Development of the variant calling algorithm, ADIScan, and its use to estimate discordant sequences between monozygotic twins.

Calling variants from next-generation sequencing (NGS) data or discovering discordant sequences between two NGS data sets is challenging. We developed a computer algorithm, ADIScan1, to call variants by comparing the fractions of allelic reads in a tester to the universal reference genome. We then created ADIScan2 by modifying the algorithm to directly compare two sets of NGS data and predict discordant sequences between two testers. ADIScan1 detected >99.7% of variants called by GATK with an additional 724 393 SNVs. ADIScan2 identified ∼500 candidates of discordant sequences in each of two pairs of the monozygotic twins. About 200 of these candidates were included in the ∼2800 predicted by VarScan2. We verified 66 true discordant sequences among the candidates that ADIScan2 and VarScan2 exclusively predicted. ADIScan2 detected many discordant sequences overlooked by VarScan2 and Mutect, which specialize in detecting low frequency mutations in genetically heterogeneous cancerous tissues. Numbers of verified sequences alone were >5 times more than expected based on recently estimated mutation rates from whole genome sequences. Estimated post-zygotic mutation rates were 1.68 × 10-7 in this study. ADIScan1 and 2 would complement existing tools in screening causative mutations of diverse genetic diseases and comparing two sets of genome sequences, respectively.

HLAscan: genotyping of the HLA region using next-generation sequencing data.

BACKGROUND: Several recent studies showed that next-generation sequencing (NGS)-based human leukocyte antigen (HLA) typing is a feasible and promising technique for variant calling of highly polymorphic regions. To date, however, no method with sufficient read depth has completely solved the allele phasing issue. In this study, we developed a new method (HLAscan) for HLA genotyping using NGS data. RESULTS: HLAscan performs alignment of reads to HLA sequences from the international ImMunoGeneTics project/human leukocyte antigen (IMGT/HLA) database. The distribution of aligned reads was used to calculate a score function to determine correctly phased alleles by progressively removing false-positive alleles. Comparative HLA typing tests using public datasets from the 1000 Genomes Project and the International HapMap Project demonstrated that HLAscan could perform HLA typing more accurately than previously reported NGS-based methods such as HLAreporter and PHLAT. In addition, the results of HLA-A, -B, and -DRB1 typing by HLAscan using data generated by NextGen were identical to those obtained using a Sanger sequencing-based method. We also applied HLAscan to a family dataset with various coverage depths generated on the Illumina HiSeq X-TEN platform. HLAscan identified allele types of HLA-A, -B, -C, -DQB1, and -DRB1 with 100% accuracy for sequences at ≥ 90× depth, and the overall accuracy was 96.9%. CONCLUSIONS: HLAscan, an alignment-based program that takes read distribution into account to determine true allele types, outperformed previously developed HLA typing tools. Therefore, HLAscan can be reliably applied for determination of HLA type across the whole-genome, exome, and target sequences.

How the necrotrophic fungus Alternaria brassicicola kills plant cells remains an enigma.

Alternaria species are mainly saprophytic fungi, but some are plant pathogens. Seven pathotypes of Alternaria alternata use secondary metabolites of host-specific toxins as pathogenicity factors. These toxins kill host cells prior to colonization. Genes associated with toxin synthesis reside on conditionally dispensable chromosomes, supporting the notion that pathogenicity might have been acquired several times by A. alternata. Alternaria brassicicola, however, seems to employ a different mechanism. Evidence on the use of host-specific toxins as pathogenicity factors remains tenuous, even after a diligent search aided by full-genome sequencing and efficient reverse-genetics approaches. Similarly, no individual genes encoding lipases or cell wall-degrading enzymes have been identified as strong virulence factors, although these enzymes have been considered important for fungal pathogenesis. This review describes our current understanding of toxins, lipases, and cell wall-degrading enzymes and their roles in the pathogenesis of A. brassicicola compared to those of other pathogenic fungi. It also describes a set of genes that affect pathogenesis in A. brassicicola. They are involved in various cellular functions that are likely important in most organisms and probably indirectly associated with pathogenesis. Deletion or disruption of these genes results in weakly virulent strains that appear to be sensitive to the defense mechanisms of host plants. Finally, this review discusses the implications of a recent discovery of three important transcription factors associated with pathogenesis and the putative downstream genes that they regulate.

Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. To identify molecular determinants of pathogenicity, we created non-pathogenic mutants of a transcription factor-encoding gene, AbPf2. The frequency and timing of germination and appressorium formation on host plants were similar between the non-pathogenic ∆abpf2 mutants and wild-type A. brassicicola. The mutants were also similar in vitro to wild-type A. brassicicola in terms of vegetative growth, conidium production, and responses to a phytoalexin, reactive oxygen species and osmolites. The hyphae of the mutants grew slowly but did not cause disease symptoms on the surface of host plants. Transcripts of the AbPf2 gene increased exponentially soon after wild-type conidia contacted their host plants . A small amount of AbPf2 protein, as monitored using GFP fusions, was present in young, mature conidia. The protein level decreased during saprophytic growth, but increased and was located primarily in fungal nuclei during pathogenesis. Levels of the proteins and transcripts sharply decreased following colonization of host tissues beyond the initial infection site. When expression of the transcription factor was induced in the wild-type during early pathogenesis, 106 fungal genes were also induced in the wild-type but not in the ∆abpf2 mutants. Notably, 33 of the 106 genes encoded secreted proteins, including eight putative effector proteins. Plants inoculated with ∆abpf2 mutants expressed higher levels of genes associated with photosynthesis, the pentose phosphate pathway and primary metabolism, but lower levels of defense-related genes. Our results suggest that AbPf2 is an important regulator of pathogenesis, but does not affect other cellular processes in A. brassicicola.

Transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. Several A. brassicicola genes have been characterized as affecting pathogenesis of Brassica species. To study regulatory mechanisms of pathogenesis, we mined 421 genes in silico encoding putative transcription factors in a machine-annotated, draft genome sequence of A. brassicicola. In this study, targeted gene disruption mutants for 117 of the transcription factor genes were produced and screened. Three of these genes were associated with pathogenesis. Disruption mutants of one gene (AbPacC) were nonpathogenic and another gene (AbVf8) caused lesions less than half the diameter of wild-type lesions. Unexpectedly, mutants of the third gene, Amr1, caused lesions with a two-fold larger diameter than the wild type and complementation mutants. Amr1 is a homolog of Cmr1, a transcription factor that regulates melanin biosynthesis in several fungi. We created gene deletion mutants of Δamr1 and characterized their phenotypes. The Δamr1 mutants used pectin as a carbon source more efficiently than the wild type, were melanin-deficient, and more sensitive to UV light and glucanase digestion. The AMR1 protein was localized in the nuclei of hyphae and in highly melanized conidia during the late stage of plant pathogenesis. RNA-seq analysis revealed that three genes in the melanin biosynthesis pathway, along with the deleted Amr1 gene, were expressed at low levels in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that a gene important for survival in nature negatively affected virulence, probably by a less efficient use of plant cell-wall materials. We speculate that the functions of the Amr1 gene are important to the success of A. brassicicola as a competitive saprophyte and plant parasite.

Transcriptional responses of the Bdtf1-deletion mutant to the phytoalexin brassinin in the necrotrophic fungus Alternaria brassicicola.

Brassica species produce the antifungal indolyl compounds brassinin and its derivatives, during microbial infection. The fungal pathogen Alternaria brassicicola detoxifies brassinin and possibly its derivatives. This ability is an important property for the successful infection of brassicaceous plants. Previously, we identified a transcription factor, Bdtf1, essential for the detoxification of brassinin and full virulence. To discover genes that encode putative brassinin-digesting enzymes, we compared gene expression profiles between a mutant strain of the transcription factor and wild-type A. brassicicola under two different experimental conditions. A total of 170 and 388 genes were expressed at higher levels in the mutants than the wild type during the infection of host plants and saprophytic growth in the presence of brassinin, respectively. In contrast, 93 and 560 genes were expressed, respectively, at lower levels in the mutant than the wild type under the two conditions. Fifteen of these genes were expressed at lower levels in the mutant than in the wild type under both conditions. These genes were assumed to be important for the detoxification of brassinin and included Bdtf1 and 10 putative enzymes. This list of genes provides a resource for the discovery of enzyme-coding genes important in the chemical modification of brassinin.

