NMR chemical shift backbone assignment of the viral protein P1 encoded by the African Rice Yellow Mottle Virus

International audienceRNA silencing describes a pan-eukaryotic pathway of gene regulation where doubled stranded RNA are processed by the RNAse III enzyme Dicer or homologs. In particular, plants use it as a way to defend themselves against pathogen invasions. In turn, to evade the plant immune response, viruses have developed anti-RNA silencing mechanisms. They may indeed code for proteins called "viral suppressor of RNA silencing" which block the degrading of viral genomic or messenger RNA by the plant. The Rice Mottle Virus is an African virus of the sobemovirus family, which attacks the most productive rice varieties cultivated on this continent. It encodes P1, a cysteine-rich protein described as a potential RNA silencing suppressor. P1 is a 157 amino-acid long protein, characterized by a high propensity to aggregate concomitant with a limited stability with time in the conditions used in structural studies. To overcome this problem, shorter fragments were also studied. This strategy enabled the assignment of more than 90% backbone resonances of P1. This assignment should set the base of future NMR investigation of the protein structure and of its interactions with rice cellular partners

Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in vivo function of reductase and thioredoxin domains

Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive thioredoxin domain, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modelling of the 3-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protected pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioene

Historical Contingencies Modulate the Adaptability of Rice Yellow Mottle Virus

The rymv1-2 and rymv1-3 alleles of the RYMV1 resistance to Rice yellow mottle virus (RYMV), coded by an eIF(iso)4G1 gene, occur in a few cultivars of the Asiatic (Oryza sativa) and African (O. glaberrima) rice species, respectively. The most salient feature of the resistance breaking (RB) process is the converse genetic barrier to rymv1-2 and rymv1-3 resistance breakdown. This specificity is modulated by the amino acid (glutamic acid vs. threonine) at codon 49 of the Viral Protein genome-linked (VPg), a position which is adjacent to the virulence codons 48 and 52. Isolates with a glutamic acid (E) do not overcome rymv1-3 whereas those with a threonine (T) rarely overcome rymv1-2. We found that isolates with T49 had a strong selective advantage over isolates with E49 in O. glaberrima susceptible cultivars. This explains the fixation of the mutation T49 during RYMV evolution and accounts for the diversifying selection estimated at codon 49. Better adapted to O. glaberrima, isolates with T49 are also more prone than isolates with E49 to fix rymv1-3 RB mutations at codon 52 in resistant O. glaberrima cultivars. However, subsequent genetic constraints impaired the ability of isolates with T49 to fix rymv1-2 RB mutations at codons 48 and 52 in resistant O. sativa cultivars. The origin and role of the amino acid at codon 49 of the VPg exemplifies the importance of historical contingencies in the ability of RYMV to overcome RYMV1 resistance

Thioredoxines végétales et interactomes redox-dépendants

Les Thiorédoxines (TRX) sont des protéines ubiquitaires appartenant à la grande famille des rédoxines qui comprend également les Glutarédoxines (GRX) et les protéines disulfides isomérases (PDI). Toutes sont caractérisées par la présence au sein de leur structure protéine d’un centre redox de type CxxC/S (pour les TRX et GRX canoniques), voire de plusieurs centres pour certaines TRX-like ou GRX_like, ainsi que pour les PDI. Ces motifs protéiques procurent aux rédoxines une activité d’oxydoréductase, consistant principalement à réduire des ponts disulfures au sein de protéines cibles en ce qui concerne les TRX qui nous intéressent ici. La réduction d’une protéine cible par une TRX répond principalement à deux nécessités pour la protéine cible, l’une étant de devoir acquérir du pouvoir réducteur pour un gain (ou l’arrêt) d’une fonction biologique (souvent en lien avec une activité enzymatique), l’autre répondant à un besoin de changement conformationnel pour les mêmes raisons que celles précédemment citées. De façon très intrigante, de nombreux organismes pro- et eucaryotes ne possèdent qu’un nombre très limité de TRX, généralement égal ou inférieur à 3, et ce malgré un nombre probablement très élevé de protéines requérant potentiellement une réduction d’un ou de plusieurs de leurs ponts disulfures. A l’inverse, le séquençage de nombreux génomes de plantes et les inventaires de gènes codant des TRX et TRX-like qui ont suivi ont montré que les végétaux possèdent plusieurs dizaines d’isoformes de TRX (et presque autant de GRX), ce qui soulève de nombreuses questions quant à la spécialisation ou la redondance fonctionnelle de toutes ces isoformes. De même, la grande majorité des cibles des TRX à l’issue des programmes de séquençage restaient à identifiées. Pourtant, les outils permettant d’identifier ces cibles des TRX dans des organismes aussi complexes et variés que les plantes ont longtemps tardé à se mettre en place, probablement pris de vitesse par le développement de la génomique à haut débit. Développer des outils adéquats pour l’identification des cibles de TRX qui tiennent compte de la nécessité de déterminer le niveau de spécificité de la relation TRX-cible est ainsi apparu comme une évidence pour une meilleure compréhension des fonctions des TRX chez les plantes. Cet objectif représente également une voie prometteuse et innovante aux études mécanistiques entre TRX et cibles et d’ouvrir largement cette thématique à de nouvelles connaissances l’interconnexion potentielle entres systèmes TRX et GRX, dont on sait qu’ils peuvent être redondants dans certaines situations physiologiques sans pour autant pouvoir expliquer ce fait d’un point de vue réactionnel et structural. Faisant lien avec ce contexte scientifique, je présenterai mes activités de recherche passées au Laboratoire Génome et Développement des Plantes de Perpignan et Montpellier, qui ont concerné en grande partie la mise en place d’outils pour la compréhension des fonctions des thiorédoxines via l’identification de leurs cibles dans la plante, ainsi que les résultats les plus marquants obtenus à ce jour. Je présenterai également une activité transversale développée à l’IRD qui ouvre des perspectives de recherche sur les relations entre rédoxines de plantes et protéines effectrices de pathogènes dépourvus de systèmes redox, au travers l’étude de la flexibilité redox-dépendante d’une protéine virale. Je détaillerai enfin un nouveau projet de recherche, initié récemment à l’interface entre rédoxines, nutrition ferrique et signalisation du stress oxydant, ainsi que les outils qui devront être mis en place dans ce contexte

