Sonia Campo 1 Montserrat Carrascal 2 Maria Coca 1 Joaquin Abián 2 Blanca San Segundo 1 1 Departamento de Genética Molecular, Instituto de Biología Molecular de Barcelona, Centro de Investigación y Desarrollo, CSIC, Barcelona, Spain 2 Unidad de Espectrometría de Masas Estructural y Biológica, Departamento de Bioanalítica Médica, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Barcelona, Spain The defense response of germinating maize embryos against fungal infection: A proteomics approach Pathogen attack on plants results in numerous host-specific biochemical responses, the activation of some of them being critical for the ability of the plant to withstand disease. We have used high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins that are differentially expressed in response to fungal infection in maize embryos. Differential spots corresponding to induced or repressed proteins were apparent in silver stained 2-DE gels of proteins extracted from sterile and fungal-infected germinating embryos. Selected spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ ionization-time of flight-mass spectrometry and nanospray ion-trap tandem mass spectrometry. Among the proteins induced in response to infection are proteins involved in protein synthesis, or in protein folding and stabilization, as well as proteins involved in oxidative stress tolerance. Additionally, the accumulation of specific patho- genesis-related proteins in tissues of the fungal-infected germinating embryos was studied by 2-DE and immunoblotting. Keywords: Fungal infection / Maize embryo / Mass spectrometry / Pathogenesis-related protein / Two-dimensional gel electrophoresis PRO 0657 1 Introduction Plants have the ability to respond to invasion by patho- gens through activation of a variety of defense strategies. Most plants exhibit resistance to pathogenic attack as the outcome of a general defense strategy in which a wide range of biochemical responses are induced in a coordi- nated manner [1–4]. Defense response includes repro- gramming of cellular metabolism, accumulation of bar- rier-forming substances (reinforcement of cell walls), and production of antimicrobial compounds that act directly to prevent pathogen invasion. Knowing the molecular mechanisms that regulate the inducible plant defenses against fungal pathogens could be useful in designing protective strategies involving expression of defense genes in crop plants. Like many other complex biological processes, plant defense responses upon pathogen infection involve changes in the expression of a large number of plant genes which are then subjected to up- or down-regula- tion. A key feature underlying successful pathogen recog- nition is the engagement of the so called oxidative burst, a rapid production of reactive oxygen intermediates (ROIs), primarily superoxide (O 2 2 ) and hydrogen peroxide (H 2 O 2 ), at the site of attempted infection [5–7]. The synthesis and accumulation of pathogenesis-related (PR) proteins is also an ubiquitous plant response to pathogen infection [8–10]. Currently, PR proteins are grouped into 17 inde- pendent families, PR-1 to PR-17, and antimicrobial prop- erties have been described for some of them. The PR-2 proteins display b-1,3-glucanase (glucan endo-1,3-b-glu- cosidase) activity, whereas the PR-3 proteins (as well as PR-4, PR-8 and PR-11 proteins) have been shown to dis- play endochitinase activity [11, 12]. Concerning the func- tion of plant b-1,3-glucanases and chitinases, a role for these hydrolytic enzymes as part of the plant defense response has been demonstrated on the basis of their inhibitory effect on the growth of pathogenic fungi. More- over, transgenic plants expressing chitinase and b-1,3- glucanase genes exhibit enhanced protection against fungal pathogens. Despite the extensive information available on the accumulation of defense proteins, and particularly on PR proteins in leaves of adult plants, the Correspondence: Dr. Blanca San Segundo, Departamento de Genética Molecular, Instituto de Biología Molecular de Barce- lona, Centro de Investigación y Desarrollo (CID), CSIC, Jordi Gi- rona 18, 08034 Barcelona, Spain E-mail: bssgmb@cid.csic.es Fax: +34-93-204-59-04 Abbreviations: BMV , brome mosaic virus; eIF , eukariotic transla- tion initiation factor; PMF , peptide mass fingerprinting; PR, pathogenesis-related; SOD, superoxide dismutase Proteomics 2004, 4, 383–396 383  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200300657