Genetic Dissection of T-DNA Insertional Mutants Reveals Uncoupling of Dikaryotic Filamentation and Virulence in Sugarcane Smut Fungus

The biotrophic basidiomycetous fungus Sporisorium scitamineum causing smut disease in sugarcane is characterized by a life-cycle composed of a yeast-like nonpathogenic haploid basidiosporial stage outside the plant and filamentous pathogenic dikaryotic hyphae within the plant. Under field conditions, dikaryotic hyphae are formed after mating of two opposite mating-type strains. However, the mechanisms underlying genetic regulation of filamentation and its association with pathogenicity and development of teliospores are currently unclear. This study has focused on the characterization and genetic dissection of haploid filamentous mutants derived from T-DNA insertional mutagenesis. Our results support the existence of at least three genotypes among the six haploid filamentous mutants that differentially contribute to virulence and development of the whip and teliospore, providing a novel foundation for further investigation of the regulatory networks associated with pathogenicity and teliospore development in S. scitamineum.

Microscopy studies of Tilletia controversa teliospore development in wheat ovaries and in culture

Teliospore formation begins in wheat ovaries when they are about 0.5 mm diam. Tilletia controversa hyphal cells initially invade host cells intercellularly. During development a narrow band of sporogenous hyphal cells develop into reticulated teliospores without hyphal attachments. The host tissue is utilized in the formation of mature teliospores. Eventually a thin layer of host tissue surrounds the mature teliospores. In contrast, when the teliospores form in medium hyphal tips appear to enlarge into developing teliospores. Light microscopy, SEM, and thin sections with TEM were used to determine whether teliospores formed from enlarged hyphal tips or from sporogenous cells which fragment from hyphae.Teliospores were obtained from USDA ARS experimental plots at Logan, UT. Wheat plants were inoculated and grown in a greenhouse. Tissues were fixed for 1 h or longer in 2% glutaraldehyde, 3% acrolein, 0.1 M Na-cacodylate buffer, pH 7.3; rinsed several times in the same buffer; postfixed in 1% OsO4 in 0.1 M Na-cacodylate buffer, pH, 7.3 for 2 h in an ice bath; and rinsed in 0.1 M Na-cacodylate buffer prior to dehydration in a graded ethanol series.