Planta (2007) 226:323–333 DOI 10.1007/s00425-007-0484-4 123 ORIGINAL ARTICLE Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola Shantharaj Deepak · Sekhar Shailasree · Ramachandra K. Kini · Bettina Hause · Shekar H. Shetty · Axel Mithöfer Received: 30 October 2006 / Accepted: 19 January 2007 / Published online: 14 February 2007  Springer-Verlag 2007 Abstract Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall components involved in plant defense response to pathogen attack. In the pres- ent study, a resistant pearl millet (Pennisetum glau- cum) cultivar, IP18292, was compared with a susceptible cultivar, 7042S, to investigate the contribu- tion of HRGPs in the successful defense against the phytopathogenic oomycete S. graminicola. Northern hybridization using MeHRGP cDNA, a heterologous probe from cassava, indicated steady accumulation of HRGP transcripts, from 2 h.p.i. onwards with a maxi- mum at 6 h.p.i., in the resistant cultivar. This is fol- lowed by HRGPs accumulation at about 8 h.p.i. as revealed by Western-blot analysis. Immunocytochemi- cal localization by tissue printing and confocal immu- noXuorescence microscopy indicated cell walls of parenchymatic cells and the vascular tissue of coleop- tile as sites of HRGP deposition. In vitro studies in the presence of horseradish peroxidase and H 2 O 2 showed cross-linking of pearl millet HRGPs, which occurred parallel to isodityrosine accumulation. Inducible high isodityrosine content was also observed in vivo in the resistant cultivar. Here, H 2 O 2 was found to accumulate as twin burst at 1 and 6 h.p.i., whereas in the suscepti- ble cultivar only an early single peak was detectable. Moreover, the amount of hydroxyproline in HRGPs was about twice as high in the resistant as in the suscep- tible cultivar. These results suggest that cell wall strengthening in S. graminicola-infected resistant pearl millet is brought about by a combination of polypep- tide cross-linking of isodityrosine as well as by the high content of hydroxyproline in HRGPs, and H 2 O 2 , in contrast to the susceptible plant. Keywords Cell wall · Hydroxyproline-rich glycoproteins · Pearl millet · Plant defense · Sclerospora Abbreviations DTT Dithiothreitol h.p.i. Hours post infection HRGP Hydroxyproline-rich glycoproteins Hyp Hydroxyproline IDT Isodityrosine PAGE Polyacrylamide gel electrophoresis Introduction Plant cell walls are dynamic structures that change with physiological Xuctuations and upon external stimuli from environment. The most abundant structural pro- teins in plant cell walls are the hydroxyproline-rich gly- coproteins (HRGPs). They are induced in disease resistant responses, speciWcally in incompatible plant– pathogen interactions (Davies et al. 1997). Functionally, S. Deepak · S. Shailasree · R. K. Kini · S. H. Shetty Department of Studies in Applied Botany and Biotechnology, University of Mysore, Manasagangotri, Mysore, 570006, Karnataka, India S. Deepak · A. Mithöfer (&) Department of Bioorganic Chemistry, Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany e-mail: amithoefer@ice.mpg.de B. Hause Department of Secondary Metabolism, Leibniz Institute of Plant Biochemistry, 06120 Halle/Saale, Germany