SUMMARY
PtaRHE1 is a poplar (Populus tremula x P. alba) gene encoding a REALLY INTERESTING NEW GENE (RING) domain-containing protein. RING proteins are largely represented in plants and play important roles in the regulation of many developmental processes as well as in plant-environment interactions. In this thesis, we present a functional characterization of PtaRHE1. To gain further insight into the role of this gene, molecular and genetic alteration approaches were used. The results of in vitro ubiquitination assays indicate that PtaRHE1 protein is a functional E3 ligase and this activity was shown to be specific with the human UbCH5a, among the tested ubiquitin-conjugating enzymes. Histochemical GUS stainings showed that the PtaRHE1 promoter is induced by plant pathogens and by elicitors such as salicylic acid and cellulase and is also developmentally regulated.
In silico predictions and the transient expression of PtaRHE1-GFP fusion protein in N. tabacum epidermal cells revealed that PtaRHE1 is localized both in the plasma membrane and in the nucleus.
The localization of expression of PtaRHE1 in poplar stem by in situ hybridization indicated that
PtaRHE1 transcripts are localized within the cambial zone mainly in ray cells, suggesting a role of this
gene in vascular tissue development and/or functioning. The overexpression of PtaRHE1 in tobacco
resulted in a pleiotropic phenotype characterized by a curling of leaves, the formation of necrotic
lesions on leaf blades, growth retardation as well as a delay in flower transition. Plant genes
expression responses to PtaRHE1 overexpression provided evidence for the up-regulation of defence
and/or programmed cell death (PCD) related genes. Moreover, genes coding for WRKY transcription
factors as well as for MAPK, such as WIPK, were also found to be induced in the transgenic lines as
compared to the wild type (WT). Taken together, our results suggest that the E3 ligase PtaRHE1 plays
a role in the signal transduction pathways leading to defence responses against biotic and abiotic
stresses. Identification of PtaRHE1 target(s) is required in order to fully assess the role of this E3
ligase in the ubiquitination-mediated regulation of defence response.
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RÉSUMÉ
PtaRHE1 est un gène qui code pour une protéine possédant un domaine RING (REALLY INTERESTING NEW GENE) chez le peuplier (Populus tremula x P. alba). Les protéines de type RING sont très répandues chez les végétaux où elles jouent de rôles importants dans la régulation de plusieurs processus de développement et également dans les interactions plantes-environnement. Dans le cadre de ce travail, nous avons procédé à la caractérisation fonctionnelle du gène PtaRHE1. Dans le but de découvrir la fonction de ce gène, nous avons adopté une stratégie faisant usage d’approches moléculaires ainsi que de l’altération de l’expression génique. Les résultats obtenus montrent que la protéine PtaRHE1 est une E3 ligase et que cette activité enzymatique est spécifique à l’Ubiquitin- Conjugating enzym humaine UbCH5a. Les résultats du test histochimique GUS ont montré que le promoteur du gène PtaRHE1 est induit par des pathogènes et aussi par l’acide salicylique et la cellulase. Par ailleurs, ce promoteur est aussi régulé au cours du développement végétal. Les prédictions in silico et l’expression transitoire d’une fusion traductionnelle GFP-PtaRHE1, au niveau de l’épiderme des feuilles du tabac N. tabacum, ont révélé que la protéine PtaRHE1 se situe tant au niveau de la membrane cytoplasmique qu’au niveau du noyau. La localisation de l’expression du gène PtaRHE1, par les techniques d’hybridation in situ, montre que les transcrits de ce gène se retrouvent principalement au niveau des cellules de rayon, dans la zone cambiale, suggérant que ce gène pourrait jouer un rôle dans le développement ou la formation du tissu vasculaire. La surexpression du gène PtaRHE1 chez le tabac a conduit à l’obtention d’un phénotype pléiotropique caractérisé par un recroquevillement (incurvation) des feuilles, la formation des lésions nécrotiques sur le limbe, un retard de croissance ainsi qu’un retard dans la transition florale. L’analyse de la réponse de l’expression de différents gènes à la surexpression de PtaRHE1 a mis en évidence l’induction des gènes liés à la défense et ou à la mort cellulaire programmée. En outre, l’expression des gènes codant pour des facteurs de transcription WRKY et aussi des MAPKs, tel que WIPK, était aussi plus élevée chez les plantes transgéniques comparées au type sauvage. Les résultats de ce travail suggèrent que PtaRHE1, comme E3 ligase, pourrait jouer un rôle dans la transduction des signaux cellulaires conduisant aux réactions de défense contre les stress biotiques et abiotiques.
