Research article The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves José Chuc-Uc a , Ligia Brito-Argáez a , Blondy Canto-Canché b , Miguel Tzec-Simá b , Cecilia Rodríguez-García b , Leticia Peraza-Echeverría b , Santy Peraza-Echeverría b , Andrew James-Kay b , Carlos Alberto Cruz-Cruz b , Luis Manuel Peña-Rodríguez b , Ignacio Islas-Flores a, * a Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43, No. 130, Colonia Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico b Unidad de Biotecnología, Centro de Investigación Científica de Yucatán A.C., Calle 43, No. 130, Colonia Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico article info Article history: Received 20 February 2010 Accepted 7 February 2011 Available online 12 February 2011 Keywords: M. fijiensisebanana interaction Mycosphaerella fijiensis Plantepathogen interaction Protease activity Secretome Virulence factors abstract The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Appli- cation of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Some fungi have evolved to become pathogens to most eukaryotic organisms. In plants, fungal pathogenesis cause major yield and economical losses in many crops [21]. During interaction with the host, a fungus must implement a mechanism to establish tissue infection [25]. Successful infection requires a biochemical cross-talk between the fungal pathogen and the plant. These signals initiate at the plant surface and continue to the intra or intercellular space after pathogen penetration [20]. In some plantepathogen interactions, proteins that promote or cause the infection in the plant cell apoplast have been characterized (e. g. Cladosporium fulvumeSolanum esculentum pathosystem). However, it is always difficult to distinguish between fungal and plant proteins in many other pathosystems, especially in those whose genomes have not been sequenced [20,35]. The establishment of in vitro systems to study the initial mechanisms of plant pathogenesis provides a powerful tool to dissect the biochemical cross-talk between pathogen and host, and it is always desirable to use it for easy and quick pathogenic response assays. Recent improvements on in vitro maintenance of phytopathogenic and saprophytic fungi, and the successful estab- lishment of protocols to simulate the hostepathogen interaction in vitro, have made possible to characterize secreted proteins that enable a successful interaction of some filamentous fungi from axenic cultures [12]. For example, the phytopathogenic fungus Ustilago maydis secretes a plethora of proteins, including hydro- phobins and repellent-like proteins, which may play key roles in hyphal adhesion to the host surface and thus, promote the estab- lishment of infection [23,25]. In addition, fungal secreted proteins * Corresponding author. Tel./Fax: þ52 999 9 42 83 30 E-mail address: islasign@cicy.mx (I. Islas-Flores). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.02.006 Plant Physiology and Biochemistry 49 (2011) 572e578