Identification of cis-Active Elements in Ustilago maydis mig2 Promoters Conferring High-Level Activity During Pathogenic Growth in Maize

Abstract

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.

Thea2mating-type-locus genelga2ofUstilago maydisinterferes with mitochondrial dynamics and fusion, partially in dependence on a Dnm1-like fission component

“…The mrb1 ORF, deleted for the region encoding amino acids 1-40, extended by 998 bp of 5Ј region was generated by PCR-based mutagenesis as described (Farfsing et al, 2005) and inserted into the SbfI site of pCV11 to yield a translational fusion between mrb1 and myc. The 649-bp 3Ј region flanking the mrb1 ORF was introduced into the EcoRV-NcoI sites 3Ј of NOST.…”

Dissecting Defense-Related and Developmental Transcriptional Responses of Maize during Ustilago maydis Infection and Subsequent Tumor Formation

“…Strain JF1 is solopathogenic and thus is not relying on a mating partner to become pathogenic. In addition, based on the presence of an enhanced green fluorescent protein (eGFP) reporter construct under control of the mig2-5 promoter, hyphal growth within plant tissue can be assessed from fluorescence microscopy (Farfsing et al, 2005). This revealed that from 3 d after spotting of JF1 sporidia on the leaf surface, rare hyphal growth was observed in epidermal and underlying cells ( Fig.…”

“…hawita-gruppe.de), and plants were kept in the greenhouse with a 16-/8-hphoto (15,000-20,000 lux)/-dark period at 28°/20°C and a humidity of 35%/ 75%. Ustilago maydis strains FB1 (a1b1), FB2 (a2b2; Banuett and Herskowitz, 1989), FB1Dmrb1, FB2Dmrb1 (Bortfeld et al, 2004), AB311 (a1b1) and AB312 (a2b2; Brachmann et al, 2003), and JF1 (a1mfa2bE1bW2;Farfsing et al, 2005) were grown at 28°C in yeast/peptone/Suc (Tsukuda et al, 1988), potato dextrose (PD; DIFCO Laboratories, Detroit), or complete medium (Holliday, 1974). Sporidial cultures after overnight growth in yeast/peptone/Suc medium were adjusted to a cell density of 5 3 10 7 /mL in distilled water.…”

“…To address the interplay between U. maydis and its host, differential display analysis was performed comparing expression in leaf tumor and noninfected leaf tissue . The fungal genes retrieved from this approach were mig1 and mig2-1 to mig2-5, which display a pronounced plant-specific expression profile and encode small, secreted, Cys-containing proteins of unknown function, as well as udh1, which encodes a functional steroid 5a-reductase (Basse et al, , 2002a(Basse et al, , 2002bFarfsing et al, 2005). Here, I report the identification of maize genes that exhibit a marked differential expression profile during U. maydis infection.…”

Regulation of haplid phenotypes in Ustilago maydis by ammonium transporters and components of the b mating locus.

“…Additionally, the mig genes, encoding small, secreted, cysteine-containing proteins, are normally highly up-regulated upon penetration into host tissue (Farfsing, Auffarth, & Basse, 2005). Here, we found expression of mig 2-6 on low ammonium was dramatically reduced for FB1 wild-type and FB1Δump2 compared to their growth on nitrogen-replete medium, while expression for the ump2 overexpressor was even further reduced (see Table 4); moreover, the mutants also had reduced expression in replete medium relative to wild-type.…”