Validation of Melampsora larici-populina reference genes for in planta RT-quantitative PCR expression profiling during time-course infection of poplar leaves Stéphane Hacquard, Claire Veneault-Fourrey, Christine Delaruelle, Pascal Frey, Francis Martin, Sébastien Duplessis * INRA (Institut National de la Recherche Agronomique), Unité Mixte de Recherche 1136 INRA/Nancy Université ‘Interactions Arbres/Micro-organismes’, Centre INRA de Nancy, 54280 Champenoux, France article info Article history: Accepted 29 October 2010 Keywords: Populus Poplar rust Biotrophic fungus Gene expression Housekeeping genes Thiamine abstract The foliar rust caused by Melampsora larici-populina (Mlp) is the main disease affecting poplar planta- tions in Europe. The biotrophic status of this fungus is a major limitation to address in planta transcripts profiling. Thus, identification of reference rust genes steadily expressed during plant tissue colonization is a crucial point. A quantitative PCR approach to assess fungal ITS amplification profile and Reverse Transcription quantitative-PCR was set to compare candidate reference genes amplification profiles in poplar infected tissues. We selected two M. larici-populina genes encoding an alpha-tubulin and the elongation factor-1-alpha that showed the highest expression stability across biological samples and for which transcript levels were correlated with fungal ITS amplification during time-course infection of poplar leaves. We report the use of these reference genes to assess in planta expression profiles of two genes involved in thiamine biosynthesis (THI1 and THI2) for which specific haustorium expression was previously described in the bean rust fungus Uromyces fabae. Mlp- THI1 and Mlp-THI2 showed similar expression profiles. Trancripts were barely detectable in urediniospores as well as during the early stages of infection compared to those reported in the bean rust, whereas a strong induction was observed after haustorial formation after 24 hpi. These data are in frame with the results obtained in U. fabae and consistent with a metabolic reorientation that likely occurs after the fungus derived nutrients from its host in the haustorial structure essential for fungal biotrophy. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Rust caused by Melampsora larici-populina is the main disease affecting poplar plantations in Europe with severe economic losses [1]. Epidemiology of the disease and poplar defence responses have been previously addressed [2,3] but still very little is known about fungal molecular mechanisms during the infection process. It is crucial to increase our understanding of the biology of the path- ogen in order to develop strategies to control the disease. In the wake of the Populus genome sequencing [4], the ∼100 Mb genome of M. larici-populina has been recently sequenced by the Joint Genome Institute (JGI; http://genome.jgi-psf.org/Mellp1/Mellp1. home.html) and the analysis of this genome is a great opportu- nity to identify effector genes expressed by the rust fungus to achieve plant infection [3,5]. Availability of the genome sequences of both the plant host and the rust fungus and recent updates on Populus defence reactions upon rust infection [6,7,8], make the Populus/Melampsora pathosystem an emerging model in forest pathology to study the molecular mechanisms developed by rust to infect plant tissues and to feed on the host [3]. The biotrophic status of rust fungi precludes some routine manipulations in the laboratory. Up to now, most of the knowledge gained on rust gene expression was obtained after sequencing of Expressed Sequence Tags (EST) from urediniospores, haustorial tissues or infected plant tissues [5,9,10,11]. A synthetic system has been established to study the development of Uromyces fabae infection structure after the growth of urediniospores on poly- ethylene membranes [12]. By this means, it is possible to follow transcripts accumulation in most fungal structures usually formed Abbreviations: Cq, quantification cycle; EST, expressed sequence tag; ITS, internal transcribed spacer; LPI, leaf plastochrony index; Mlp, Melampsora larici- populina; qPCR, quantitative polymerase chain reaction; rDNA, ribosomal DNA; RT, Reverse Transcription. * Corresponding author. Tel.: þ33 383 39 40 13; fax: þ33 383 39 40 69. E-mail address: duplessi@nancy.inra.fr (S. Duplessis). Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp 0885-5765/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2010.10.003 Physiological and Molecular Plant Pathology 75 (2011) 106e112