Search

Plant viruses transmitted by insects cause tremendous losses in most important crops around the world. The identification of receptors of plant viruses within their insect vectors is a key challenge to understanding the mechanisms of transmission and offers an avenue for future alternative control strategies to limit viral spread. We here report the identification of two cuticular proteins within aphid mouthparts, and we provide experimental support for the role of one of them in the transmission of a noncirculative virus. These two proteins, named Stylin-01 and Stylin-02, belong to the RR-1 cuticular protein subfamily and are highly conserved among aphid species. Using an immunolabeling approach, they were localized in the maxillary stylets of the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae, in the acrostyle, an organ earlier shown to harbor receptors of a noncirculative virus. A peptide motif present at the C termini of both Stylin-01 and Stylin-02 is readily accessible all over the surface of the acrostyle. Competition for in vitro binding to the acrostyle was observed between an antibody targeting this peptide and the helper component protein P2 of Cauliflower mosaic virus. Furthermore, silencing the stylin-01 but not stylin-02 gene through RNA interference decreased the efficiency of Cauliflower mosaic virus transmission by Myzus persicae. These results identify the first cuticular proteins ever reported within arthropod mouthparts and distinguish Stylin-01 as the best candidate receptor for the aphid transmission of noncirculative plant viruses. IMPORTANCE Most noncirculative plant viruses transmitted by insect vectors bind to their mouthparts. They are acquired and inoculated within seconds when insects hop from plant to plant. The receptors involved remain totally elusive due to a long-standing technical bottleneck in working with insect cuticle. Here we characterize the role of the two first cuticular proteins ever identified in arthropod mouthparts. A domain of these proteins is directly accessible at the surface of the cuticle of the acrostyle, an organ at the tip of aphid stylets. The acrostyle has been shown to bind a plant virus, and we consistently demonstrated that one of the identified proteins is involved in viral transmission. Our findings provide an approach to identify proteins in insect mouthparts and point at an unprecedented gene candidate for a plant virus receptor.

BGPI : équipe 2; Cucumber mosaic virus (CMV, Bromoviridae family) and Turnip mosaic virus (TuMV, Potyviridae family) are non-persistentviruses transmitted by aphids. They are acquired during brief periods (few seconds to several minutes) when aphids feed on infected plants and are inoculated to healthy plants during subsequent feeding. Virions are thought to interact either directly to receptors on the maxillary stylet cuticle (capsid strategy used by Cucumoviruses, CMV) or via an additional viral protein the helper component HC (helper strategy used by Caulimoviruses and Potyviruses, such as TuMV).To identify the location of retention of both of these viruses within aphid stylets two approaches were conducted: (i)transmission electron microscopy (TEM) observations of cross-sections of stylets and (ii) epifluorescence microscopy on viruliferous aphid stylets or on dissected stylets incubated with fluorescently labelled HC or virions.A critical point to ensure the maximum chance of observing virus-stylet interactions is to determine the best virus strain-aphid species pair for transmission, since we suppose the viruses’ interactions with aphid receptor(s) might be more labile , than for P2 and CaMV binding. The second critical step, for in vitro interaction assays is to define a “perfect interaction buffer” to favor the retention of virions or HC. Some additional challenges still need to be solved since we mainly have results on CMV retention sites made by TEM: in vitro interaction assays on dissected stylets has not allowed identification of CMV particles on the maxillary stylets. However,CMV particles were found in the food canal and upper part of the common canal by TEM.Progress with TuMV has also been made as we recently found a TuMV/aphid pair surpassing our previous transmission rates (up to 70% with 1 aphid per plant) and we are now more confident for future experiments. A first series of in vitro interaction assays made with different buffers do not yet allow to define an optimal pH or a specific composition to reach the "perfect interaction buffer". The current results and goals of these experiments will be discussed

