Stage:80

From Master 2 Recherche Biosciences Végétales

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Identification and validation of protein partners of two transcription factors from Eucalyptus

Laboratoire d'accueil : LRSV
Equipe d'accueil : Génomique fonctionnelle de l’Eucalyptus
Encadrant(e)(s) : Marçal Soler (soler@lrsv.ups-tlse.fr) et Jacqueline Grima-Pettenati (grima@lrsv.ups-tlse.fr)

Scientific context:
Wood, the most abundant biological resource on earth, is of major environmental and economic importance, which could be even greater with the recent interest in the utilization of lignocellulosic biomass for second-generation biofuels. Wood is a complex vascular tissue that contains specialized cells with thick lignified secondary cell walls. The synthesis and assembly of these secondary cell walls require fine spatiotemporal regulation achieved through a complex network of transcriptional regulatory pathways including master switches such as MYB and NAC transcription factors. In Eucalyptus EgMYB1 and EgMYB2 regulate the biosynthesis of lignin and impact secondary cell wall formation in an opposite way: EgMYB1 acts as a repressor (Legay et al. 2010) and EgMYB2 as an activator (Goicoechea et al. 2005). It is believed that secondary cell wall is regulated with the participation of multiple complexes that might share specific sets of transcription factors, thus offering a rich palette of combinatorial diversity for the cross-regulation of different metabolic pathways. These two MYB transcription factors very likely function within combinatorial complexes with other still unknown transcription factors. Hence, the discovery of the protein partners of EgMYB1 and EgMYB2 is the main objective of the project.

Subject of the stage:
Protein interactions of EgMYB1 and EgMYB2 will be screened using the Two-hybrid approach in yeast against a library of eucalyptus xylem cDNA. Screening of both genes is planned to be performed in the host lab before December 2011, and a list of putative candidate interactions will arise from this work. The master student will select the most promising candidate interactions (according to bibliographic data and expression pattern analysis when necessary) and test them using a targeted Two-hybrid approach in yeast. In a second step, the interactions will be validated in planta using the FRET-FLIM (Fluorescence Resonance Energy Transfer – Fluorescence Lifetime Imaging Microscopy) technology by transient expression in Nicotiana leaves.

Competences that the master student will acquire:
Basic techniques of molecular biology and microbiology: cloning, digestion, plasmid purification, PCR…
Gene expression analyses: real time RT-PCR
Plant transformation and transient expression techniques
Fluorescent microscopy imaging
Bioinformatics: sequence alignment and gene prediction

References:
Legay S, Sivadon P, Blervacq AS, Pavy N, Baghdady A, Tremblay L, Levasseur C, Ladouce N, Lapierre C, Séguin A, Hawkins S, Mackay J, Grima-Pettenati J (2010). EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar. New Phytol 188: 774-778.

Goicoechea M, Lacombe E, Legay S, Mihaljevic S, Rech P, Jauneau A, Lapierre C, Pollet B, Verhaegen D, Chaubet-Gigot N, Grima-Pettenati J (2005). EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant J 43:553-567.