Mutation in a heat-regulated hsp70 gene of Ustilago maydis.

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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 In approximately 50% of transformants examined, one of the two wild‐type ums2 alleles had been replaced by the mutated sequence, demonstrating the feasibility of using one‐step gene disruption to create heterozygous diploids in Ustilago.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.