VeA and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius A. Crespo-Sempere ⁎, S. Marín, V. Sanchis, A.J. Ramos Applied Mycology Unit, Food Technology Department, University of Lleida, UTPV-XaRTA, Agrotecnio Center, Av. Rovira Roure 191, 25198 Lleida, Spain abstract article info Article history: Received 22 May 2013 Received in revised form 18 July 2013 Accepted 29 July 2013 Available online 7 August 2013 Keywords: Aspergillus carbonarius VeA LaeA Ochratoxin production Conidiation Light Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic and immunotoxic properties which represents a serious risk for human and animal health. Aspergillus carbonarius is considered the main OTA-producing species in grapes and products such as raisins, wine or juices, although it has also been isolated from coffee, cocoa and cereals. Till now not much information is available about regulatory mechanisms of OTA production by A. carbonarius. A better understanding of how environmental factors influence OTA production and which genes are involved in its regulation could help us design new control strategies. In this study, we have evaluated the role of VeA and LaeA transcriptional factors, which have been shown to regulate secondary metabolism in response to light in A. carbonarius. To this aim, veA and laeA genes were deleted in an ochratoxigenic A. carbonarius strain by targeted gene replacement using Agrobacterium tumefaciens-mediated transformation. Loss of veA and laeA in A. carbonarius yields to an organism with slight differences in vegetative growth but a strong reduction in conidial production. A drastic decrease of OTA production that ranged from 68.5 to 99.4% in ΔveA and ΔlaeA null mutants was also observed, which was correlated with a downregulation of a nonribosomal peptide synthetase involved in OTA biosynthesis. These findings suggest that VeA and LaeA have an important role regulating conidiation and OTA biosynthesis in response to light in A. carbonarius in a similar way to other fungi where functions of VeA and LaeA have been previously described. This is the first report of a transcriptional factor governing the production of OTA by A. carbonarius. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium genera that may pose a serious health hazard due to its nephrotoxic, teratogenic and immunotoxic properties (Creppy, 1999; Kuiper-Goodman and Scott, 1989; Petzinger and Ziegler, 2000; Pfohl-Leszkowicz and Manderville, 2007). OTA has been classified as a possible human renal carcinogen (group 2B) by the International Agency for Research on Cancer (IARC, 1993). Aspergillus carbonarius is considered the main OTA producer species in grapes and products such as raisins (Battilani et al., 2006; Cabañes et al., 2002; Perrone et al., 2007). Addi- tionally, A. carbonarius has been found in coffee (Joosten et al., 2001; Taniwaki et al., 2003), cocoa (Mounjouenpou et al., 2008), peanuts (Magnoli et al., 2007) and maize (Shah et al., 2010). Frequently, genes responsible for mycotoxin biosynthesis are clustered on a chromosome and co-regulated by transcriptional factors, as has been described for sterigmatocystin in Aspergillus nidulans (Brown et al., 1996), aflatoxins in Aspergillus parasiticus (Yu et al., 2004), fumonisins in Gibberella moniliformis (Proctor et al., 2003; Seo et al., 2001) or tricothecenes in Fusarium (Brown et al., 2004). Till now not much information is available about the OTA biosynthetic pathway in A. carbonarius, since only the implication of a nonribosomal peptide synthetase and a polyketide synthase has been reported (Gallo et al., 2009, 2012). Clusters often include a gene encoding a transcrip- tional factor that regulates the mycotoxin biosynthesis, such as aflR, an in-cluster pathway regulator which encodes a Zn(2)-Cys(6) that ac- tivates transcription by binding to DNA sequences in the promoters of aflatoxin and sterigmatocystin biosynthesis genes (Ehrlich et al., 1999; Fernandes et al., 1998). However, mycotoxin production is also respon- sive to environmental factors that could be regulated by transcription factors located outside the mycotoxin gene cluster. It has been reported that OTA production in A. carbonarius is influenced by carbon and nitro- gen sources (Abbas et al., 2009; Ferreira and Pitout, 1969; Medina et al., 2008) and pH (Esteban et al., 2005; O'Callaghan et al., 2006). Carbon signaling may be mediated by the transcription factor CreA (Dowzer and Kelly, 1989), while nitrogen signaling could be regulated by AreA (Hynes, 1975) and pH signaling by PacC (Tilburn et al., 1995). Recently, the heterotrimeric velvet complex VelB/VeA/LaeA that couples in A. nidulans secondary metabolism with fungal development, including asexual and sexual growth, in response to light was identified (Bayram et al., 2008a). VeA is a global regulator which is transported from cytoplasm to nucleus in response to illumination. VeA interacts with LaeA in the nucleus regulating production of secondary metabo- lites and with VelB inducing sexual development (Palmer et al., 2013). Studies of diverse filamentous fungi have identified several structural International Journal of Food Microbiology 166 (2013) 479–486 ⁎ Corresponding author. E-mail address: ana.crespo@udl.cat (A. Crespo-Sempere). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.07.027 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro