The Ustilago maydis Cys2His2-type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stage.

The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys(2)His(2)-type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2-like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b-dependently regulated Cys(2)His(2)-type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings.

Promoters and their regulation in Ustilago maydis and other phytopathogenic fungi.

The lifestyle of phytopathogenic fungi is strongly determined by their environment. This implies that mechanisms providing for versatile gene regulation in response to external signals or during host associations exist. In Ustilago maydis, central players of pathogenic development are the high mobility group box protein Prf1 that binds to the pheromone response element and the homeodomain transcription factor b, which recognizes an hsg-like consensus motif known from yeast Mata1-Matalpha2 DNA binding. Transcription of prf1 is influenced by multiple inputs and this is reflected by its modular promoter structure. Analysis of the U. maydis mig promoters provides a link to transcriptional regulation during biotrophic growth. Furthermore, recognition of repeated GATA sequences as well as of triplet motifs by transcription factors with binuclear Zn(II)(2)Cys(6) DNA-binding domains appears to mediate diverse transcriptional responses relevant for phytopathogenic fungi. Although present studies shed some light on the complexity of transcriptional processes operating in phytopathogenic fungi, further investigation of promoter structures including the involvement of ubiquitous promoter elements is needed. Confronted with increasing genome-wide analysis, knowledge of promoter structures not only allows predicting transcriptional regulation, but might also advance our understanding of transcriptional networks.

Identification of cis-active elements in Ustilago maydis mig2 promoters conferring high-level activity during pathogenic growth in maize.

The Ustilago maydis mig2 cluster comprises five highly homologous genes that display a pronounced plant-specific expression profile. A 350-bp mig2-5 promoter fragment contained all elements sufficient to confer differential promoter activity. Mutational analysis of this region, fused to the green fluorescent protein reporter gene, allowed dissecting core promoter elements required for high-level promoter activity from elements conferring inducible expression in planta. In particular, the presence of several 5'-CCA-3' motifs within a short stretch of the mig2-5 promoter was decisive for inducible promoter activity. On this basis, we reconstituted an artificial promoter whose inducible activity specifically relied on multiple CCA motifs. In addition, we identified a novel mig2 homologous gene, mig2-6, that is not part of the mig2 cluster, but displayed the strongest differential expression profile among mig2 genes. The deletion of all six mig2 genes did not compromise the ability to induce tumor formation in infected maize plants. Comparative sequence analysis including the mig2-6 promoter revealed an over-representation of the consensus motif 5'-MNMNWNCCAMM-3'. We discuss putative transcriptional activators involved in mig2 regulation.