Marilyne Uzest

SummaryHybridization chain reaction (HCR) has made it possible to overcome the lack of high-resolution RNA (co-)localization techniques in non-model species. Here, we optimized a protocol for multiplexed whole-mount HCR RNA fluorescence in situ hybridization in the pea aphid Acyrthosiphon pisum asexual and sexual embryos. We describe all steps from biological material production, probes design and hybridization, HCR probes detection to confocal imaging. This protocol sets out HCR steps in details for gene expression detection at any stage of development for embryos that will develop into sexual or asexual adult morphs.

Hundreds of plant viruses are transmitted by aphid vectors, among which non-circulative ones are acquired and inoculated from one host to another within seconds. These viruses are retained on receptors located at the surface of the cuticle of aphid mouthparts. Members of the Potyviridae family are the most abundant RNA viruses infecting plants, and they cause significant economic losses. Among them, viruses of the Potyvirus genus are transmitted by aphids in a stylet-borne manner. Their receptors in aphid stylets remain poorly characterized. Using turnip mosaic virus (TuMV, Potyvirus rapae ) as a model, we developed complementary approaches to investigate potyvirus-aphid interactions in three vector species. Immunofluorescence detection and transmission electron microscopy revealed the presence of TuMV in the distal part of aphid maxillary stylets, both in the food canal and on the acrostyle. This cuticular organ houses Stylin proteins, including Stylin-01, the receptor for the cauliflower mosaic virus ( Caulimovirus tesselobrassicae ). Using CRISPR-Cas9-edited Stylin-01 mutant lines in the pea aphid, we demonstrated that this protein plays an important role in TuMV transmission. Complementary RNA interference silencing experiments revealed that Stylin-04/04bis also mediate TuMV transmission. Furthermore, our findings reveal that targeting simultaneously Stylin-01 and Stylin-04/04bis more strongly impaired the aphids’ ability to transmit TuMV, suggesting that virus transmission relies on a multi-component stylin interface rather than a single receptor. In conclusion, these results highlight that in complex interactions between potyviruses and their aphid vectors, Stylin proteins are key actors, underscoring their importance in the transmission of stylet-borne viruses.

Insect vectors play a key role in the transmission of plant viruses, acting as major drivers of crop losses worldwide. Noncirculative plant viruses rely on brief and transient interactions with their insect vectors for successful transmission. Here, we focused on aphids (Aphidoidea), whiteflies (Aleyrodoidea), and mealybugs (Coccoidea), hemipterans of the suborder Sternorrhyncha, known for transmitting 95% of viruses in a noncirculative mode. Based on the current knowledge, we compared specific morphological and behavioral traits related to their stylets, the cuticular needle‐like structures. This comparison shed some light on the leading role of aphids as vectors, owing to their highly specialized mouthparts and unique intracellular probing behavior. The review also discusses known key molecular interactions at the virus–stylet interface. Comparing these features across the 3 hemipteran superfamilies underscores how specific behavioral and anatomical traits set aphids apart, enabling their remarkable capacity to transmit stylet‐borne, noncirculative viruses.

Specific binding sites for non‐circulative viruses within the mouthparts of aphid vectors have been investigated with limited success. Such sites have been described more precisely for cauliflower mosaic virus (CaMV, Caulimovirus ), and shown to be restricted to the acrostyle, a specific anatomical structure located in the common duct of the aphid stylets. However, the nature and precise location of binding sites of other non‐circulative viruses (e.g., cucumoviruses and potyviruses), and whether distinct viral species compete for the same sites remains unknown. In this study, competition between non‐circulative viruses for binding sites was assessed for viruses that differ in their transmission mode: cucumber mosaic virus (CMV, Cucumovirus ), transmitted by the capsid strategy, and three viruses using the helper strategy for their transmission—turnip mosaic virus (TuMV, Potyvirus ), zucchini yellow mosaic virus (ZYMV, Potyvirus ), and CaMV. In this work, we performed competition and sequential acquisition experiments, with contrasting results obtained depending on the virus species used and the sequence of acquisition/inoculation of the potentially competing viruses. Our results showed that potyviruses and caulimoviruses do not appear to compete for the same binding sites and/or receptors within aphid stylets, as no modifications in their transmission rates were observed regardless of their acquisition sequence. However, a decrease of CMV transmission rate when ZYMV was previously acquired, suggests that potyviruses and cucumoviruses might compete for binding sites in their aphid vectors. Moreover, we observed that potyviruses and cucumoviruses can be co‐acquired and co‐inoculated in the same plant cell during a single intracellular puncture, demonstrating co‐infection of individual cells by more than a single virus species.

