Characterization of a Ustilago maydisGene Specifically Induced during the Biotrophic Phase: Evidence for Negative as Well as Positive Regulation

Abstract

The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle. To investigate the molecular events during infection, we used differential display to identify plant-induced U. maydis genes. We describe the U. maydis gene mig1 (for "maize-induced gene"), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection. mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development. Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication. mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP. Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation. Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation. During fungal growth in planta, sequence elements between positions ؊148 and ؊519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control. We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes.The phytopathogen Ustilago maydis belongs to the fungal class Basidiomycetes and causes smut disease in maize (4). All aerial parts of the host plant can be infected. Disease is initially characterized by tissue chlorosis and anthocyanin pigmentation and culminates in the development of plant tumors filled with masses of black teliospores. U. maydis adopts two different morphologies during its life cycle. Haploid sporidia grow yeastlike and can be propagated on artificial media. After fusion of two compatible sporidia, a filamentous dikaryon is generated; this structure is infectious. The dikaryon depends on the plant for further proliferation. Cell fusion, the morphologic switch, and pathogenicity are governed by two unlinked mating-type loci termed a and b (4). The a locus exists in two alleles, a1 and a2, each encoding a pheromone precursor and the receptor recognizing the pheromone of opposite mating type (7, 38). The multiallelic b locus contains two divergently transcribed genes, bE and bW, encoding a pair of homeodomain proteins (11,21,35). In pairwise combinations, bE and bW proteins from different alleles can dimerize and trigger subsequent pathogenic development (19).In nature, compatible haploid sporidia fuse on the leaf surface and the filamentous dikaryon differentiates in an appressorium-like structure that penetrates the host cell wall (36). Penetration through stomata has also been reported (5). From the infection site, the fungus spreads...

Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells

“…The function of these features is still unclear. For other plant induced genes, like MIG1 and LtmM, serial deletions in the promoter region provided information on the different effects of subregions on expression levels (Basse et al, 2000;May et al, 2008). LtmM expression was also shown to vary depending on the type of infected tissue (May et al, 2008).…”

“…For Avr9 and Mpg1 it was shown that expression (partly) depends on a general nitrogen response factors (NRF1 and NPR1, respectively) (Lau and Hamer, 1996;Perez-Garcia et al, 2001;Soanes et al, 2002). However, expression of other in planta induced genes does not respond to changes in nitrogen availability, for example AVR4, ECP1 and ECP2 (C. fulvum), AvrLm1 (Leptosphaeria maculans), MIG1 and the MIG2 gene cluster (Ustilago maydis) (Basse et al, 2002(Basse et al, , 2000Gout et al, 2006;Van den Ackerveken et al, 1994;Wubben et al, 1994). Even for the genes that are expressed in an environment with limited nitrogen, it is not known which circumstances induce expression in planta, since there are indications that within the host plant, nitrogen is not limiting Oliver, 2001, 2002).…”

Ustilago maydis – a valuable model system for the study of fungal dimorphism and virulence

“…It encodes secreted small proteins that contribute to the hydrophobicity of the fungal cell wall. Another gene, mig1, which is highly expressed during growth in the plant, was identified by differential display (Basse et al, 2000). From its derived amino acid sequence it was concluded that mig1 encodes a small, highly charged protein of unknown function which contains a functional Nterminal secretion sequence.…”

“…The promoter of this gene has been analysed for regulatory elements that are responsible for the expression pattern. It has been shown that this gene is subject to both negative and positive control (Basse et al, 2000). The detailed analysis of additional genes that exhibit a similar expression pattern will help to elucidate common regulatory elements involved in the activation of hostinduced genes.…”

“…From its derived amino acid sequence it was concluded that mig1 encodes a small, highly charged protein of unknown function which contains a functional Nterminal secretion sequence. Again, deletion of this gene did not result in loss of virulence (Basse et al, 2000). The promoter of this gene has been analysed for regulatory elements that are responsible for the expression pattern.…”