Temperature stress and redox homeostasis: The synergistic network of redox and chaperone system in response to stress in plants

A remarkable number of strategies has been developed by living organisms to mitigate conflict with environmental changes. The global environment rising with ambient temperature has a wide range of effects on plant growth, and therefore activation of various molecular defenses before the appearance of heat damage. Evidence revealed key components of stress that trigger enhanced tolerance, and some determinants for plant tolerance have been identified. The interplay between heat shock proteins (HSP) and redox proteins is supposed to be vital for the survival under extreme stress conditions. Any circumstance in which cellular redox homeostasis is disrupted can lead to the generation of reactive oxygen species (ROS) that are continuously generated in cells as an unavoidable consequence of aerobic life. Integrative network analysis of synthetic genetic interactions, protein-protein interactions, and functional annotations revealed many new functional processes linked to heat stress (HS) and oxidative stress (OS) tolerance, implicated upstream regulators activated by the either HS or OS, and revealed new connections between them. We present different models of acquired stress resistance to interpret the condition-specific involvement of genes. Considering the basic concepts and the recent advances, the following subsections provide an overview of calcium ion (Ca2+) and ROS interplay in abiotic signaling pathways; further we introduce several examples of chaperone and redox proteins that respond the change of cellular redox status under environmental circumstances. Thus, the involvement or contribution of redox proteins through the functional switching in conjunction with the HSP that prevent heat- and oxidative-induced protein aggregation in plants

The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing

In plants RNA silencing is a host defense mechanism against viral infection, in which double-strand RNA is processed into 21-24-nt short interfering RNA (siRNA). Silencing spreads from cell to cell and systemically through a sequence-specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according to the isolates that they originated from. Here, we show that P1 proteins from two RYMV isolates displaying contrasting suppression strength reduced local silencing induced by single-strand and double-strand RNA in Nicotiana benthamiana leaves. This suppression was associated with a slight and a severe reduction in 21- and 24-nt siRNA accumulation, respectively. Unexpectedly, cell-to-cell movement and systemic propagation of silencing were enhanced in P1-expressing Nicotiana plants. When transgenically expressed in rice, P1 proteins induced specific deregulation of DCL4-dependent endogenous siRNA pathways, whereas the other endogenous pathways were not affected. As DCL4-dependent pathways play a key role in rice development, the expression of P1 viral proteins was associated with the same severe developmental defects in spikelets as in dcl4 mutants. Overall, our results demonstrate that a single viral protein displays multiple effects on both endogenous and exogenous silencing, not only in a suppressive but also in an enhancive manner. This suggests that P1 proteins play a key role in maintaining a subtle equilibrium between defense and counter-defense mechanisms, to insure efficient virus multiplication and the preservation of host integrity

Looking for the function(s) of the plastidial BolA1 and BolA4 proteins in Arabidopsis thaliana

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AtCXXS: atypical members of the Arabidopsis thaliana thioredoxin h family with a remarkably high disulfide isomerase activity

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