Analysis of gene transcripts during Ustilago maydis teliospore dormancy and germination

Ustilago maydis is a pathogenic basidiomycete that infects maize and its ancestor teosinte, causing tumour formation in the developing stems, leaves, tassels and ears of the plant. This pathogen can have a significant economic impact on corn production and serves as a model for less tractable fungal pathogens. The dispersal agent for U. maydis is the dormant diploid teliospore and it requires the plant for development. Teliospores are critical to disease spread and assessing gene expression at specific stages of their germination will provide knowledge of how to inhibit dispersal of these fungi. However, teliospores germinate asynchronously, therefore RNA isolated from teliospores induced to germinate contains mRNA from genes expressed at different morphological stages of germination. The focus of this research was to physically separate teliospores at different morphological stages of germination using counterflow centrifugal elutriation (CCE) and size-selective sieving. CCE was unsuccessful; however size-selective sieving resulted in an approximate 20% decrease in the number of haploid cells in teliospore samples induced to germinate. Varying the host corn variety and isolation of teliospores from a single tumour versus a mixture of tumours also had little effect on teliospore germination synchrony although rates of germination did vary between tumours. Without separation of the stages of germination, only gross changes in gene expression could be detected above background changes in transcript levels. Bioinformatic investigations identified genes that were candidates for expression during teliospore germination. Semi-quantitative RT-PCR results suggested that some of these genes may be involved in protecting the teliospore during dormancy and/or early stages of germination. While transcript levels determined using RT-qPCR supported roles for: 1) the U. maydis cdc5 ortholog in exit from the pachytene checkpoint, 2) the rad51 and rad2 orthologs in the early stages of germination, 3) a rsv2/rpn4 otholog in stimulating stress related gene expression during early germination and 4) Mcg1 in influencing gene expression during the exit from the pachytene checkpoint and the later stages of meiosis.;Keywords: smut fungi, teliospores, centrifugal elutriation, filtration, semi-quantitative RT-PCR, quantitative RT-PCR, dormancy, germination.