The Bdtf1 gene in Alternaria brassicicola is important in detoxifying brassinin and maintaining virulence on Brassica species.

Brassinin is an antifungal compound induced in Brassica plants after microbial infection. Molecular evidence is incomplete, however, in supporting the importance of brassinin in plant resistance to pathogens. To test the importance of brassinin in plant defense, we studied the functions of the gene Bdtf1 in the necrotrophic fungus Alternaria brassicicola. Several strains of mutants of this gene were weakly virulent on Brassica species, causing lesions 70% smaller in diameter than the wild type on three Brassica species. These mutants, however, were as virulent as the wild type on Arabidopsis thaliana. They were similar to the wild type in spore germination, colony morphology, and mycelial growth in nutrient-rich media, both with and without stress-inducing chemicals. Unlike wild-type A. brassicicola, however, the mutants failed to germinate and their hyphal growth was arrested in the presence of 200 µM brassinin. When grown in a medium containing 100 µM brassinin, wild-type mycelium entirely converted the brassinin into a nontoxic derivative, of which the precise chemical nature was not established. Mutants of the Bdtf1 gene were unable to perform this conversion. Our results support the hypothesis that the ability of A. brassicicola to detoxify brassinin is necessary for successful infection of Brassica species.

Characteristics of the Ni-B film in printed circuit board for high-power light-emitting diodes.

To develop metal printed circuit boards for a high-power light-emitting diode package using electroless plated Ni-B films on an all-in-one Al2O3-Al substrate with a 10 nm pore size, the growth mode of an electroless Ni-B film on a screen-printed Ag pattern/Al2O3-Al substrate was studied. So as not to damage the Al2O3-AI substrate, a nearly neutral Ni plating solution bath (pH 6.5) included dimethylamine borane was used. It was confirmed that the Ni-B film was selectively grown on the printed Ag paste layer, without growth on the Al2O3. The structure of the electroless plated Ni-B film was amorphous, and the deposition rate of the film was 1.64 +/- 0.078 nm/sec. According to the increase in plating time, the grain sizes of the electroless plated Ni-B film became bigger, and the surface morphology gradually became flatter. In addition, both the mass difference and the film thickness were changed linearly. From these results, it can be concluded that the electroless Ni-B film on printed Ag paste grows immediately from the beginning, and then grows linearly with increasing plating time.

Assessment of a 50:50 mixture of two Bacillus subtilis strains as growth promoters for finishing pigs: productivity improvement and noxious gas reduction.

In this study, we aimed to assess the potential of a 50:50 mixture of two Bacillus subtilis strains in improving the productivity and health of finishing pigs and reducing noxious gases in their feces. These strains were found to abundantly secrete surfactin which has been shown to alleviate the effects of lipopolysaccharides in vitro. For the 10-wk experiment, 200 finishing pigs ([Landrace × Yorkshire] × Duroc) with an average body weight of 54.15 ±â€…1.70 kg were divided into four groups. Each group was fed with a basal diet supplemented with an equal amount of spores from the two B. subtilis strains at different levels: control group, no addition; treatment group 1, 0.5 × 109; treatment group 2, 1.0 × 109; treatment group 3, 1.5 × 109 cfu·kg-1 addition. During the 10-wk feeding period, dietary supplementation of 0.5 × 109, 1.0 × 109, and 1.5 × 109 cfu·kg-1 of the spore cells from these two strains resulted in a 0.9%, 1.9%, and 2.5% increase in body weight, respectively (linear P < 0.095). During the final 5 wk, the average daily gain (ADG) in weight was increased by the strains at amounts of 0.5 × 109, 1.0 × 109, and 1.5 × 109 cfu·kg-1 with a clear dosage effect (linear P < 0.05). However, neither the gain-to-feed ratio, the average daily feed intake, nor nutrient digestibility was affected by the supplementation. In blood, the endotoxin lipopolysaccharides, and two liver toxicity indicator enzymes; aspartate aminotransferase and lactate dehydrogenase were decreased (P < 0.05) in the 1.0 × 109 cfu·kg-1 spores-feeding group. Furthermore, four noxious gases were reduced by 8 to 20% in feces excreted by pigs fed with 1.5 × 109 cfu·kg-1 spores with a linear dosage effect (linear P < 0.001 to 0.05) during the final 5 wk. Our findings suggest that the mixture of B. subtilis strains may enhance the productivity of finishing pigs by reducing the risk of mild endotoxemia, rather than increasing digestibility or daily feed intake. Therefore, these Bacillus strains have the potential to act as growth promoters for pigs, leading to improved animal health and productivity. These results have significant implications for pig farmers seeking to optimize the health and growth of their animals.

