Search

International audience; Alighting aphids probe a new host plant by intracellular test punctures for suitability. These induce immediate calcium signals that emanate from the punctured sites and might be the first step in plant recognition of aphid feeding and the subsequent elicitation of plant defence responses. Calcium is also involved in the transmission of non-persistent plant viruses that are acquired by aphids during test punctures. Therefore, we wanted to determine whether viral infection alters calcium signalling. For this, calcium signals triggered by aphids were imaged on transgenic Arabidopsis plants expressing the cytosolic FRET-based calcium reporter YC3.6-NES and infected with the non-persistent viruses cauliflower mosaic (CaMV) and turnip mosaic (TuMV), or the persistent virus, turnip yellows (TuYV). Aphids were placed on infected leaves and calcium elevations were recorded by time-lapse fluorescence microscopy. Calcium signal velocities were significantly slower in plants infected with CaMV or TuMV and signal areas were smaller in CaMV-infected plants. Transmission tests using CaMV-infected Arabidopsis mutants impaired in pathogen perception or in the generation of calcium signals revealed no differences in transmission efficiency. A transcriptomic meta-analysis indicated significant changes in expression of receptor-like kinases in the BAK1 pathway as well as of calcium channels in CaMV- and TuMV-infected plants. Taken together, infection with CaMV and TuMV, but not with TuYV, impacts aphid-induced calcium signalling. This suggests that viruses can modify plant responses to aphids from the very first vector/host contact.

De nombreux virus développent des corps d'inclusion dans les cellules végétales ou animales infectées, et leur synthèse, composition et morphologie varient amplement suivant l'espèce virale considérée. Ces structures sont souvent le résultat de réarrangements des membranes de différents compartiments cellulaires, mais certaines sont indépendantes du système membranaire de l'hôte. Les corps d'inclusion dépourvus de membranes sont en général assimilés à des agrésomes ou à des granules de stress mais, là encore, certains d'entre eux n'ont pas de relation apparente avec une structure cellulaire. Dans les cas les mieux caractérisés, ces corps d'inclusion forment un " organite viral ", lieu propice à la réplication du génome et à l'assemblage des particules, à l'abri des défenses de l'hôte, et dénommé " usine virale ". Progressivement des fonctions multiples et diverses ont été associées à ces usines virales. Un exemple spectaculaire est le cas du virus de la mosaïque du chou-fleur ou Cauliflower mosaic virus (CaMV) qui fait l'objet de cette revue. De manière inattendue, les usines virales du CaMV interviennent au-delà de la réplication et de l'assemblage des virions dans une autre étape clé du cycle viral : la transmission par les pucerons vecteurs.

BGPI : équipe 2; International audience; Many viruses form inclusion bodies in infected plant and mammalian cells. Their formation often requires membrane rearrangement of various organelles, but some inclusions form in the cytoplasm independently of the endomembrane system. In the latter case, they may resemble aggresomes or stress bodies but many inclusions do not seem to be related to any cellular structures. Synthesis, composition and size of these inclusions change with virus species. The best characterized inclusions create a “viral organelle” protecting viruses from host defenses and optimizing viral replication and assembly. These inclusions are also called viral factories. Recently, more complex and original functions were described for viral factories. This is exemplified here for Cauliflower mosaic virus (CaMV) factories. Unexpectedly, besides replication, CaMV factories also participate in another crucial step of the viral cycle: vector-transmission by aphids.; De nombreux virus développent des corps d’inclusion dans les cellulesvégétales ou animales infectées, et leur synthèse, composition et morphologie varient amplement suivant l’espèce virale considérée. Ces structures sont souvent le résultat de réarrangements des membranes de différents compartiments cellulaires, mais certaines sont indépendantes du système membranaire de l’hôte. Les corps d’inclusion dépourvus de membranes sont en général assimilés à des agrésomes ou à des granules de stress mais, là encore, certains d’entre eux n’ont pas de relation apparente avec une structure cellulaire. Dans les cas les mieux caractérisés, ces corps d’inclusion forment un « organite viral », lieu propice à la réplication du génome et à l’assemblage des particules, à l’abri des défenses de l’hôte, et dénommé « usine virale ». Progressivement des fonctions multiples et diverses ont été associées à ces usines virales. Un exemple spectaculaire est le cas du virus de la mosaïque du chou-fleur ou Cauliflower mosaic virus (CaMV) qui fait l’objet de cette revue. De manière inattendue, les usines virales du CaMV interviennent au-delà de la réplication et de l’assemblage des virions dans une autre étape clé du cycle viral : la transmission par les pucerons vecteurs.

