The gene encoding a development-specific protein (Ssp1) was identified; it previously was described as the major protein present in mature sclerotia of Sclerotinia sclerotiorum. To determine the developmental specificity of ssp1 gene expression in relation to protein accumulation we examined transcript and protein accumulation during various growth and development stages of the lifecycle. We found that ssp1 transcript accumulated exclusively within developing sclerotium tissue and not in any other examined stage of growth or development. In contrast high levels of Sspl protein were detectable by western blot and tandem mass spectrometry analyses in all stages of sclerotium as well as apothecium development. Immunolocalization further indicated that Ssp1 protein bodies were depleted from the sclerotium tissue surrounding the site of apothecium germination, but by this method Sspl was not detected in the apothecium. Together these findings suggest that Sspl is not metabolized during carpogenic germination, instead it is translocated from the sclerotium to the apothecium in an antigenically novel form. Outside the Sclerotiniaceae ssp1 homologs were found only from the sclerotium-forming Aspergillus species A. flavus and A. oryzae. Further studies concerning the regulation and function of this gene and its occurrence in other species have the potential to inform our understanding of sclerotium development and the evolution of sclerotia and other forms of fungal stroma.
Transcripts encoding Sclerotinia sclerotiorum γ-glutamyl transpeptidase (Ss-Ggt1) were found to accumulate specifically during sclerotium, apothecium, and compound appressorium development in S. sclerotiorum. To determine the requirement of this protein in these developmental processes, gene deletion mutants of Ss-ggt1 were generated and five independent homokaryotic ΔSs-ggt1 mutants were characterized. All deletion mutants overproduced sclerotial initials that were arrested in further development or eventually produced sclerotia with aberrant rind layers. During incubation for carpogenic germination, these sclerotia decayed and failed to produce apothecia. Total glutathione accumulation was approximately 10-fold higher and H(2)O(2) hyperaccumulated in ΔSs-ggt1 sclerotia compared with the wild type. Production of compound appressoria was also negatively affected. On host plants, these mutants exhibited a defect in infection efficiency and a delay in initial symptom development unless the host tissue was wounded prior to inoculation. These results suggest that Ss-Ggt1 is the primary enzyme involved in glutathione recycling during these key developmental stages of the S. sclerotiorum life cycle but Ss-Ggt1 is not required for host colonization and symptom development. The accumulation of oxidized glutathione is hypothesized to negatively impact these developmental processes by disrupting the dynamic redox environment associated with multicellular development.
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