In a previous study, we discovered two new strains of Bacillus subtilis that showed high surfactin secretion during growth in culture media. This surfactin proved effective in reducing endotoxin effects, particularly lipopolysaccharides, in vitro. To explore their potential as pig growth promoters, we administered 50:50 bacteria blend to 200 finishing pigs, dividing them into four groups for a 10-wk trial. Results showed that supplementing the pigs' diet with 0.5, 1.0, or 1.5 billion bacteria per kilogram led to weight gains of 0.9%, 1.9%, and 2.5%, respectively, with a dosage effect. The weight gain was notably higher during the final 5 wk. However, there were no significant differences in feed intake or nutrient digestibility. Blood analysis revealed reduced lipopolysaccharides and liver toxicity indicators, suggesting improved animal health. Moreover, the pigs that received the bacterial mixture showed reduced noxious gas levels in their feces with a dosage effect. These findings suggest that these new B. subtilis strains could serve as effective growth promoters for pigs by minimizing the risk of mild endotoxemia, leading to enhanced animal health and productivity. These results could have valuable implications for pig farmers seeking to optimize the health and growth of their animals.

A zinc-finger-family transcription factor, AbVf19, is required for the induction of a gene subset important for virulence in Alternaria brassicicola.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall-degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19-reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

Throughput screening of Bacillus subtilis strains that abundantly secrete surfactin in vitro identifies effective probiotic candidates.

Since the prohibition of antibiotics as animal growth promoters, demand for effective probiotic strains has steadily increased. The goal is to maintain productivity and mitigate environmental concerns in the livestock industry. There are many probiotic animal-diet supplements available, over 2,000 products in the Republic of Korea alone, with little explanation about the desirable properties of each probiotic strain. The purpose of this study was to describe the underlying logic and methods used to select two novel strains of probiotic candidates. To economically screen these candidates, the abundance of surfactin secreted was used as an in vitro marker. We used a modified oil-misting method to screen ~2,000 spore-forming bacteria for novel strains of Bacillus subtilis. Of these, 18 strains were initially selected based on the semiquantitative criterion that they secreted more surfactin than B. subtilis ATCC21322 on Luria-Berani (LB) agar plates. The whole genome sequence was determined for two of the 18 strains to verify their identity. A phylogeny of 1,162 orthologous genes, genome contents, and genome organization confirmed them as novel strains. The surfactin profiles produced by these two strains consisted of at least four isoforms similar to standard surfactin and enhanced cellulase activities up to 50%. Four fractionated individual isoforms of surfactin suppressed inflammation induced by lipopolysaccharides. The half-maximal inhibitory concentration (IC50) was about 20 µM for each isoform. Both selected strains were susceptible to seven important antibiotics. Our results implied that an abundant secretion of surfactin was a useful biomarker in vitro and could be utilized for mining probiotic candidates through high-throughput screening of environmental samples.

TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.

Identification of novel virulence factors associated with signal transduction pathways in Alternaria brassicicola.