National audience

National audience; Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise mechanism of TB-mediated virus acquisition by aphids is unknown. We have shown that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting the TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results reveal a fascinating, and hitherto unforeseen mechanism whereby CaMV shares the horst’s perception of the aphid, translating it into an independent response. The unattended capability of viruses to react, via the host, to the outside world opens new horizons, i.e. investigating the impact of “perceptive behaviors” on other steps of the infection cycle. We try to understand which mechanisms and signalization pathway are involved in this phenomenon. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of ‘perceptive behaviors’ on other steps of the infection cycle

International audience; Viruses rely totally on the host to achieve every step of the infection cycle. Much is known about how viruses interfere with cellular processes to put them at their use and it is clear that they intercept intracellular and intra-host communication and processes to optimise interaction with the host. Here we unprecedentedly that show viruses are also able to use the host sensorial system to very rapidly perceive and react on cues from the world outside the host, in a way disconnected from the reaction of the host itself. Cauliflower mosaic virus (CaMV) is transmitted from plant-to-plant by aphids, and previous work has shown that the virus-aphid interaction is not an accidental process but depends on the presence of the virusinduced Transmission Bodies (TBs) in infected cells, containing the CaMV transmissible complexes. Our results demonstrate that TBs react on the presence and feeding of the insect vector by rapidly and reversibly dispersing their contents on cortical microtubules throughout the cell. If this TB reaction is perturbed, transmission rates drop; if this reaction is artificially enhanced, transmission rates rise. This shows that CaMV intercepts the host's perception of the aphid and immediately translates it in an appropriate response that optimises its chances of acquisition, everything going back to normal standby state a few minutes later.

International audience; The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt-stressed root cells are discussed.

International audience; Transmission of Cauliflower mosaic virus (CaMV) by aphids depends on the presence of viral inclusions, the Transmission Bodies (TB), in infected plant cells. TB contain the aphid transmission factor, the viral protein P2, and the viral protein P3. When TB do not form, no transmission occurs even when infected cells contain functional P2 (Khelifa et al. 2007). Thus, TB are structures specialised for transmission, hence our interest to study their formation and function (Martinière et al. 2009). We detected that stress induces import of apparently soluble tubulin into TB. FRAP experiments indicated a high turnover rate of TB-contained tubulin. In aphid transmission experiments, we found that aphids fed on stressed infected leaves transmitted CaMV better than aphids fed on control leaves, that there was a positive correlation between tubulin entry in TB and transmission efficiency, and that aphid punctures themselves might induce rapid (within seconds) tubulin influx into TB. The Ca2+ ionophor A23187 induced tubulin influx into TB; the Ca2+ channel blocker La3+ completely inhibited transmission. The microtubule depolymeriser oryzalin inhibited transmission indicating involvement of microtubules in CaMV transmission. Finally, incubation of infected protoplasts with NaN3 induced disintegration of TB and relocalisation of P2 and virions on microtubules, concomitant with drastically increased CaMV transmission. Preliminary data indicate that also ROS might induce TB disintegration. Taken together, our results indicate that a Ca2+ signalling cascade, which might be triggered as an early plant defence response to exploratory intracellular stylet punctures of the aphid vector, “activates” the otherwise “dormant” TB for transmission by causing massive entry of tubulin in TB, possibly followed in a second step by redistribution of P2 and virions on microtubules all over the cell.Thus it seems that CaMV deflects host perception and signalling pathways to perceive the presence of the aphid vector and to actively prepare its own acquisition.