L’identification de la (des) cible(s) de PtaRHE1 est indispensable pour la compréhension du rôle de
cette protéine dans la régulation des réponses de défense par l’intermédiaire de l’ubiquitination.
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LIST OF ABBREVIATIONS
ABA Abscisic acid
ABRC Arabidopsis Biological Resource Center AFLP Amplified Fragment Length Polymorphism
AMP Adenosine Monophosphate
ATP Adenosine Triphosphate
BLAST Basic Local Alignment Search Tool
bp base pair
CaMV Cauliflower Mosaic Virus cDNA Complementary DNA DEPC Diethyl Pyrocarbonate DNA Deoxyribonucleic Acid
dNTP deoxy Nucleotide Triphosphate DTT Dithiothreitol
EDTA Ethylene Diamine Tetraacetic Acid EGFP Enhanced GFP
EST Expressed Sequence Tag GFP Green Fluorescent Proteins GST Glutathione S-Transferase GUS β-glucuronidase gene
H
2Odd double distilled water (deionized water)
HEPES 4-(2-Hydroxy Ethyl)-1-Piperazine Ethane Sulfonic acid INRA Institut National de la Recherche Agronomique
kDa kiloDalton
Mb Megabase
MES 2-(N-Morpholino) Ethane Sulfonic acid
mRNA messenger RNA
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MS Murashige and Skoog
Ni-NTA Nickel Nitrilo-triacetic Acid NTE NaCl Tris-HCl EDTA PBS Phosphate Buffered Saline RNA Ribonucleic Acid
RT-PCR Retro-Transcription Polymerase Chain Reaction RT-qPCR Real Time quantitative Polymerase Chain Reaction SA Salicylic Acid
SDS-PAGE Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis SSC NaCl Sodium Citrate
TAIR The Arabidopsis Information Resource TDF Trancript-Derived Fragment
T-DNA Transfert DNA
Ub Ubiquitin
tRNA Transfer RNA
UV Ultra Violet
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LIST OF FIGURES
Figure 1.1. cDNA-AFLP sampling and anatomical features of cross-sections along a poplar stem. ... 9
Figure 1.2. In situ RT-PCR localization of transcripts in hand-made transverse sections of 1-month-old poplar stems. ... 10
Figure 1.3. Alignment of the PtaRHE1 deduced amino acid sequence with the four most similar predicted proteins found in the databases ... 11
Figure 1.4. Structure of RING domains. ... 14
Figure 1.5. RING and modified RING domains: consensus and number of each type of RING domain identified in Arabidopsis. ... 15
Figure 1.6. Arabidopsis RING-H2 proteins. ... 15
Figure 1.7. Schematic representation of the conserved domains in ATL proteins. ... 16
Figure 1.8. Phylogeny of Arabidopsis and rice ATL proteins. ... 17
Figure 1.9. The ubiquitination pathway. ... 22
Figure 1.10. Amino acid alignment of Ub from various model organisms. ... 23
Figure 1.11. The three-dimensional structure of plant Ub. ... 23
Figure 1.12. A list of known Ub genes ... 25
Figure 1.13. E3 ligases in plants. ... 30
Figure 1.14. Ubiquitin modifications and their cellular functions. ... 34
Figure 1.15. Regulation of protein transport by ubiquitin signals. ... 37
Figure 1.16. The generation of a polyubiquitin chain. ... 39
Figure 1.17. Polyubiquitination modifications. ... 40
Figure 2.1. Sequences of PtaRHE1 full length and PtaRHE1-Ct ... 48
Figure 2.2. Genetic construction for PtaRHE1 and PtaRHE1-Ct proteins production ... 48
Figure 2.3. Expression analysis of recombinant PtaRHE1-Ct by SDS-PAGE after production in E. coli TOP 10 strain. ... 49
