Functional analysis of genes involved in cAMP-mediated signaling in the wide host range necrotroph Sclerotinia sclerotiorum (Lib.) de Bary.

摘要

The filamentous ascomycete phytopathogen, Sclerotinia sclerotiorum (Lib.) de Bary, is a broad host range necrotroph with global distribution. Dissemination and survival of this fungus is attributed to resting structures termed sclerotia that can remain viable in the soil for years. A sclerotium can give rise to multiple mushroom-shaped apothecia that are the source of forcibly-discharged ascospores that serve as the primary source of inoculum for Sclerotinia diseases. Genetic regulators of sclerotial development and pathogenesis were determined by functionally characterizing, a cAMP-dependent protein kinase A catalytic subunit gene (pka1) and an adenylate cyclase gene (sac1), that are involved in cAMP-mediated signal transduction. Contrary to our original hypothesis, pka1 loss-of-function mutants were cAMP-responsive, produced wild type like sclerotia, and were pathogenic. This finding prompted the making of a new hypothesis that a second PKA catalytic subunit gene, pka2 , contributes the majority of PKA activity in the cell. Mining recently available data from the S. sclerotiorum genome project uncovered the second hypothesized pka catalytic subunit gene, pka2. In a second approach to determine the biological role of cAMP in S. sclerotiorum, an adenylate cyclase gene, sac1, was mutated by targeted deletion. Adenylate cyclase knock-out (AC-KO) mutants revealed several morphological defects including a reduced rate of mycelial expansion and the production of aberrant sclerotia in concentric rings in culture. Sclerotia were competent for myceliogenic germination, but did not carpogenically germinate and form apothecia under standard conditions. AC-KO mutants produced 90°-branched hyphae in contrast to acutely branched hyphae observed in wild type. Cyclic AMP levels were greatly reduced in the AC-KO and it was non-pathogenic on detached tomato leaflets. The AC loss-of-function mutant was capable of colonizing mechanically wounded leaves, but the rate of lesion expansion was much slower than wild type. Loss of pathogenicity may be attributed to the lack of infection cushion formation in the AC-KO strain as determined by in vitro morphogenesis assays. Slower growth rate observed in culture and in planta appears to account for the reduced virulence in wounded plants. Taken together, these results demonstrate that cAMP plays important roles in pathogenicity, growth and vegetative development in S. sclerotiorum.