BGPI : équipe 2; Nanoviruses are multipartite ssDNA viruses transmitted by aphid vectors. Their mode of transmission is believed to be of the circulative non-propagative type [1, 2], but a recent analysis of the relative frequency of the distinct genome segments in aphids versus in plants indicated a more complex relationship [3]. Previous microscopy studies using immunofluorescence against the coat protein of Banana bunshy top virus (BBTV) confirmed that the virus is detected solely in gut and principal salivary gland cells of its aphid vector Pentalonia nigronervosa [4]. However, the subcellular localization and the cell compartment(s) with which the virus associates during transcytosis could not be determined [5]. Another unresolved question is whether, as demonstrated in monopartite viruses, nanoviruses are submitted to strong bottleneck during aphid transmission (see the abstract by Romain Gallet). In that case, when the virus particles traverse cellular barrier, different particles containing different segments may be separated, increasing the chance of loosing genomic information (loosing segments) and thus aborting transmission. We have revisited the cycle of a nanovirus, the Faba bean necrotic stunt virus, within the body of its aphid vector, Acyrthosiphon pisum, using a combination of FISH (Fluorescent In Situ Hybridization), immunolabelling, confocal and electron microscopy. FISH labeling of viral DNA indicates that the virus massively accumulates in all cells of the midgut but is virtually absent in all other regions of the gut. At the salivary glands, the virus is very much restricted not only to the principal salivary glands but also to a single cell type within these glands. No signal could ever be detected in any other organ. When using DNA-FISH probes specific to distinct segments, we could demonstrate that all segments are always colocalized within the midgut, and to a lesser extent also within the salivary gland cells. This observation contrasts with the situation in infected plant cells (see abstract from Anne Sicard), and indicate that the size of the viral population traversing cellular barriers within the aphid is often sufficiently large to prevent the loss of rare genome segments. Finally, we have indication that the genome segment N controls internalization of the virus within aphid gut cells. We currently investigate the subcellular compartment with which the virus associates, and the mode of action of the NSP protein (product of gene N)

Nanoviruses are multipartite ssDNA viruses transmitted by aphid vectors. Their mode of transmission is believed to be of the circulative non-propagative type [1, 2], but a recent analysis of the relative frequency of the distinct genome segments in aphids versus in plants indicated a more complex relationship [3]. Previous microscopy studies using immunofluorescence against the coat protein of Banana bunshy top virus (BBTV) confirmed that the virus is detected solely in gut and principal salivary gland cells of its aphid vector Pentalonia nigronervosa [4]. However, the subcellular localization and the cell compartment(s) with which the virus associates during transcytosis could not be determined [5]. Another unresolved question is whether, as demonstrated in monopartite viruses, nanoviruses are submitted to strong bottleneck during aphid transmission (see the abstract by Romain Gallet). In that case, when the virus particles traverse cellular barrier, different particles containing different segments may be separated, increasing the chance of loosing genomic information (loosing segments) and thus aborting transmission. We have revisited the cycle of a nanovirus, the Faba bean necrotic stunt virus, within the body of its aphid vector, Acyrthosiphon pisum, using a combination of FISH (Fluorescent In Situ Hybridization), immunolabelling, confocal and electron microscopy. FISH labeling of viral DNA indicates that the virus massively accumulates in all cells of the midgut but is virtually absent in all other regions of the gut. At the salivary glands, the virus is very much restricted not only to the principal salivary glands but also to a single cell type within these glands. No signal could ever be detected in any other organ. When using DNA-FISH probes specific to distinct segments, we could demonstrate that all segments are always colocalized within the midgut, and to a lesser extent also within the salivary gland cells. This observation contrasts with the situation in infected plant cells (see abstract from Anne Sicard), and indicate that the size of the viral population traversing cellular barriers within the aphid is often sufficiently large to prevent the loss of rare genome segments. Finally, we have indication that the genome segment N controls internalization of the virus within aphid gut cells. We currently investigate the subcellular compartment with which the virus associates, and the mode of action of the NSP protein (product of gene N).