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Figure 2.4. Detection of recombinant PtaRHE1 by western blot using anti-V5-HRP antibody. ... 49
Figure 2.5. In vitro E3 Ub ligase activity of PtaRHE1 and PtaRHE1-Ct proteins. ... 51
Figure 2.6. Alignment of sequences of human ubiquitin conjugating enzymes (E2) used in in vitro ubiquitination assay. ... 52
Figure 2.7. PtaUbC5a coding sequence (from 1 to 447) and deduced amino acids sequence of PtaUbC5a protein ... 54
Figure 2.8. Alignment of the P. tremula x P. alba UbC5a amino acids sequences ... 55
Figure 2.9. Genetic construction for PtaUbC5a protein production ... 56
Figure 2.10. Expression analysis of recombinant poplar UbC5a by Western-blot using anti-V5-HRP antibody 56 Figure 3.1. Probe synthesis and in situ hybridization reaction scheme. ... 64
Figure 3.2. Redox reaction of BCIP and NBT ... 70
Figure 3.3. Genetic construct for the fusion of pPtaRHE1 with GFP and GUS (in pKGWFS7) ... 73
Figure 3.4. Nucleotide sequence of the 5’-flanking promoter region and putative cis-acting elements of the PtaRHE1 promoter. ... 75
Figure 3.5. Identification of pPtaRHE1 driven GUS transgenic homozygous lines during seedling development in tobacco. ... 78
Figure 3.6. pPtaRHE1 response to various biotic and abiotic stresses. ... 79
Figure 3.7. pPtaRHE1 driven expression during plant development ... 81
Figure 3.8. Localization of PtaRHE1 mRNA by in situ hybridization... 82
Figure 3.9. Hydropathy profile of PtaRHE1 ... 84
Figure 3.10. Hydropathy plot of PtaRHE1 deduced protein sequence... 84
Figure 3.11. Genetic construct for PtaRHE1-GFP fusion protein localization (in pK7WGF2) ... 85
Figure 3.12. Intracellular localization of PtaRHE1-GFP fusion proteins in lower epidermal cells of tobacco leaf agroinfiltrated with PtaRHE1-GFP construct. ... 85
Figure 4.1. Genetic construct of PtaRHE1 coding sequence ... 96
Figure 4.2. Identification of homozygous transgenic lines overexpressing PtaRHE1. ... 96
Figure 4.3. Phenotype of transgenic lines overexpressing PtaRHE1 ... 98
Figure 4.4: RT-PCR expression analysis of PtaRHE1 in 3 individual plants ... 99
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List of tables
Table 1. E1 genes numbers in plants where the sequence of the genome is available ... 26
Table 2: E2 genes numbers in plants where the sequence of the genome is available ... 28
Table 3: RING E3 genes numbers in plants where the sequence of the genome is available ... 30
Table 4. Some known single RING E3 ligases involved in plants growth and development ... 32
Table 2.1. Identities and similarities between three human UbCH5 E2 enzymes ... 52
Table 2.2. Identities and similarities of P. tremula x P. alba E2 enzyme with some homologs. ... 55
Table 3.1. Annotation and functions of putative Cis-acting elements identified in pPtaRHE1. ... 76
Table 4.1.Primer sequences and amplicon size for RT-PCR and RT-qPCR. ... 92 Table 4.2. Relative expression (fold) of a selection of genes associated with various types of cell processes linked to defence and/or cell death in two lines of PtaRHE1 overexpressors compared with the wild type. 101
