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Author(s): Jörg Kämper (corresponding author) [1]; Regine Kahmann (corresponding author) [1]; Michael Bölker (corresponding author) [2]; Li-Jun Ma [3]; Thomas Brefort [1]; Barry J. Saville [4, 27]; Flora Banuett [5]; [...]

As a biotrophic fungus, Ustilago maydis, the causal agent of common smut of maize, must establish and maintain a relationship with the host throughout the pathogenic cycle. Functional characterization of the U. maydis transcription factor Zfp1 suggests a role in modulating pathogenic development. Deletion of zfp1 resulted in attenuated pathogenic hyphal growth, reduced infection frequency, arrested disease symptom development, and decreased anthocyanin production. Complementation with Zfp1 constructs lacking conserved domains indicated it may function with and without DNA binding and suggests a role for the Fungal_trans domain in enabling full virulence. RNA‐sequencing revealed the transcription of approximately 30% of U. maydis genes, including those encoding predicted and confirmed effectors, were altered in the zfp1 deletion strain during pathogenic growth. Together, the results suggest that Zfp1 is a Zn(II)2Cys6 transcription factor that is required for the full induction of infection, anthocyanin production, and virulence. [ABSTRACT FROM AUTHOR]

Rhizobium tropici type B strains induce foliar chlorosis if applied to seeds of symbiotic host and non-host dicotyledonous plants. CT4812, a Tn5-induced mutant of R. tropici strain CIAT899, unable to induce chlorosis, has been isolated. The Tn5 insertion was found to be located in a similar gene to glnD, encoding uridylyltransferase/uridylyl-removing enzyme in enteric bacteria. A mutation in mviN, the Salmonella typhimurium virulence gene, had no effect on CIAT899's ability to induce chlorosis in bean plants.

The emergence of new fungal pathogens threatens sustainable crop production worldwide. One mechanism by which new pathogens may arise is hybridization. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they both infect Zea mays, can hybridize, and tools are available for their analysis. The hybrid dikaryons of these fungi grew as filaments on plates but their colonization and virulence in Z. mays were reduced compared to the parental dikaryons. The anthocyanin induction caused by the hybrid dikaryon infections was distinct, suggesting its interaction with the host was different from that of the parental dikaryons. Selected virulence genes previously characterized in U. maydis and their predicted S. reilianum orthologs had altered transcript levels during hybrid infection of Z. mays. The downregulated U. maydis effectors, tin2, pit2, and cce1, and transcription factors, rbf1, hdp2, and nlt1, were constitutively expressed in the hybrid. Little impact was observed with increased effector expression; however, increased expression of rbf1 and hdp2, which regulate early pathogenic development by U. maydis, increased the hybrid's capacity to induce symptoms including the rare induction of small leaf tumors. These results establish a base for investigating molecular aspects of smut fungal hybrid pathogen emergence. [ABSTRACT FROM AUTHOR]

Abstract Background Ustilago maydis is the basidiomycete fungus responsible for common smut of corn and is a model organism for the study of fungal phytopathogenesis. To aid in the annotation of the genome sequence of this organism, several expressed sequence tag (EST) libraries were generated from a variety of U. maydis cell types. In addition to utility in the context of gene identification and structure annotation, the ESTs were analyzed to identify differentially abundant transcripts and to detect evidence of alternative splicing and anti-sense transcription. Results Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 × 521) and haploid cells of strain 521 grown under nutrient rich, carbon starved, and nitrogen starved conditions. Using the genome sequence as a scaffold, the 15,901 ESTs were assembled into 6,101 contiguous expressed sequences (contigs); among these, 5,482 corresponded to predicted genes in the MUMDB (MIPS Ustilago maydis database), while 619 aligned to regions of the genome not yet designated as genes in MUMDB. A comparison of EST abundance identified numerous genes that may be regulated in a cell type or starvation-specific manner. The transcriptional response to nitrogen starvation was assessed using RT-qPCR. The results of this suggest that there may be cross-talk between the nitrogen and carbon signalling pathways in U. maydis. Bioinformatic analysis identified numerous examples of alternative splicing and anti-sense transcription. While intron retention was the predominant form of alternative splicing in U. maydis, other varieties were also evident (e.g. exon skipping). Selected instances of both alternative splicing and anti-sense transcription were independently confirmed using RT-PCR. Conclusion Through this work: 1) substantial sequence information has been provided for U. maydis genome annotation; 2) new genes were identified through the discovery of 619 contigs that had previously escaped annotation; 3) evidence is provided that suggests the regulation of nitrogen metabolism in U. maydis differs from that of other model fungi, and 4) Alternative splicing and anti-sense transcription were identified in U. maydis and, amid similar observations in other basidiomycetes, this suggests these phenomena may be widespread in this group of fungi. These advances emphasize the importance of EST analysis in genome annotation.