Vol. 26, No. 6, 2013 / 695 MPMI Vol. 26, No. 6, 2013, pp. 695–708. http://dx.doi.org/10.1094/MPMI-01-13-0003-R. Infection Structure-Specific Reductive Iron Assimilation Is Required for Cell Wall Integrity and Full Virulence of the Maize Pathogen Colletotrichum graminicola Emad Albarouki 1 and Holger B. Deising 1,2 1 Interdisziplinäres Zentrum für Nutzpflanzenforschung and 2 Institut für Agrar- und Ernährungswissenschaften, Phytopathologie und Pflanzenschutz, Martin-Luther-Universität Halle-Wittenberg, Betty-Heimann-Str. 3, D-06120 Halle (Saale), Germany Submitted 5 January 2013. Accepted 20 February 2013. Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxi- dase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colleto- trichum graminicola. Complementation of growth defects of the ferroxidase-deficient Saccharomyces cerevisiae strain Δfet3fet4 showed that both Fet3-1 and Fet3-2 of C. graminicola represent functional ferroxidases. Expression of enhanced green fluorescent protein fusions in yeast and C. graminicola indicated that both ferroxidase proteins localize to the plasma membrane. Transcript abundance of FET3-1 increased dramatically under iron-limiting conditions but those of FET3-2 were hardly detectable. Δfet3-1 and Δfet3-2 single as well as Δfet3-1/2 double-dele- tion strains were generated. Under iron-sufficient or defi- cient conditions, vegetative growth rates of these strains did not significantly differ from that of the wild type but Δfet3-1 and Δfet3-1/2 strains showed increased sensitivity to reactive oxygen species. Furthermore, under iron-limit- ing conditions, appressoria of Δfet3-1 and Δfet3-1/2 strains showed significantly reduced transcript abundance of a class V chitin synthase and exhibited severe cell wall defects. Infection assays on intact and wounded maize leaves, quantitative data of infection structure differentiation, and infection stage-specific expression of FET3-1 showed that reductive iron assimilation is required for appresso- rial penetration, biotrophic development, and full viru- lence. Iron is the most prominent redox element in living cells and a co-factor of enzymes catalyzing rapid redox reactions such as iron-sulfur proteins and cytochromes. Thus, iron is consid- ered an indispensable element for almost all organisms (Haas 2003; Kosman 2003). Paradoxically, although an essential ele- ment, iron can be harmful to cells, due to its participation in production of hydroxyl radicals by the Fenton/Haber Weiss re- action (Halliwell and Gutteridge 1984, 1992). Therefore, iron homeostasis must be tightly controlled by iron concentration- dependent regulation of iron uptake, transport and, storage within cells and organisms. Although iron is the fourth most abundant element in Earth’s crust, its bioavailability is extremely low. At neutral pH, the solubility product of Fe 3+ , the main oxidation state in an aerobic environment, is less than 10 –18 M (Neilands et al. 1987). Compared with iron-rich terrestrial environments, the availability of iron may be significantly lower for pathogenic fungi growing in plant or animal hosts (Expert 1999; Weinberg 1978, 2009), due to tight iron sequestration by ferritin, trans- ferrins, or several other iron-binding proteins (Arosio and Levi 2002; Ratledge and Dover 2000). Because iron-dependent gen- eration of reactive oxygen species (ROS) contributes to the first line of plant defense, and because iron is required for detoxification of ROS by the pathogen (Hidalgo et al. 1997; Welinder 1992), competitive iron uptake by the invading hypha serves different functions (i.e., weakening of plant defense, protection of infection hyphae from ROS, and provision with an essential redox element required for fungal development) (Haas et al. 2008). Thus, the ability to efficiently take up iron can be regarded as a key factor for pathogenicity. Four different iron uptake systems (i.e., reductive iron assimi- lation [RIA], siderophore-mediated Fe 3+ uptake, heme uptake, and direct Fe 2+ uptake) have been identified in fungi, with RIA and siderophore-mediated Fe 3+ uptake representing high-affinity iron acquisition systems (Haas et al. 2008; Philpott 2006). Iron uptake via RIA is a two-step process. First, plasma membrane- localized iron reductases catalyze extracellular reduction of insoluble or chelator-complexed ferric (Fe 3+ ) to soluble ferrous (Fe 2+ ) iron. Subsequently, Fe 2+ is bound by a bipartite high-affin- ity iron transport complex, consisting of a multicopper ferroxi- dase (Fet3), and an iron permease (Ftr1), transferred across the plasma membrane, and delivered into the cytoplasm as Fe 3+ (Eide 1997; Haas et al. 2008; Shi et al. 2003). Thus, high-affin- ity translocation requires Fet3-mediated oxidation of iron. The advantage gained by this mechanism of iron uptake is unknown but one may speculate that it confers specificity for iron, be- cause other divalent ions are not prone to changes in their redox status and, therefore, are excluded from the RIA-mediated up- take system (Eide 1997; Haas et al. 2008; Kosman 2003). In the biotrophic maize smut fungus Ustilago maydis, two components of the high-affinity RIA system (i.e., the high-affin- ity iron permease Fer2 and the iron multicopper oxidase Fer1) were studied. Both Δfer2 and Δfer1 deletion mutants were severely affected in virulence, demonstrating the importance of this iron-uptake pathway for biotrophic development of U. maydis on maize (Eichhorn et al. 2006). Likewise, mutants of Corresponding author: H. B. Deising; Telephone: +49-345-5522660; E- mail: holger.deising@landw.uni-halle.de * The e -Xtra logo stands for “electronic extra” and indicates that four sup- plementary figures and one supplementary table are published online. © 2013 The American Phytopathological Society e - Xt ra *