Regulation of Ustilago maydis sporulation in maize

Abstract

The plant pathogenic fungus Ustilago maydis causes corn smut disease on maize plants. The lifecycle of this semi-obligate fungus from phylum basidiomycota is completed when it releases teliospores from the galls it produces on its host. U. maydis is a model system for other obligate pathogens, such as the rusts and smuts, which are responsible for huge losses in agriculture worldwide. To better understand the development of these important fungal diseases of plants, we have studied the maize-dependent sporulation of U. maydis by undertaking two projects. In the first project, we examined the function of the fungal-specific velvet family in U. maydis. This gene family is known to regulate many aspects of growth and development in from ascomycota phylum. We found three members of the velvet family in the U. maydis genome. These genes, umv1, umv2 and umv3 were deleted individually and the resulting mutants were analyzed. In the umv1 deletion mutants, disease was blocked in maize seedlings before the teliospore formation stage. When umv2 was deleted, there was a delay in the formation of teliospores in planta and a reduction in virulence. The umv3 deletion mutants did not show any effect on disease. When the umv1 and umv2 deletion mutants were complemented with native copies of each gene, virulence rose back to normal levels. Thus, we show that two members of the velvet gene family function in teliospore development and disease progression in the basidiomycota fungus U. maydis. In the second project, we cloned and attempted to alter expression of a maize gene predicted to influence sexual development in U. maydis. This gene, called ZmfluG, is homologous to fluG in the saprobic fungus Aspergillus nidulans and nodGS in Arabidopsis thaliana. In A. nidulans, FluG secretes an extracellular factor required for asexual sporulation, while in A. thaliana NodGS expression increases during biotic stress. The absence of a FluG homolog in U. maydis led us to hypothesize that maize ZmfluG might produce an extracellular signal essential for U. maydis sporulation in planta. To find evidence of this gene’s role, first we introduced ZmfluG into U. maydis, but we did not see any effect on U. maydis’ growth or pathogenicity. Second, maize plants were transformed with a ZmfluG RNAi construct. When we analyzed the resulting maize lines for herbicide resistance, we found that leaf sensitivity to the herbicide spray did not match with the results from a PCR screen for the herbicide resistance gene. Out of the 12 ZmFluG RNAi lines that were grown, three lines did not germinate at all. We also made constructs that overexpress ZmfluG, which can be transformed into maize in the future. Future analysis to confirm silencing of ZmfluG in the RNAi lines would allow us to examine U. maydis’ sporulation in them.