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue-cultured plantlets grown in vitro. Six mutants of strain XaFL07-1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly-β-hydroxybutyrate than the wild-type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non-ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild-type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.

UMR BGPI: équipe 7; International audience; Cowpea, (Vigna unguiculata L. (Walp)) is an annual tropical grain legume. Often referred to as "poor man's meat", cowpea is one of the most important subsistence legumes cultivated in West Africa due to the high protein content of its seeds. However, African cowpea production can be seriously constrained by viral diseases that reduce yields. While twelve cowpea- infecting viruses have been reported from Africa, only three of these have so-far been reported from Burkina Faso. Here we use a virion-associated nucleic acids (VANA)-based metagenomics method to screen for the presence of cowpea viruses from plants collected from the three agro-climatic zones of Burkina Faso. Besides the three cowpea-infecting virus species which have previously been reported from Burkina Faso (Cowpea aphid borne mosaic virus [Family Potyviridae], the Blackeye cowpea mosaic virus-a strain of Bean common mosaic virus D [Family Potyviridae] and Cowpea mottle virus [Family Tombusviridae]) five additional viruses were identified: Southern cowpea mosaic virus (Sobemovirus genus), two previously uncharacterised polerovirus-like species (Family Luteoviridae), a previously uncharacterised tombusvirus-like species (Family Tombusviridae) and a previously uncharacterised mycotymovirus-like species (Family Tymoviridae). Overall, potyviruses were the most prevalent cowpea viruses (detected in 65.5% of samples) and the Southern Sudan zone of Burkina Faso was found to harbour the greatest degrees of viral diversity and viral prevalence. Partial genome sequences of the two novel polerovirus-like and tombusvirus-like species were determined and RT-PCR primers were designed for use in Burkina Faso to routinely detect all of these cowpea-associated viruses.

International audience

International audience

Begomoviruses (family Geminiviridae) are transmitted by the whitefly Bemisia tabaci in a circulative non propagative manner. B. tabaci is a species complex composed of at least 24 morphocryptic species which differ in host range, insecticide resistance, endosymbionts and virus transmission. Begomoviruses are supposed to cross the gut barrier at the midgut level and salivary gland barrier in the principal salivary gland (PSG) cells because of the highest virus concentrations in these organs. Thus, the critical steps of the virus circulation in the insect body are (i) the exit of virion from the midgut, (ii) their preservation in the hemocoel and (iii) their entry in the PSG. Thus, we proposed that the efficiency of viral transfer from midgut to PSG may be assessed by measuring the viral content in both compartments and that the deduced viral content ratio may be correlated to viral transmission efficiency by the vector. Our predicition was tested with two invasive B. tabaci species, Middle East-Minor Asia 1 (MEAM1), and Mediterranean (MED), and three begomoviruses: the invasive species Tomato yellow leaf curl virus-Mild (TYLCV-Mld), Tomato leaf curl Comoros virus (ToLCKMV), indigenous from Mayotte and R4, a recombinant between TYLCV and ToLCKMV. In a first approach, PSG and midgut were separated by a cross section through the prothorax and viral loads were estimated in both sections by measuring viral DNA using real time PCR. As the midgut of B. tabaci was reported to be sometimes pushed through the diaphragm separating the abdomen and the thorax, the estimation of the viral content ratio between PSG and midgut may be biased by thorax sectioning. The simple cross sectioning was however validated because the ratio determined with such sections and the ratio determined after a careful gut dissection was similar. Using the simple cross section, the viral content ratio between head and abdomen was higher for MEAM1 than for Med for the three begomoviruses. As predicted, the transmission efficiency was higher with MEAM1 than Med Q2 for the three viruses. These results indicate that viral content ratio may be a reliable predictor of the relative transmission efficiency between different B. tabaci populations. Measuring transmission efficiency is time consuming, involves technically difficult experiments with acquisition and inoculation steps and needs specialized cage and containment equipment. However measuring viral content ratios needs only a few cages for the acquisition step, a binocular lens and an access to the commonly used qPCR machines. This approach might be extended to estimate the relative transmission efficiency of other circulative non propagative viruses.