Aphids are major agricultural pests, notably because they transmit nearly 30% of known plant viruses, including non-circulative ones. They can be collected and dispersed rapidly among crops while aphids feed on infected plants. Most of these viruses are retained on receptors located on the cuticle of the stylet tip. The acrostyle, a cuticular micro-territory at the apex of aphid stylets, has been identified for its ability to retain the cauliflower mosaic virus (CaMV). The acrostyle displays cuticular proteins, known as stylins, with exposed domains accessible at the virus-vector interface. RNAi-mediated silencing of Stylin-01 designated this protein as the prime candidate receptor of CaMV. However, the results were incomplete due to the transient effect and highlighted the need for stable mutants to advance our knowledge and validate these putative virus receptors. Here, we characterized the phenotype of two pea aphid Stylin-01 mutant lines, the first generated with CRISPR/Cas9 in this hemipteran. We showed that Stylin-01 mutations significantly disrupt CaMV transmission and impair the acrostyle’s ability to bind the CaMV helper protein P2. Stylin-01 mutations also reshape the distribution of other stylins on the surface of mutant aphid stylets. In addition, Stylin-02, the putative ortholog of Stylin-01, is overexpressed in the mutant lines, pointing out a potential partial complementation of Stylin-01 in its structural role but not for virus transmission. In conclusion, this study, using the first stable aphid mutant lines, allows the characterization of the central role of Stylin-01 virus receptor in CaMV transmission.

Potyvirids are the largest group of plant RNA viruses. Pelota, a core component of RNA quality controls (RQC), promotes the degradation of potyvirids’ genomic RNA by recognizing a specific G_1-2A_6-7 motif. Here we demonstrate that the viral RNA-dependent RNA polymerase, NIb, acts as a SUMOylation decoy to effectively reduce Pelota SUMOylation by competing with SCE1 to inhibit Pelota-mediated RQC. TuMV NIb is comprised of two functional SUMO interacting motif (SIM): SIM2 and SIM3. The former is identified as the key site for NIb’s SUMOylation by SUMO3, whereas the latter is responsible for the interaction with SCE1. These two SIMs are conserved among the majority of potyvirids-encoded NIbs. Thus, virus protein-mediated SUMOylation decoy strategy to suppress host defense may be a common feature in plant virus pathosystems. These findings highlight a dynamic interplay between plant defense mechanism and viral counter-strategy by orchestrating the post-translational modifications of virus and host defense components.

Detecting RNA molecules within their natural environment inside intact arthropods has long been challenging, particularly in small organisms covered by a tanned and pigmented cuticle. Here, we have developed a methodology that enables high‐resolution analysis of the spatial distribution of transcripts of interest without having to dissect tiny organs or tissues, thereby preserving their integrity. We have combined an in situ amplification approach based on hybridization chain reaction, which enhances the signal‐to‐noise ratio, and a clearing approach that allows the visualization of inner organs beneath the cuticle. We have implemented this methodology for the first time in Hemiptera, mapping two salivary aphid (Acyrthosiphon pisum) transcripts, the effector c002 and the salivary sheath protein SHP. With a multiplex approach, we could simultaneously detect different mRNAs in mounted pea aphid head‐thorax samples and show that they were distributed in distinct secretory cells of salivary glands.Research Highlights: Combining hybridisation chain reaction and clearing allows the detection of transcripts in intact aphids heads. The transcripts of the two salivary proteins c002 and SHP are compartmentalized in distinct secretory cells of the principal glands.

To avoid the activation of plant defenses and ensure sustained feeding, aphids are assumed to use their mouthparts to deliver effectors into plant cells. A recent study has shown that effectors detected near feeding sites are differentially distributed in plant tissues. However, the precise process of effector delivery into specific plant compartments is unknown. The acrostyle, a cuticular organ located at the tip of maxillary stylets that transiently binds plant viruses via its stylin proteins, may participate in this specific delivery process. Here, we demonstrate that Mp10, a saliva effector released into the plant cytoplasm during aphid probing, binds to the acrostyles of Acyrthosiphon pisum and Myzus persicae. The effector probably interacts with Stylin-03 as a lowered Mp10-binding to the acrostyle was observed upon RNAi-mediated reduction in Stylin-03 production. In addition, Stylin-03 and Stylin-01 RNAi aphids exhibited changes in their feeding behavior as evidenced by electrical penetration graph experiments showing longer aphid probing behaviors associated with watery saliva release into the cytoplasm of plant cells. Taken together, these data demonstrate that the acrostyle also has effector binding capacity and supports its role in the delivery of aphid effectors into plant cells.

Background: Aedes aegyptimosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance inAe. aegyptihas not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity.Results: BW- and FW-Ae. aegyptiwere compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerantAe. aegyptiwere also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults.Conclusions: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerantAe. aegyptiare expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerantAe. aegyptiand genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerantAe. aegyptiand requires appropriate mitigating measures. The findings inAe. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.