Alternaria brassicicola is an important, necrotrophic fungal pathogen that causes black spot disease on Brassicas. In order to study pathogenicity mechanisms, gene deletion mutants were generated for 21 putative regulatory genes including kinases and transcription factors subjectively selected from the annotated A. brassicicola genome. Except for Ste12, the deletion of the SNF1 kinase, XlnR, and CreA homologues that control cell wall-degrading enzyme production did not significantly affect virulence in contrast to other pathogenic fungi. Only deletion of XlnR but not CreA, Ste12 or SNF1 impaired the fungus' ability to utilize sole carbon sources suggesting Alternaria regulates expression of cell wall-degrading enzymes in a novel manner. In addition, two novel virulence factors encoding a transcription factor (AbPro1) and a two-component histidine kinase gene (AbNIK1) were discovered. Deletion of AbPro1 resulted in a 70% reduction in virulence and a 25% reduction in vegetative growth rates in vitro. Deletion of AbNIK1 resulted in a near complete loss of virulence, increased sensitivity to osmotic stress, and no changes in vegetative growth rates in vitro. Interestingly, addition of long polypeptides to spores of both Deltaabste12 and Deltaabnik1 during inoculations resulted in a complete restoration of pathogenicity through a yet to be defined mechanism.

Frequent, phylogenetically local horizontal transfer of the cox1 group I Intron in flowering plant mitochondria.

Horizontal gene transfer is surprisingly common among plant mitochondrial genomes. The first well-established case involves a homing group I intron in the mitochondrial cox1 gene shown to have been frequently acquired via horizontal transfer in angiosperms. Here, we report extensive additional sampling of angiosperms, including 85 newly sequenced introns from 30 families. Analysis of all available data leads us to conclude that, among the 640 angiosperms (from 212 families) whose cox1 intron status has been characterized thus far, the intron has been acquired via roughly 70 separate horizontal transfer events. We propose that the intron was originally seeded into angiosperms by a single transfer from fungi, with all subsequent inferred transfers occurring from one angiosperm to another. The pattern of angiosperm-to-angiosperm transfer is biased toward exchanges between plants belonging to the same family. Illegitimate pollination is proposed as one potential factor responsible for this pattern, given that aberrant, cross-species pollination is more likely between close relatives. Other potential factors include shared vectoring agents or common geographic locations. We report the first apparent cases of loss of the cox1 intron; losses are accompanied by retention of the exonic coconversion tract, which is located immediately downstream of the intron and which is a product of the intron's self-insertion mechanism. We discuss the many reasons why the cox1 intron is so frequently and detectably transferred, and rarely lost, and conclude that it should be regarded as the "canary in the coal mine" with respect to horizontal transfer in angiosperm mitochondria.

Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola.

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola.

Mechanism of the natural product moracin-O derived MO-460 and its targeting protein hnRNPA2B1 on HIF-1α inhibition.

Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3'-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.

Prevalence of Rare Genetic Variations and Their Implications in NGS-data Interpretation.

Next-generation sequencing (NGS) technology has improved enough to discover mutations associated with genetic diseases. Our study evaluated the feasibility of targeted NGS as a primary screening tool to detect causal variants and subsequently predict genetic diseases. We performed parallel computations on 3.7-megabase-targeted regions to detect disease-causing mutations in 103 participants consisting of 81 patients and 22 controls. Data analysis of the participants took about 6 hours using local databases and 200 nodes of a supercomputer. All variants in the selected genes led on average to 3.6 putative diseases for each patient while variants restricted to disease-causing genes identified the correct disease. Notably, only 12% of predicted causal variants were recorded as causal mutations in public databases: 88% had no or insufficient records. In this study, most genetic diseases were caused by rare mutations and public records were inadequate. Most rare variants, however, were not associated with genetic diseases. These data implied that novel, rare variants should not be ignored but interpreted in conjunction with additional clinical data. This step is needed so appropriate advice can be given to primary doctors and parents, thus fulfilling the purpose of this method as a primary screen for rare genetic diseases.

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs.

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.

A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola.

Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development.