ABotrytis cinereaEmopamil Binding Domain Protein, Required for Full Virulence, Belongs to a Eukaryotic Superfamily Which Has Expanded in Euascomycetes

Abstract

A previous transcriptomic analysis of 3,032 fungal genes identified the Botrytis cinerea PIE3 (BcPIE3) gene to be up-regulated early in planta (A. Gioti, A. Simon, P. Le Pêcheur, C. Giraud, J. M. Pradier, M. Viaud, and C. Levis, J. Mol. Biol. 358:372-386, 2006). In the present study, BcPIE3 was disrupted in order to determine its implication in pathogenicity. BcPIE3 was shown to be a virulence factor, since the ⌬BcPIE3 mutant was blocked during the colonization of tomato and bean leaves, giving lesions reduced in size by at least 74%. Within the emopamil binding domain (EBD), BcPIE3 shows significant structural similarities to mammalian emopamil binding proteins (EBPs). Mammalian EBPs function as sterol isomerases, but an analysis of the sterol content and the results of growth inhibition experiments with the ⌬BcPIE3 strain indicated that BcPIE3 is dispensable for ergosterol biosynthesis. The systematic identification of EBD-containing proteins included in public databases showed that these proteins constitute a protein superfamily present only in eukaryotes. Phylogenetic analysis showed that the ancestral EBD-encoding gene was duplicated in the common ancestor of animals and fungi after the split from plants. Finally, we present evidence that the EBP phylogenetic clade of this superfamily has further expanded exclusively in euascomycetes, especially in B. cinerea, which contains three copies of the EBP gene.Botrytis cinerea is a ubiquitous fungus capable of infecting a wide range of plants, fruits, and ornamentals at any stage of their development (15,30). In order to identify novel pathogenicity determinants, we recently studied the expression profiles of B. cinerea genes during the infection of Arabidopsis thaliana leaves. Among the genes shown to be induced in planta, one called B. cinerea PIE3 (BcPIE3) was found to be highly expressed 24 to 29 h after the inoculation of the fungus onto the plant leaves (18). To gain insight into early infection events, this gene was further studied.BcPIE3 showed strong similarity to mammalian emopamil binding proteins (EBPs). EBPs are integral, nonglycosylated proteins of the endoplasmic reticulum membrane, characterized by the presence of four transmembrane (TM) segments forming a binding domain for the drug emopamil, a phenylalkylamine Ca 2ϩ antagonist. This domain is called the emopamil binding domain (EBD). Mammalian EBPs (43, 25) have received increasing attention due to their binding properties. Indeed, the binding of sigma ligands but also of numerous, structurally diverse drugs and fungicides on the EBD (36) exerts anti-ischemic effects in animal models (43). Mammalian EBPs have been further studied because of a number of syndromes caused by EBP gene mutations, such as the genetic disorder X-linked dominant chondrodysplasia punctata, Conradi-Hünermann syndrome (5, 26), CHILD (congenital hemidysplasia with ichthyosiform erythroderma and limb defects) syndrome (21), and the "tattered" mouse phenotype (13).According to the above-mentioned studies, the molecular patholo...

A Xenobiotic Detoxification Pathway through Transcriptional Regulation in Filamentous Fungi

“…Additional parts of the ShXDR1 regulon are four genes encoding enopamil binding, phospholipase B1, cellulose-binding family II, and a hypothetical protein. Emopamil binding proteins (EBPs) are integral membrane proteins of the endoplasmic reticulum and bind sigma ligands and structurally diverse drugs and fungicides on the emopamil binding domain (EBD) ( 37 ). The protein may contribute to phase III of the xenobiotic detoxification system because EBPs share similar structural features with drug transporters, especially due to a high content of aromatic amino acid residues in transmembrane segments ( 38 ), which have been suggested to be involved in the drug transport by the P-glycoprotein ( 39 ).…”

LongSAGE gene-expression profiling of Botrytis cinerea germination suppressed by resveratrol, the major grapevine phytoalexin

“…When this work was begun, microarrays and serial analysis of gene expression (SAGE) were the two high-throughput methods being used to analyze complete transcriptomes in microbial systems. In B. cinerea, macroarrays containing 3032 genes were used to monitor infection of Arabidopsis thaliana leaves, confirming the value of transcriptomic analysis under conditions of infection for the identification of fungal genes related to pathogenicity (Gioti et al , 2008. More recently, the genome of B. cinerea has been completely sequenced in two different genetic backgrounds (Fillinger et al 2007).…”

Characterizing co-expression networks underpinning maize stalk rot virulence inFusarium verticillioidesthrough computational subnetwork module analyses

“…In mammalian systems, this protein family is known to be associated with endoplasmic reticulum and plays a critical role in sterol isomerization and lipoprotein internalization 23 . An emopamil binding protein BcPIE3 in Botrytis cinerea which shares significant structural similarities to mammalian EBPs was shown to be important for virulence 24 . FvEBP1 has four direct edges to FVEG_03416, FVEG_04142, FVEG_08818 and FVEG_09111.…”