, while VGII has expansions in the stress-related heat shock proteins relative to the other lineages. We also characterized genes uniquely absent in each lineage, including a copper transporter absent from VGIV, which influences Cryptococcus survival during pulmonary infection and the onset of meningoencephalitis. Through inclusion of population-level data for an additional 37 isolates, we identified a new transcontinental clonal group that we name VGIIx, mitochondrial recombination between VGII and VGIII, and positive selection of multidrug transporters and the iron-sulfur protein aconitase along multiple branches of the phylogenetic tree. Our results suggest that gene expansion or contraction and positive selection have introduced substantial variation with links to mechanisms of pathogenicity across this species complex. IMPORTANCE: The genetic differences between phenotypically different pathogens provide clues to the underlying mechanisms of those traits and can lead to new drug targets and improved treatments for those diseases. In this paper, we compare 16 genomes belonging to four highly differentiated lineages of Cryptococcus gattii, which cause pulmonary infections in otherwise healthy humans and other animals. Half of these lineages have not had their genomes previously assembled and annotated. We identified 15 ancestral rearrangements in the genome and over 700 genes that are unique to one or more lineages, many of which are associated with virulence. In addition, we found evidence for recent transcontinental spread, mitochondrial genetic exchange, and positive selection in multidrug transporters. Our results suggest that gene expansion/contraction and positive selection are diversifying the mechanisms of pathogenicity across this species complex.

Genome Sequences of Three Phytopathogenic Species of the Magnaporthaceae Family of Fungi.

Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi cause widespread disease and loss in cereal and grass crops, including rice blast disease (M. oryzae), take-all disease in wheat and other grasses (G. graminis), and summer patch disease in turf grasses (M. poae). Here, we present the finished genome sequence for M. oryzae and draft sequences for M. poae and G. graminis var. tritici. We used multiple technologies to sequence and annotate the genomes of M. oryzae, M. poae, and G. graminis var. tritici. The M. oryzae genome is now finished to seven chromosomes whereas M. poae and G. graminis var. tritici are sequenced to 40.0× and 25.0× coverage respectively. Gene models were developed by the use of multiple computational techniques and further supported by RNAseq data. In addition, we performed preliminary analysis of genome architecture and repetitive element DNA.

Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal.

BACKGROUND: The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents. METHODS AND FINDINGS: We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937-1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974-1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988-1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe. CONCLUSIONS: In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.

Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus.

BACKGROUND: The genus Microbotryum includes plant pathogenic fungi afflicting a wide variety of hosts with anther smut disease. Microbotryum lychnidis-dioicae infects Silene latifolia and replaces host pollen with fungal spores, exhibiting biotrophy and necrosis associated with altering plant development. RESULTS: We determined the haploid genome sequence for M. lychnidis-dioicae and analyzed whole transcriptome data from plant infections and other stages of the fungal lifecycle, revealing the inventory and expression level of genes that facilitate pathogenic growth. Compared to related fungi, an expanded number of major facilitator superfamily transporters and secretory lipases were detected; lipase gene expression was found to be altered by exposure to lipid compounds, which signaled a switch to dikaryotic, pathogenic growth. In addition, while enzymes to digest cellulose, xylan, xyloglucan, and highly substituted forms of pectin were absent, along with depletion of peroxidases and superoxide dismutases that protect the fungus from oxidative stress, the repertoire of glycosyltransferases and of enzymes that could manipulate host development has expanded. A total of 14% of the genome was categorized as repetitive sequences. Transposable elements have accumulated in mating-type chromosomal regions and were also associated across the genome with gene clusters of small secreted proteins, which may mediate host interactions. CONCLUSIONS: The unique absence of enzyme classes for plant cell wall degradation and maintenance of enzymes that break down components of pollen tubes and flowers provides a striking example of biotrophic host adaptation.