SummaryDevelopment of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by pathogens are called 'avirulence' proteins. Their identification is important for being able to assess the usefulness and durability of resistance genes in agricultural settings.We have used genome sequencing of a set of strains of the melon wilt fungus Fusarium oxysporum f. sp. melonis (Fom), bioinformatics-based genome comparison and genetic transformation of the fungus to identify AVRFOM2, the gene that encodes the avirulence protein recognized by the melon Fom-2 gene.Both an unbiased and a candidate gene approach identified a single candidate for the AVRFOM2 gene. Genetic complementation of AVRFOM2 in three different race 2 isolates resulted in resistance of Fom-2-harbouring melon cultivars. AvrFom2 is a small, secreted protein with two cysteine residues and weak similarity to secreted proteins of other fungi.The identification of AVRFOM2 will not only be helpful to select melon cultivars to avoid melon Fusarium wilt, but also to monitor how quickly a Fom population can adapt to deployment of Fom-2-containing cultivars in the field.
Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes.
Here we present SMAP, a software package that implements a suite of computational tools to extract multi-allelic haplotypes using read-backed haplotyping. SMAP tools first perform accurate read processing and analyze read mapping distributions across sample sets. Then, two complementary modules can be invoked for haplotype calling: SMAP haplotype-sites combines known Single Nucleotide Polymorphisms (SNPs) and/or read mapping position polymorphisms (SMAPs) to reconstruct compressed, read-reference-encoded haplotype strings. In contrast, SMAP haplotype-window works independent of prior knowledge of polymorphisms, groups reads by locus, defines a window enclosed between two custom border sequences, and retains the entire corresponding DNA sequence as haplotype. Haplotype-window is, among many applications, especially useful for high-throughput CRISPR/Cas mutation screens. Either way, SMAP creates a single integrated haplotype call table across all loci and samples. SMAP haplotyping is extremely versatile and can be applied to highly multiplex amplicon sequencing (HiPlex), Shotgun (e.g. whole genome shotgun (WGS) sequencing, probe capture and RNA-Seq), or Genotyping-by-Sequencing (GBS) data; and to Illumina short reads, PacBio and MinION long reads. SMAP creates discrete genotype calls for individuals of any ploidy or quantitative haplotype frequency spectra for Pool-Seq data, and can scale from tens to thousands of loci and/or samples. SMAP, including the source code written in Python is available at https://gitlab.com/truttink/smap, and a detailed user manual and guidelines for accurate read processing is available at https://ngs-smap.readthedocs.io/, under the GNU Affero General Public License v3.0.
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