Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs.

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene's function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5' and 3' flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens.

The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms' pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses identified a set of proteins that are shared among all three wilt pathogens, and present in few other fungal species. One of these is a homolog of a bacterial glucosyltransferase that synthesizes virulence-related osmoregulated periplasmic glucans in bacteria. Pathogenicity tests of the corresponding V. dahliae glucosyltransferase gene deletion mutants indicate that the gene is required for full virulence in the Australian tobacco species Nicotiana benthamiana. Compared to other fungi, the two sequenced Verticillium genomes encode more pectin-degrading enzymes and other carbohydrate-active enzymes, suggesting an extraordinary capacity to degrade plant pectin barricades. The high level of synteny between the two Verticillium assemblies highlighted four flexible genomic islands in V. dahliae that are enriched for transposable elements, and contain duplicated genes and genes that are important in signaling/transcriptional regulation and iron/lipid metabolism. Coupled with an enhanced capacity to degrade plant materials, these genomic islands may contribute to the expanded genetic diversity and virulence of V. dahliae, the primary causal agent of Verticillium wilts. Significantly, our study reveals insights into the genetic mechanisms of niche adaptation of fungal wilt pathogens, advances our understanding of the evolution and development of their pathogenesis, and sheds light on potential avenues for the development of novel disease management strategies to combat destructive wilt diseases.

One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi.

Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Previously we reported a method called DelsGate for rapid preparation of deletion constructs for protoplast-mediated fungal transformation systems, which is based on Gateway® technology. However, over the past several years Agrobacteriumtumefaciens-mediated transformation (ATMT) has become the preferred genetic transformation method for an increasing number of fungi. Therefore, we developed a method for One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), to rapidly create deletion constructs for ATMT systems. The OSCAR methodology involves PCR amplification of the upstream and downstream flanks of the gene of interest, using gene specific primers each with a 5' extension containing one of four different attB recombination sites, modified from the Invitrogen MultiSite Gateway® system. Amplified gene flanks are then mixed with specifically designed marker and binary vectors and treated with BP clonase, generating the deletion construct in a single cloning step. The entire process of deletion construct preparation can be accomplished in just 2days. Using OSCAR we generated eight targeted deletion constructs and used two of them to generate deletion mutants in Verticillium dahliae by ATMT. In summary, OSCAR methodology combines PCR and Gateway® technology to rapidly and robustly generate precise deletion constructs for fungal ATMT and homologous gene replacement.

DelsGate: a robust and rapid method for gene deletion.

Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of generation of deletion mutants by greatly simplifying the production of gene deletion constructs. With this purpose we have developed a method, which we named DelsGate (Deletion via Gateway), that combines PCR and Gateway cloning technology together with the use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. DelsGate consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for the transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. The PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. We describe here the entire process, from the generation of the deletion construct with DelsGate to the analysis of the fungal transformants to test for gene replacement, with the Basidiomycete fungus Ustilago maydis. Application of DelsGate to other fungal species is also underway. Additionally, we describe how this basic approach can be adapted to other genetic manipulations with minor changes. We specifically describe its application to create unmarked deletions in Ralstonia solanacearum, a Gram-negative phytopathogenic bacterium.

Synthesis and antifungal activity of beta-trifluoroalkyl aminovinyl ketone derivatives.

Ten beta-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effects on several phytopathogenic fungi, an oomycete and plants were assessed. The various compounds were fungitoxic at the 10-100 microM range, with (Z)-3-amino-4,4,4-trifluoro-1-(4-chlorophenyl)but-2-en-1-one exhibiting the highest inhibitory effect on most of the test pathogens. Alternaria alternata and Neurospora crassa were the most tolerant and sensitive fungi to the compounds, respectively. We propose that (Z)-3-amino-4,4,4-trifluoro-1-phenylbut-2-en-1-one is the minimal structural requirement for a beta-trifluoroalkyl aminovinyl ketone fungitoxic derivative.