International audience

The vast majority of plant viruses being transmitted from host to host by vectors, studying the process of transmission is of interest to both basic and applied research. While the molecular interactions between viruses and their vectors are the object of many studies, the participation of the host in the transmission mechanism seems to have been neglected. Thus, the host cell is often considered as a mere “sac” from which the viruses are accidentally acquired by the vector. However, the CaMV forms in infected host cells a structure specialised and mandatory for transmission, the electron-lucent inclusion body (EL). Consequently, the issues addressed in this thesis were (i) to determine how ELs form in infected host cells, and (ii) to determine how the CaMV is acquired by its vector. To address the first question, we searched to identify cellular partners involved in EL formation. Our results show that microtubules are required for their formation and suggest that the kinesin TBK5 transports the CaMV proteins via microtubules from their site of synthesis to emerging ELs. Concerning the mechanisms of CaMV acquisition by its aphid vector, we show that the wounding caused by the intracellular puncture of the aphid provokes a cellular stimulus triggering a calcium-mediated signaling cascade. After this stimulus, an important influx of tubulin into the EL. This process might “activate” the EL, since it is accompanied by a highly significant increase of the transmission rate of CaMV by its vector. Taken together, our results indicate that the process of transmission of CaMV is finely tuned on the level of each infected cell, which not only participates in EL formation but also in its “activation”., La grande majorité des phytovirus étant transmis d’hôte en hôte par vecteur, l’étude des processus de transmission présente un intérêt tant fondamental qu’appliqué. Alors que les interactions moléculaires entre le virus et son vecteur font l’objet de nombreuses études, les mécanismes cellulaires impliqués dans la transmission ont été peu étudiés. La cellule est ainsi souvent considérée comme un sac dans lequel les virus sont prélevés passivement par le vecteur. Le CaMV développe pourtant dans les cellules hôtes un « corps d’inclusion clair », structure spécialisée et indispensable à la transmission. De ce fait, le présent travail met la cellule hôte au centre des recherches. Tout d’abord, nous avons cherché à identifier les partenaires cellulaires permettant la formation des corps clairs. Les résultats montrent que les microtubules sont indispensables à leur formation et suggèrent que la kinésine TBK5 transporte des protéines viral sur les microtubules, de leur site de synthèse vers les corps clairs émergeant. Nous nous sommes, ensuite, focalisé sur les évènements cellulaires se produisant lors de l’acquisition du virus par le puceron. La blessure provoquée par la piqûre intracellulaire du puceron, provoque un stimulus déclenchant une cascade de signalisation via la voie calcique. Après cette blessure, la tubuline afflue massivement dans les corps clairs. Ce processus semble alors les « activer » car il augmente très significativement la transmission des virus par leurs vecteurs. Nos résultats indiquent que le processus de transmission du CaMV par vecteur est finement régulé au sein de chaque cellule infectée, celle-ci participant non seulement à la synthèse du corps clair mais également à son « activation ».

National audience; Afin d'assurer leur cycle viral, les virus détournent spécifiquement la physiologie de la cellule hôte. Ce phénomène a été de nombreuses fois décrit durant la réplication et le mouvement de cellule à cellule. En revanche, des interactions entre l'hôte et le virus contrôlant spécifiquement la transmission par vecteur n'ont jamais été décrites. Chez le Cauliflower Mosaic Virus (CaMV), il apparaît que la transmission peut être préparée bien avant la rencontre virus-vecteur. En effet, ce virus forme au sein des cellules hôtes des corps d'inclusion clairs (CC) spécialisés dans la transmission, mais non obligatoires au développement du virus (Espinoza et al., 1991; Khelifa et al., soumis). Ces CC sont composés majoritairement de deux protéines virales, d'une part P2 qui est le facteur helper de la transmission, et d'autre part P3, une protéine multifonctionnelle (Drucker et al., 2002). La formation des CC reste énigmatique, car tous les produits viraux sont synthétisés en un autre lieu, dans les corps denses (CD). Nous avons donc suivi les cinétiques de la formation des CC dans des protoplastes transfectés avec le CaMV. Par immunofluorescence, les protéines virales constituant les CC (P2 et P3) ont été suivies durant l'infection. Nos résultats montrent que (i) ces protéines apparaissent d'abord au niveau des CD, (ii) ensuite, elles co-localisent de manière transitoire avec le réseau microtubulaire de la cellule, et (iii) enfin elles s'accumulent dans un seul CC par cellule. Des drogues modifiant la dynamique du cytosquelette d'actine (latrunculine B) et de tubuline (taxol et oryzaline) montrent que seule la dépolymérisation des microtubules aboutit à l'inhibition de la formation des CC. La présence d'un réseau microtubulaire intact est donc nécessaire à la formation des CC et permet ainsi la transmission du CaMV. Un modèle de formation des corps d'inclusion clairs sera présenté.

International audience

National audience

International audience

National audience

National audience