International audience; Hundreds of plant virus species with major agricultural and economical impacts are transmitted by aphids in a non-circulative manner : they are retained on attachment sites within the insect maxillary stylets, and subsequently released when Aphids feed for inoculation into new host plants. Highly specific interactions between viruses and their vectors are involved in this reversible attachment process. Working on aphid stylets still represents a major bottleneck to the identification of receptors, and despite considerable efforts only those from the Cauliflower mosaic virus were partially characterized to date. These receptors are located in a newly identified organ, the Acrostyle, present in the common canal at the extreme tip of the maxillary stylets. We showed that this organ contains cuticular proteins from the RR2 family. Its physiological role remains totally unknown. However, because it’s the only distinctive feature present at this location, and because we have shown that at least one virus can use it as a natural affinity chromatography device, for binding and release during transmission, we propose the Acrostyle could transiently promote the binding and release of other compounds, from the plant (eg:toxic molecules) or from the aphid saliva (eg: effectors counteracting plant defences), and thereby play a role in aphid/plant compatibility. Research is currently underway to determine whether this organ plays a role in the transmission of the Cucumber Mosaic virus. The Most Recent Advances Will Be Presented And discussed.

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.

Two types of interactions have been described during the Aeschynomene-photosynthetic Bradyrhizobium symbioses-a classical one which is Nod factors (NF) dependent and an atypical one which is NF independent. It is the Aeschynomene sp. that determines the signaling modus. Some photosynthetic strains such as ORS285 that do contain the canonical nodABC genes are able to use bath strategies depending on the hast plant. Whereas the nod-Iacking strain such as BTAi 1 and ÛRS278 nodulate only some Aeschynomene species according a Nod-independent process. In this study, we investigate the role of lipopolysaccharides (LPS) in the establishment of the Aeschynomene-Bradyrhizobium symbiosis. Structural analysis of the LPS structure of photosynthetic Bradyrhizobium strains (BTAi1 and ORS278) reveals two original features : i) The only component of the 0- O-antigen region consists of a unique monosaccharide that had never been described before in nature. This new sugar was named Bradyrhizose (Alba S et al. 20 Il), il) the lipid A region displays the covalent attachment of an hopanoid molecule to a very long chain fatty acid. Screening of a mutant library of ûRS285 s train for clones showing an aberrant colony morphology has permitted to identify a mutant affected in a putative 0-antigen ligase that lacks the O-antigen part. Analysis of the symbiotic properties of this mutant shows that this region is important for the establishment of the Nod-dependent process but not for the Nod-independent one. To investigate the biological significance of the Hopanoid-lipid A, we cOl1structed a mutant in the squalene hopene cyclase (shc) gene that catalyzes a key step in hopanoid biosynthesis. This mutant was perfectly able to engage in an efficient symbiotic relationship with the plant but very rapidly the nodules lost their ability to fix nitrogen and senesce prematurely. Altogether these data indicate that Photosynthetic bradyrhizobia display a unique LPS structure that plays an important role in the establishment of the Nod-dependent symbiosis and in the chronic infection of the plant cells.

National audience

Equipe 2; 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

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.

