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Disruption analysis of genes encoding PKA C-subunit in Ustilago maydis

University of British Columbia

Ustilago maydis is a dimorphic fungus that undergoes a transition from yeast-like budding growth to filamentous growth upon compatible mating interactions or in response to certain environmental conditions. Previous studies have shown that unregulated (elevated) PKA (protein kinase A) activity, for example, in mutants defective for the regulatory subunit of PKA, results in a multiple-budding phenotype. Presumably, this phenotype is due to defects in cell separation and bud site selection. On the other hand, cells with low PKA activity, resulting from inactivation in adenylate cyclase, display constitutively filamentous growth. These results lead to the prediction that disruption of the catalytic (C) subunit of PKA would result in a constitutively filamentous phenotype like that observed in mutants defective in adenylate cyclase. In this study, it was demonstrated that disruption of the adrl gene, previously isolated from U. maydis and predicted to encode a PKA C-subunit, indeed resulted in a constitutively filamentous phenotype. Cells carrying the disrupted adrl gene also showed reduced virulence, as was seen for adenylate cyclase mutants in previous studies. In addition, mutant cells defective in both the adrl and ubcl (encoding the regulatory subunit of PKA) genes displayed hyphal growth, identical to the growth pattern seen with a mutant disrupted only in adrl. Interestingly, when another putative PKA C-subunit-encoding gene (ukal), was isolated and disrupted, the resulting mutant exhibited no detectable phenotypic differences in morphology, mating and virulence compared with wild type. When both the ukal and ubcl genes were disrupted, a modified multiple-budding pattern was detected, where cells not only produced multiple buds but also commonly formed chains of cells. Mutants carrying disruptions of the two putative PKA C-subunit-encoding genes displayed somewhat attenuated filamentous growth compared to the single adrl gene disruption mutant. This reduction of "fuzziness" was more noticeable in terms of colony morphology, but less obvious for cellular morphology. The results described in this thesis provide further evidence for a role of the cAMP/PKA signal transduction pathway in the morphological transition of 17. may dis. Similar observations have been made in some other dimorphic fungi. Moreover, the results presented here illustrate that despite the fact that both the ukal and adrl genes were predicted to encode PKA C-subunits in U. maydis, each PKA C-subunit seems to have a different cellular function; Adrl appeared to play a major role in morphogenesis while Ukal plays a more minor role.

Thesis/Dissertation

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2009-02-12

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http://hdl.handle.net/2429/4531

Microbiology and Immunology

University of British Columbia

UBCV

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