Yangrae Cho

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.

At Death's Door: Alternaria Pathogenicity Mechanisms

Christopher B. Lawrence*, Thomas K. Mitchell, Kelly D. Craven, Yangrae Cho, Robert A. Cramer Jr. and Kwang-Hyung Kim Virginia Bioinformatics Institute and Department of Biological Sciences, Blacksburg, VA 24061, USA Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA Department of Plant and Environmental Protection Sciences, University of Hawaii, Honolulu, HI 96822, USA Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA (Received on April 14, 2008; Accepted on May 23, 2008)

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

ABSTRACT 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.

Functional analysis of the Alternaria brassicicola non-ribosomal peptide synthetase gene AbNPS2 reveals a role in conidial cell wall construction

SUMMARY Alternaria brassicicola is a necrotrophic pathogen causing black spot disease on virtually all cultivated Brassica crops worldwide. In many plant pathosystems fungal secondary metabolites derived from non-ribosomal peptide synthetases (NPSs) are phytotoxic virulence factors or are antibiotics thought to be important for niche competition with other micro-organisms. However, many of the functions of NPS genes and their products are largely unknown. In this study, we investigated the function of one of the A. brassicicola NPS genes, AbNPS2. The predicted amino acid sequence of AbNPS2 showed high sequence similarity with A. brassicae, AbrePsy1, Cochliobolus heterostrophus, NPS4 and a Stagonospora nodorum NPS. The AbNPS2 open reading frame was predicted to be 22 kb in length and encodes a large protein (7195 amino acids) showing typical NPS modular organization. Gene expression analysis of AbNPS2 in wild-type fungus indicated that it is expressed almost exclusively in conidia and conidiophores, broadly in the reproductive developmental phase. AbNPS2 gene disruption mutants showed abnormal spore cell wall morphology and a decreased hydrophobicity phenotype. Conidia of abnps2 mutants displayed an aberrantly inflated cell wall and an increase in lipid bodies compared with wild-type. Further phenotypic analyses of abnps2 mutants showed decreased spore germination rates both in vitro and in vivo, and a marked reduction in sporulation in vivo compared with wild-type fungus. Moreover, virulence tests on Brassicas with abnps2 mutants revealed a significant reduction in lesion size compared with wild-type but only when aged spores were used in experiments. Collectively, these results indicate that AbNPS2 plays an important role in development and virulence.

Bioinformatic analysis of expressed sequence tags derived from a compatible Alternaria brassicicola-brassica oleracea interaction

SUMMARY Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on members of the Brassicaceae plant family. In order to identify candidate fungal pathogenicity genes and characterize a compatible host response, a suppression subtractive hybridization (SSH) cDNA library enriched for A. brassicicola and Brassica oleracea genes expressed during the interaction was created, along with a fungal cDNA library representing genes expressed during nitrogen starvation (NS). A total of 3749 and 2352 expressed sequence tags (ESTs) were assembled into 2834 and 1264 unisequence sets for the SSH and NS libraries, respectively. We compared two methods to identify the origins (plant vs. fungal) of ESTs in the SSH library using different classification procedures, with and without the availability of a database representing the A. brassicicola whole genome sequence and Brassicaceae-specific genes. BLASTX analyses of the 2834 unisequence set using the GenBank non-redundant database identified 114 fungal genes. Further BLASTN analyses of the genes with unidentifiable origin using a database consisting of the 1264 fungal unisequence set from the nitrogen-starved library identified 94 additional fungal genes. By contrast, BLASTN analyses of the same SSH unisequence set using a partially assembled A. brassicicola whole genome draft sequence identified a total of 310 unisequenes of fungal origin. Our results indicated that even a small number of organism-specific EST sequences can be very helpful to identify pathogen genes in a library derived from infected tissue, partially overcoming the limitation of the public databases for little studied organisms. However, using the whole genome draft sequence of A. brassicicola we were able to identify approximately 30% more fungal genes in the SSH library than without utilizing this resource. The putative role of specific fungal and plant genes identified in this study in a compatible interaction is discussed.

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.

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.

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.