National audience; Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, is a pathogen that experienced genome reduction during its speciation. Additionally, this xanthomonad is notably missing the Hrp type III secretion system and the xanthan gene cluster that are commonly found in pathogenic Xanthomonas species. X. albilineans was up to now considered as limited to the xylem of sugarcane. However, recently published studies suggested that X. albilineans was able to invade tissues other than the xylem of sugarcane leaves but the occurrence of X. albilineans outside the xylem has not been clearly proven. In this study, we used confocal microscopy and transmission electron microscopy to investigate the localization of this pathogen in diseased leaves and stalks of sugarcane. Three sugarcane cultivars with different levels of resistance to leaf scald were inoculated with the green fluorescent protein labelled X. albilineans strains XaFL07-1 (from Florida) and GPE PC73 (from Guadeloupe). Sections of sugarcane leaves and stalks were examined 8-60 days after inoculation in order to localize X. albilineans in the different plant tissues. Confocal microscopy observation of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and the metaxylem, however, X. albilineans was also observed in the phloem, the parenchyma and the bulliform cells of the leaves. Similarly, the protoxylem and the metaxylem of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in the intercellular spaces between these cells. Several of these observations made by confocal microscopy have been confirmed by transmission electron microscopy. X. albilineans can therefore no longer be considered as a xylem-limited pathogen. To our knowledge, this is the first description of a plant pathogenic bacterium invading apparently intact non-vascular plant tissue and multiplying in parenchyma cells. The mechanisms and virulence factors used by X. albilineans to enter and invade different tissues of sugarcane remain to be identified

Cauliflower mosaic virus (CaMV) is transmitted by aphids. CaMV forms in infected cells many viralfactories, which contain a lot of CaMV particles, and a single “transmission body” (TB). The TBcontains only few viral particles and the aphid transmission protein P2. P2 is absolutely requiredfor virus transmission because it binds CaMV particles to a receptor localized in the stylets of theaphid vector. (...)

National audience

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; Transmission of Cauliflower mosaic virus (CaMV) by aphids depends on the presence of viral 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 functions. 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. 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 by 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; Nearly all plant viruses use specific vectors to spread from one host to another, the most common vectors being insects, especially aphids. The predominant strategy for virus–vector interaction is the so-called non-circulative transmission, in which the virus is taken up by a vector on an infected plant, attached to unknown receptors somewhere in the feeding apparatus, and subsequently released to inoculate a new host plant. The most precise data on these enigmatic binding sites in insect mouthparts came from a recent pioneering study using Cauliflower mosaic virus (CaMV), a non-circulative virus, to search for receptor molecules in the aphid vectors. A novel in vitro system allowed rapid visualization of the interaction between dissected aphid stylets and the CaMV ligand protein. This provided the first direct evidence for the existence, the precise localization and the chemical nature of a true receptor molecule used by a plant virus in the vector’s mouthparts. The receptor molecules are concentrated exclusively in a tiny area located in the common duct at the extreme distal tip of the aphid maxillary stylets, and are non-glycosylated proteins strongly linked to and deeply embedded into the chitin matrix (Uzest et al., 2007). Using transmission and scanning electron microscopy for further investigating the ultra-structure of this area, we uncovered an intriguing anatomical zone, lining the bed of the common duct in each maxillary stylet, that had thus far been overlooked. This area appears as a swelling of the cuticle surface that perfectly matches the area where the CaMV is specifically binding. It is present at all developmental stages of the aphid vectors, and also found in aphid species that do not transmit CaMV. We are currently investigating the protein contents, the ultrastructure, the biochemical and biological properties of this peculiar structure, in order ultimately to elucidate its physiological function, presumably in the feeding process of aphids or in their relationship with the host plant.

National audience; Transmission of Cauliflower mosaic virus (CaMV) by aphids depends on the presence of viral electron-lucent inclusion bodies (EL) in infected plant cells. EL contain the aphid transmission factor, the viral protein P2, and the viral protein P3. When EL do not form, no transmission occurs even when infected cells contain functional P2 (Khelifa et al., 2007, J Gen Virol 88, 2872-2880). Thus, EL are structures specialised in transmission, hence our interest to study their formation and functions. We detected that stress induces import of apparently soluble tubulin into EL. FRAP experiments indicated a high turnover rate of EL-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 EL and transmission efficiency, and that aphid punctures themselves might induce tubulin influx into EL. The Ca2+ ionophore A23187 induced tubulin influx into EL ; the Ca2+ channel blocker La3+ completely inhibited transmission. Finally, incubation of infected protoplasts with NaN3 induced disintegration of EL and relocalisation of P2 and virions on microtubules, concomitant with drastically increased CaMV transmission. Taken together, our results indicate that a Ca2+ signalling cascade, which might be triggered as an early plant defence response by exploratory intracellular stylet punctures of the aphid vector, “activates” the otherwise “dormant” EL for transmission by causing massive entry of tubulin in EL, possibly followed by redistribution of P2 and virions on microtubules all over the cell. Thus it seems that CaMV might deflect a host defence pathway to perceive the presence of the aphid vector and to prepare its acquisition.

