Rodriguez C1., Gereige D.1, Chapuis S.2, Ziegler-Graff V.2, Revers F3., Brault V.1
1
UMR INRA-UDS Virus-Vection group 28 rue de Herrlisheim 68021 Colmar France
2
Institut de Biologie Moléculaire des Plantes du CNRS, Virologie Végétale Integrative, 12 rue du Général Zimmer, 67084 Strasbourg, France
3
UMR BFP INRA-UB2, Centre de Bordeaux, BP 81, 33883 Villenave d’Ornon cedex, France Veronique.Brault@colmar.inra.fr
Turnip yellows virus is a polerovirus (Luteoviridae family) which cycle is restricted to
phloem cells namely the companion cells, the phloem parenchyma cells and the enucleate sieve elements. In order to identify cellular partners of TuYV, a companion cell-specific cDNA library from Arabidopsis thaliana was constructed from FACS-sorted fluorescent protoplasts and screened by yeast two-hybrid system (Y2H) using viral protein domains as baits.
Among the TuYV putative partners, we found a CIPK (calcineurin B-like protein-interacting protein kinase) protein-interacting with the non-structural C-terminal part of the readthrough protein of TuYV involved in virus movement. The CIPK identified belongs to a multigenic family in A. thaliana comprising 26 different CIPK that can interact with 10 CBL isoforms to decode calcium signals. We confirmed by Y2H that the identified CIPK does interact with different CBLs (calcineurin B-like proteins). When co-expressed in Nicotiana
benthamiana both proteins co-localized in different sub-cellular compartments (cytoplasm,
nucleus, nucleolus and filaments). Preliminary assays confirmed by FLIM the in vivo interaction between both partners in the cytoplasm. Experiments are underway to assess potential interactions in the other sub-cellular organelles. In addition, we showed that expression of the CIPK mRNA is transiently up-regulated early during infection and that transient expression of the kinase in N. benthamiana induces an increase in virus accumulation. These results suggest that the CIP kinase may be involved in TuYV cycle but the mechanism by which the kinase is acting is still unknown and needs further investigations.
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Nucleocytoplasmic shuttling of two proteins encoded by Grapevine fanleaf
virus
Berthold F., Sshmitt-Keichinger C., Herranz-Gordo MC., Ackerer L., Hemmer C., Ritzenthaler C.
Institut de Biologie Moléculaire des Plantes, CNRS / Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.
francois.berthold@ibmp-cnrs.unistra.fr
Grapevine fanleaf virus (GFLV) is a bipartite positive-sense RNA virus that replicates
on endoplasmic reticulum membranes (Ritzenthaler et al., 2002). Each of the two viral RNAs encodes a single polyprotein. Polyprotein P1, encoded by RNA 1, is processed by the viral proteinase into five proteins of which protein 1A is of unknown function. Polyprotein P2, encoded by RNA 2, gives rise to protein 2A involved in RNA 2 replication, protein 2BMP necessary for tubule-guided movement and the capsid protein 2CCP.
To get insight into the subcellular localization and the function of proteins 1A and 2A, we tagged them with fluorescent proteins. Fusions of protein 1A at its N-terminus as well as fusion of protein 2A at its C-terminus are compatible with local and systemic infection on
Chenopodium quinoa, Nicotiana benthamiana and Arabidopsis thaliana, thus indicating that
functionality of 1A and 2A is not hindered in this context. This important result allowed us to study the subcellular localization and show the interaction of both proteins using confocal microscopy and Fluorescence Lifetime Imaging (FLIM). Upon ectopic expression, fluorescent protein-tagged 1A exclusively accumulates in the nucleus, whereas fluorescent protein-tagged 2A is exclusively cytoplasmic. When transiently coexpressed as well as in viral context, both recombinant proteins colocalize in both cellular compartments, further demonstrating their interaction.
In order to get a better understanding of the intrinsic properties of each protein as well as the interplay between them, a collection of deletion mutants of 1A and 2A proteins has been generated. These mutants have been studied by transient expression in fusion to fluorescent proteins. This resulted in the identification of two domains in 2A involved in its particular subcellular localization upon ectopic expression and two interaction domains, each of them being sufficient to interact with 1A. One particular mutant of 2A also appeared to mimic 1A subcellular localization even when expressed alone. This mutant also colocalizes and possibly interacts with the structural protein of Cajal bodies, coilin.
Together these results suggest a nucleocytoplasmic shuttling of 1A and 2A. Possible functions for this interplay will be discussed.
Ritzenthaler, C., Laporte, C., Gaire, F., Dunoyer, P., Schmitt, C., Duval, S., Piequet, A., Loudes, A.M., Rohfritsch, O., Stussi-Garaud, C., and Pfeiffer, P. (2002). Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes. Journal of Virology 76: 8808–8819.
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Interests of Next Generation Sequencing, gene synthesis and codon
Bruyninx M., Potier N.
Eurofins MWG Operon, 9 avenue de la Laponie 91978 Les Ulis Marc.Bruyninx@eurofins.com
Nathalie.Potier@eurofins.com
The talk will be divided into two parts:
- Gene synthesis and codon optimization: expression of heterologous genes in plant cells. Following a short introduction on the main applications of synthetic genes, the talk will focus on sequence optimization and codon usage adaptation, demonstrating how these latter can be useful, or even necessary, for the expression of genes in heterologous organisms, notably in plant cells. The main optimization algorithms will be reviewed, describing their respective advantages and drawbacks.
- Use of high-throughput sequencing in plant genomics.
The second part of the talk will be dedicated to high-throughput sequencing technologies in plant genomics, notably plant genome sequencing via multi-libraries approaches (shotgun + Long Jumping Distance).
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