Marilyne Uzest

A protein key to plant virus transmission at the tip of the insect vector stylet

Hundreds of species of plant viruses, many of them economically important, are transmitted by noncirculative vector transmission (acquisition by attachment of virions to vector mouthparts and inoculation by subsequent release), but virus receptors within the vector remain elusive. Here we report evidence for the existence, precise location, and chemical nature of the first receptor for a noncirculative virus, cauliflower mosaic virus, in its insect vector. Electron microscopy revealed virus-like particles in a previously undescribed anatomical zone at the extreme tip of the aphid maxillary stylets. A novel in vitro interaction assay characterized binding of cauliflower mosaic virus protein P2 (which mediates virus–vector interaction) to dissected aphid stylets. A P2-GFP fusion exclusively labeled a tiny cuticular domain located in the bottom-bed of the common food/salivary duct. No binding to stylets of a non-vector species was observed, and a point mutation abolishing P2 transmission activity correlated with impaired stylet binding. The novel receptor appears to be a nonglycosylated protein deeply embedded in the chitin matrix. Insight into such insect receptor molecules will begin to open the major black box of this scientific field and might lead to new strategies to combat viral spread.

Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission. Hence, unlike P2, P3 and virions cannot be sequentially acquired by the vector. Immunogold labeling revealed the predominance of spatially separated P2:P3 and P3:virion complexes in infected plant cells. This specific distribution indicates that the transmissible complex, P2:P3:virion, does not form primarily in infected plants but in aphids. A model, describing the regulating role of P3 in the formation of the transmissible CaMV complex in planta and during acquisition by aphids, is presented, and its consequences are discussed.

New research horizons in vector-transmission of plant viruses.

Understanding the mechanisms controlling vector-transmission of plant viruses requires integrating information from at least three different viewpoints: virus-vector interactions, plant-vector interactions and virus-plant interactions. While some of these aspects have been covered by past and present investigations, others have been bypassed completely, because of technical bottlenecks or conceptual lacunas. Here, we highlight recent advances and needs in hitherto poorly documented aspects of vector transmission, such as characterization of the vector molecules responsible for initial viral recognition, and the role of vector saliva in inoculation and initial onset of infection in a new plant. We also propose and discuss some novel conceptual and complementary questions that are opening up fascinating new horizons in this field. We explore the possible existence of viral morphs with specific properties that facilitate acquisition by vectors, and discuss the dynamics/genetics of such viral subpopulations, which could differentiate and specialize in different host compartments.

A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus.

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.

Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

ABSTRACT Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.

A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species.

ABSTRACT Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the “noncirculative” mode of transmission dominant in plant viruses (designated “mechanical vector transmission” in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the “helper component” (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.

Biochemical Characterization of the Helper Component of Cauliflower Mosaic Virus

ABSTRACT The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% α-helical. Most α-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

Proteomic composition of the acrostyle: Novel approaches to identify cuticular proteins involved in virus–insect interactions

The acrostyle is a distinct anatomical region present on the cuticle at the inner face of the common food/salivary canal at the tip of aphid maxillary stylets. This conserved structure is of particular interest as it harbors the protein receptors of at least 1 plant virus, Cauliflower mosaic virus, and presumably has other roles in plant–insect interactions. Previously we reported immunolabeling of a highly conserved motif of cuticular proteins from the CPR family (named for the presence of a Rebers and Riddiford consensus) within the acrostyle. Here we report the development of novel tools to further study the proteomic composition of this region and to identify proteins involved in insect‐virus interactions. Using a series of antibodies against cuticular proteins from the RR‐2 subfamily, we identified additional peptides present within the acrostyle. Our results demonstrated that the acrostyle is a complex structure containing multiple domains of cuticular proteins accessible for interaction. In addition, an array of overlapping peptides, which covers the diversity of the majority of the RR‐2 subfamily, was developed as a generic tool to characterize cuticular protein/pathogen interactions. Upon probing this array with Cucumber mosaic virus particles, consensus peptide sequences from hybridizing peptides were identified. Use of these novel tools has extended our knowledge of the proteomic composition of insect maxillary stylets and identified sequences that could be involved in virus binding, thus contributing to further elucidation of the various properties and functions of the acrostyle.

A clonally reproducing generalist aphid pest colonises diverse host plants by rapid transcriptional plasticity of duplicated gene clusters

Background The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception, and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. Results To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively up-regulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced up-regulation of these genes. Conclusions Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.

Fasting alters aphid probing behaviour but does not universally increase the transmission rate of non-circulative viruses

A fasting period prior to non-circulative virus acquisition has been shown to increase the rate of transmission by aphids. However, this effect has only been studied for a few virus-vector combinations, and there are contradictory results in the literature as to the role of fasting on virus acquisition. We analysed the influence of fasting on the transmission of three non-circulative viruses, Cucumber mosaic virus, Zucchini yellow mosaic virus and Cauliflower mosaic virus, by two aphid vector species: Myzus persicae Sulzer (Hemiptera: Aphididae) and Aphis gossypii Glover (Hemiptera: Aphididae). All variables tested, including the virus species and isolate, and the species of aphid, influenced the effect of a fasting period on virus transmission efficiency. Furthermore, when aphids were subjected to an overnight feeding period on a sucrose solution, the fasting effect disappeared and the probing behaviour of these aphids was markedly different to plant-reared aphids. The electrical penetration graph (EPG) technique revealed that fasting altered the probing behaviour of M. persicae and A. gossypii, with fasted aphids beginning to feed sooner and having a significantly longer first intracellular puncture, measured as a potential drop. Significantly longer sub-phase II-3 of the potential drop and more archlets during this sub-phase were also observed for fasted aphids of both species. However, these behavioural changes were not predictive of increasing virus transmission following a fasting period. The impacts of pre-acquisition fasting on aphid probing behaviour and on the mechanisms of non-circulative virus transmission are discussed.