National audience; La grande majorité des virus de plante utilise un vecteur pour passer d’un hôte à un autre, et les pucerons sont de loin les vecteurs qui transmettent le plus de virus. La transmission non- circulante est la stratégie majoritaire : les virus acquis sur une plante infectée ne sont pas internalisés dans l’organisme de l’insecte vecteur, mais sont retenus au niveau de ses pièces buccales, d’où ils pourront être relargués lors de nouvelles piqûres, initiant ainsi l’infection de nouvelles plantes hôte. Nous avons utilisé le modèle Cauliflower mosaic virus (CaMV)-puceron pour caractériser les sites d’attachement d’un virus non-circulant dans les stylets de son insecte vecteur. Nous avons développé un système d’interaction in vitro, sur les stylets du puceron disséqué, qui nous a permis d’y localiser précisément les protéines cuticulaires que le CaMV utilise comme des récepteurs spécifiques. Les sites d’attachement du CaMV sont très concentrés et restreints à une petite zone, non décrite jusque là, qui tapisse le fond du canal commun (alimentaire/salivaire) située à l’extrême pointe des stylets maxillaires1. Des études sont en cours pour identifier les molécules réceptrices du CaMV, et nous présenterons des données préliminaires qui indiquent que des protéines cuticulaires à motif R&R seraient présentes au niveau cette zone. L’utilisation de la microscopie électronique et de la microscopie à balayage nous a permis de mettre en évidence une ultra-structure singulière, présente dans le fond du canal commun, qui correspond parfaitement à la zone d’accrochage du CaMV, et qui n’avait jamais été mise à jour par les travaux antérieurs d’anatomie. Nous discuterons des fonctions putatives de cet « organe » très intrigant qui semble présent chez toutes les espèces de pucerons testées (y compris une espèce non-vectrice du CaMV) et à tous les stades du développement.

International audience

UMR BGPI - Equipe 2; National audience; Du fait de l’immobilité de leurs hôtes, l’immense majorité des virus de plante utilisent des vecteurs spécifiques pour passer d’un hôte à un autre. Ces « véhicules de transport » sont principalement des arthropodes et en grande majorité des pucerons, qui sont des insectes de type piqueur-suceur [1]. Pour les interactions virus-vecteur, la stratégie la plus communément utilisée par les virus de plante est la transmission dite non circulante, où les particules virales prélevées lors d’un repas dans les cellules infectées seront retenues au niveau de sites d’attachement dans les pièces buccales antérieures de l’insecte sans effectuer de passage à l’intérieur de son organisme. Ces particules virales seront ensuite relarguées de ces sites d’attachement lors de piqûres sur de nouvelles cellules hôtes et induiront ainsi l’infection dans de nouvelles plantes [2]. Si les mécanismes moléculaires de la transmission non circulante sont bien documentés en ce qui concerne le partenaire viral, les sites d’attachement correspondants dans les stylets du vecteur demeurent la principale « boîte noire » pour laquelle aucune donnée n’est disponible. Malgré l’importance de ce mode de transmission, l’existence même d’un récepteur spécifique n’a jamais été prouvée pour aucun virus. Dans une étude très récemment publiée [3], nous avons localisé précisément le récepteur du Caulifower mosaic virus (CaMV) et déterminé sa nature chimique.

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.