An activating mutation of the Sclerotinia sclerotiorum pac1 gene increases oxalic acid production at low pH but decreases virulence

The production of oxalic acid by Sclerotinia sclerotiorum is regulated by the ambient pH environment. This regulation and that of a few investigated pH-responsive genes is mediated in part by the zinc finger transcription factor encoded by pac1, an orthologue of the Aspergillus nidulans pacC gene. We manipulated the pac1 sequence by site-directed mutagenesis to create a dominant activating pac1(c) mutation and introduced this allele into a pac1 loss-of-function (Delta pac1) strain. Consistent with a constitutive activation of Pac1 function, oxalic acid accumulation in recovered Delta pac1-pac1(c) strains was largely independent of ambient pH. Likewise, all three Delta pac1-pac1(c) strains accumulated detectable pac1 transcripts in a pH 3 environment; however, accumulation of pac1 transcripts remained alkaline-inducible, but much reduced relative to wild-type in two of the three Delta pac1-pac1(c) strains. Surprisingly, the accumulation of pg1 and acp1 transcripts, normally favoured by low pH conditions, were up-regulated across the range of ambient pH conditions examined (pH 3.4-7.2). Accumulation of neutral pH-expressed enclopolygalacturonase-6 (pg6) transcripts, however, did not differ from wild-type. In pathogenicity assays using Arabidopsis and detached tomato leaflets, Delta pac1-pac1(c) strains were reduced in virulence despite the ability to accumulate oxalic acid independent of the prevailing ambient pH environment. These results support the hypothesis that appropriate gene regulation in response to ambient pH is important for full S. sclerotiorum virulence independent of oxalic acid accumulation.