Comment[ArrayExpressAccession] E-GEOD-36781 MAGE-TAB Version 1.1 Public Release Date 2013-02-19 Investigation Title The transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola Comment[Submitted Name] The transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola Experiment Description Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. Several A. brassicicola genes have been characterized as virulence factors 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. Targeted gene disruption mutants for 114 of the genes with proteins predicted to contain at least one putative zinc-finger domain were produced and functionally analyzed. Six of these genes were associated with pathogenesis. Disruption mutants corresponding to five of the genes were ≥50% less virulent than the wild type. Unexpectedly, the mutants of one gene (designated Amr1) were 100% more virulent. Amr1 is a homolog of Cmr1, a transcription factor previously found to regulate melanin biosynthesis in several fungi. Gene deletion mutants (Δamr1) were created and their phenotypes characterized. The Δamr1 mutants utilized 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 not expressed in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at much higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that only a small number of transcription factors with zinc finger domains are needed to maintain strong virulence. Furthermore, a gene important for survival in nature negatively affected virulence, probably by less efficient use of pectin. We speculate that the functions of the Amr1 gene are important to the success of A. brassicicola as a competitive saprophyte and plant parasite gene expression profile comparisons between wild type and a transcription factor mutant during the late stage of host infection Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Cho Cho Ohm Person First Name Yangrae Yangrae Robin Person Email yangrae@hawaii.edu Person Affiliation University of Hawaii at Manoa Person Phone 808-956-5305 Person Fax 808-956-2832 Person Address Plant and Environmental Protection Sciences, University of Hawaii at Manoa, 3190 Maile Way, St John 317, Honolulu, HI, USA Person Roles submitter Protocol Name P-GSE36781-1 P-GSE36781-3 P-GSE36781-2 P-GSE36781-4 Protocol Description Conidia were harvested and washed three times with water. 2,000 conidia in 10ul water were inoculated. Libaray was constructed with TruSeq (Illumina kit) with mRNA enriched with Agencourt AMPureXP (Beckman Coulter) All host plant, Brassica oleracea were grown under 14 hours light 10 hour dark cycle. Both wild type and Δamr1-4 mutant were grown on PDA plates for 7 days before conidium harvest Sequence tags were mapped to the genome sequence of A. brassicicola using the programs TopHat 1.3.1 [69] and Bowtie 0.12.7 [70]. Default settings were used, except the segment length was set at 25 nucleotides and the number of allowed segment mismatches was set at 1 nucleotide. Additionally, intron length was designated as a minimum of 10 nucleotides and a maximum of 400 nucleotides. The program Cuffdiff, version 1.0.3, which is part of Cufflinks [71], was used to identify reads overlapping with previously predicted genes. The expression levels of each predicted gene were determined and normalized by the mapped Fragments Per Kilobase of exon model per Million (FPKM). Differentially expressed genes between the wild type and the mutant were determined by comparing FPKMs from three biological replicates for both the wild type and the mutant. We also applied a cutoff of at least a two-fold change in expression value for differential expression. The bias correction method was used while running Cuffdiff [72]. Custom scripts were written in Python to analyze the data. Processed data linked as supplementary files on Series record. Genome_build: version 1; http://jgi.doe.gov/Abrassicicola Supplementary_files_format_and_content: Processed data files describing proteinId, gene name, expression value of WT, expression value of Δamr1, fold-change, significant difference below P-value 0.05, biological sample that show higher expreesion, PFAM annotation, and GO annotation. Protocol Type specified_biomaterial_action nucleic acid library construction protocol grow feature_extraction Experimental Factor Name VARIATION Experimental Factor Type variation Publication Title Transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola. Publication Author List Cho Y, Srivastava A, Ohm RA, Lawrence CB, Wang KH, Grigoriev IV, Marahatta SP PubMed ID 23133370 Publication DOI 10.1371/journal.ppat.1002974 Comment[SecondaryAccession] GSE36781 Comment[GEOReleaseDate] 2013-02-19 Comment[ArrayExpressSubmissionDate] 2012-03-25 Comment[GEOLastUpdateDate] 2013-02-19 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE36781_WT_Mut_expression_submitted.txt Comment[AdditionalFile:Data2] GSE36781_gene_exp_diff.txt Comment[AdditionalFile:Data3] GSE36781_genes_fpkm_tracking.txt Comment[SecondaryAccession] SRP011934 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR448374-SRR448383 SDRF File E-GEOD-36781.sdrf.txt