Downloaded from www.microbiologyresearch.org by IP: 54.198.95.179 On: Fri, 09 Sep 2016 03:08:11 Fusarium verticillioides GAP1, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence Uma Shankar Sagaram, Brian D. Shaw and Won-Bo Shim Correspondence Won-Bo Shim wbshim@tamu.edu Department of Plant Pathology and Microbiology, Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, TX 77843-2132, USA Received 1 March 2007 Revised 3 May 2007 Accepted 9 May 2007 Fusarium verticillioides is an important pathogen of maize that causes ear rot and produces the mycotoxins known as fumonisins. To date, knowledge of pathogenicity and the regulation of fumonisin biosynthesis in F. verticillioides is limited. Here, the molecular characterization of GAP1, a gene encoding a putative 540 aa protein that belongs to a glycolipid-anchored surface (GAS) protein family, is presented. F. verticillioides GAP1 was identified as an expressed sequence tag (EST) upregulated in a culture condition conducive to conidiation and fumonisin B 1 (FB 1 ) production. GAP1 null mutants GAM126 (Dgap1 :: HYG) and GAG8 (Dgap1 :: GEN) exhibited restricted growth, with more aerial hyphae than their wild-type progenitor on solid media. No defect in mycelial mass or filamentous growth was observed when the GAM126 and GAG8 strains were grown in liquid media under shaking conditions. When grown in suspended conditions, GAM126 and GAG8 strains produced significantly fewer conidia and produced comparatively densely branched hyphae. Concanavalin A staining indicated that the GAP1 deletion altered the cell wall carbohydrate composition/deposition process. Deletion of GAP1 did not affect the production level of FB 1 or F. verticillioides virulence on maize seedlings and stalks. Complementation of GAM126 with the wild-type GAP1 gene restored growth, conidiation and cell wall abnormality phenotypes. The results suggest that GAP1 is associated with growth, development and conidiation in F. verticillioides, but not with pathogenicity or regulation of FB 1 . INTRODUCTION Fusarium verticillioides (Sacc.) Nirenberg (teleomorph: Gibberella moniliformis Wineland) causes rots of maize (Zea mays) stalks and ears (Munkvold & Desjardins, 1997). Stalk rot of maize is widespread in dry, warm climates (White, 1999), and can cause losses in several different ways, such as stalk breakage, lodging, interruption of the normal grain-filling process, and sometimes premature death of the plant (Dodd, 1980; Michaelson, 1957; White, 1999). On maize ears, the fungus can produce polyketide-derived carcinogenic mycotoxins called fumo- nisins (Nelson et al., 1993; Marasas, 2001). Since the discovery of fumonisins, several efforts have been made to understand the regulation of fumonisin biosynthesis in the F. verticillioides–maize system (reviewed by Sagaram et al., 2006). Briefly, fumonisins are synthesized by a cluster of 22 FUM genes, of which 15 are co-regulated (Proctor et al., 1999, 2003). Recently, a putative regulatory gene within the FUM cluster, FUM21, has been identified and characterized (Brown et al., 2007). However, FUM21 gene deletion is not sufficient to completely block fumonisin biosynthesis, suggesting that transcriptional regulation of FUM genes is also affected by regulatory genes outside the cluster. Fumonisin biosynthesis is also impacted by several environmental factors, including available nitrogen, ambi- ent pH and maize kernel constitution environment (reviewed by Sagaram et al., 2006). The first regulatory gene identified by the restriction enzyme-mediated integration (REMI) strategy was FCC1, which encodes a C-type cyclin (Shim & Woloshuk, 2001). Mutation in FCC1 leads to a severe reduction in Abbreviations: CFW, Calcofluor white; ConA, concanavalin A; CR, Congo red; EST, expressed sequence tag; FB 1 , fumonisin B 1 ; GAS protein, glycolipid-anchored surface protein; GPI, glycosylphosphatidylinositol; SSH, suppression subtractive hybridization. The GenBank/EMBL/DDBJ accession no. for the 350 bp GAP1 gene sequence from F. verticillioides is DQ458798. Figures showing the F. verticillioides seedling rot assay on hybrid sweetcorn, and Northern analysis of GAP2 and GAP3 expression in F. verticillioides, are available as supplementary data with the online version of this paper. Microbiology (2007), 153, 2850–2861 DOI 10.1099/mic.0.2007/007708-0 2850 2007/007708 G 2007 SGM Printed in Great Britain