Templeton, MChristie, DMesarich, Carl2012-03-252011https://hdl.handle.net/2292/15253Fungi secrete a variety of proteins to mediate cell-to-cell, cell-to-substrate and cell-to-host adhesion. Many of these are secreted tandem-repeat proteins (STRPs). This research has focused on investigating the structure and function of ViCin1, a novel STRP from the apple scab fungus Venturia inaequalis, which has seven or eight cysteine-rich repeats of about 60 amino acids. Two paralogs and three orthologs of ViCin1 were identified in the genomes of V. inaequalis and the related pear pathogen V. pirina, but no other fungi, suggesting restriction to the Venturia genus. A ViCin1::EYFP promoter fusion was constructed to monitor ViCin1 expression. Expression was high in subcuticular runner hyphae and stromata in planta, and similar infection-like structures in vitro within cellophane membranes; specifically those undergoing processes of aggregation/adhesion and division. A high level of expression was also observed in aerial hyphae, with lower expression in conidiophores and conidia. A ViCin1::EYFP protein fusion corroborated this expression profile, with localization to an extracellular matrix material between cells and hyphal filaments of stroma-like structures and subcuticular-like runner hyphae, the interface between adherent aerial hyphae, and to a lesser extent, the surface of immature conidiophores and conidia. Localization to hyphal tips and septa suggested secretion by the apical- and subapical-directed exocytosis pathways, respectively. ViCin1 was silenced by RNAi; however transformants retained pathogenicity and were unaffected in the differentiation and morphology of infection and infection-like structures. This may suggest functional redundancy or a level of ViCin1 expression above the threshold required for an observable change in phenotype. Structures of one- and two-domain truncated versions of the ViCin1 protein (ViCin1-D1 and ViCin1-D1D2, respectively) were solved using solution-state heteronuclear NMR, and represent the first fungal STRP structures to be solved. Each repeat domain has a predominantly α-helical structure based on a core helix-loop-helix (HLH) motif, and is reminiscent of several repeat-domain proteins involved in mediating proteinprotein interactions; although protein-carbohydrate interactions are also possible. A multiple-sequence line-up between the ViCin1 repeat domains and the corresponding paralog and ortholog domains revealed several conserved and hyper-variable residues that, when plotted on the ViCin1-D1D2 structure, suggest importance in structural maintenance vi and/or function. Together, these results suggest that ViCin1 forms part of an extracellular matrix material involved in cell-to-cell adhesion, with a potential function involving protein-protein or protein-carbohydrate interactions.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmhttps://creativecommons.org/licenses/by-nc-sa/3.0/nz/ThesisCopyright: The authorQ112887240