Mutation in a heat-regulated hsp70 gene of Ustilago maydis

Abstract

Four genes (ums1, ums2, ums3 and ums4) representing an hsp70-related gene family were isolated from a genomic library of Ustilago maydis. All four genes are transcriptionally active during normal growth. Following a heat shock, the mRNA levels of ums1 and ums2 increase by approximately 5-fold, whereas the ums3 transcript becomes less abundant. The amount of ums4 mRNA remains relatively unchanged after heat treatment. The nucleotide sequence of the 5' non-coding and a portion of the ums2 coding region was determined. The sequence encoding the first 90 amino acids is 73% identical to corresponding regions of the Drosophila and yeast (SSA1) hsp70 genes. To investigate the effect of a mutation in ums2, a plasmid was constructed in which most of the transcriptional unit of ums2 was deleted and substituted with the Escherichia coli hygromycin B (hygB) phosphotransferase gene. Transcription of this gene is controlled by the ums2 promoter, allowing the expression of hygB resistance in Ustilago. The marker was introduced into diploid cells as a linear sequence with termini homologous to the 5' and 3' flanking regions of ums2. In approximately 50% of transformants examined, one of the two wild-type ums2 alleles had been replaced by the mutated sequence, demonstrating the feasibility of using one-step gene disruption to create heterozygous diploids in Ustilago. The ums2/ums2::hygBr heterozygote produced teliospores after injection into corn plants, but only cells carrying functional ums2 were found among their meiotic progeny. Therefore ums2::hygBr segregates as a recessive lethal, which strongly suggests that ums2 is essential for growth in Ustilago.