Antigen-functionalized turnip mosaic virus nanoparticles increase antibody sensing in saliva. A case study with SARS-CoV-2 RBD.

Nanoparticles derived from plant viruses play an important role in nanomedicine due to their biocompatibility, self-assembly and easily-modifiable surface. In this study, we developed a novel platform for increasing antibody sensing using viral nanoparticles derived from turnip mosaic virus (TuMV) functionalized with SARS-CoV-2 receptor binding domain (RBD) through three different methods: chemical conjugation, gene fusion and the SpyTag/SpyCatcher technology. Even though gene fusion turned out to be unsuccessful, the other two constructs were proven to significantly increase antibody sensing when tested with saliva of patients with different infection and vaccination status to SARS-CoV-2. Our findings show the high potential of TuMV nanoparticles in the fields of diagnostics and immunodetection, being especially attractive for the development of novel antibody sensing devices.

A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP-cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research.

The Fifth Residue of the Coat Protein of Turnip Mosaic Virus Is Responsible for Long-Distance Movement in a Local-Lesion Host and Aphid Transmissibility in a Systemic Host.

HC-Pro and coat protein (CP) genes of a potyvirus facilitate cell-to-cell movement and are involved in the systemic movement of the viruses. The interaction between HC-Pro and CP is mandatory for aphid transmission. Two turnip mosaic virus (TuMV) isolates, RC4 and YC5, were collected from calla lily plants in Taiwan. The virus derived from the infectious clone pYC5 cannot move systemically in Chenopodium quinoa plants and loses aphid transmissibility in Nicotiana benthamiana plants, like the initially isolated virus. Sequence analysis revealed that two amino acids, P5 and A206, of YC5 CP uniquely differ from RC4 and other TuMV strains. Recombination assay and site-directed mutagenesis revealed that the fifth residue of leucine (L) at the N-terminal region of the CP (TuMV-RC4), rather than proline (P) (TuMV-YC5), is critical to permit the systemic spread in C. quinoa plants. Moreover, the single substitution mutant YC5-CPP5L became aphid transmissible, similar to RC4. Fluorescence microscopy revealed that YC5-GFP was restricted in the petioles of inoculated leaves, whereas YC5-CPP5L-GFP translocated through the petioles of inoculated leaves, the main stem, and the petioles of the upper uninoculated leaves of C. quinoa plants. In addition, YC5-GUS was blocked at the basal part of the petiole connecting to the main stem of the inoculated C. quinoa plants, whereas YC5-CPP5L-GFP translocated to the upper leaves. Thus, a single amino acid, the residue L5 at the N-terminal region right before the 6DAG8 motif, is critical for the systemic translocation ability of TuMV in a local lesion host and for aphid transmissibility in a systemic host.

Subcellular Colocalization Assay of Host Factors with Viral Replication Complex in the dsRNA Reporter Nicotiana benthamiana.

As obligate pathogens, plant viruses co-opt several host factors for viral replication. Double-stranded RNA (dsRNA) plays important roles in plants, including eliciting innate immune responses and RNA interference. dsRNA also represents the genetic entities of a number of viruses and is a marker of infection by positive-sense single-stranded RNA viruses. Previous detection methods for RNA viruses basically relied on immunological methods, but later researchers discovered that the dsRNA-binding domain of the Flock house virus B2 protein is a perfect alternative to the J2 mAb for sensitive and rapid detection of long dsRNA in vitro and in vivo, and developed B2:GFP transgenic Nicotiana benthamiana line. This method describes in detail how to visualize host factors in the viral replication complex in time and space with the help of B2:GFP transgenic plants, exemplified by Turnip mosaic virus (TuMV), a representative virus member of the Potyviruses.

Co-immunoprecipitation-Based Isolation of Double-Stranded RNA-Associated Protein Complexes in Nicotiana benthamiana.

Double-stranded RNA (dsRNA) is associated with most viral infections, and is generated in host cells during viral replication. Viral RNA replication occurs within the viral factories called the viral replication complexes (VRCs). In addition to viral genome, viral-derived dsRNA and replicase, the VRCs composition remains largely unexplored. The dsRNA binding domain of the B2 protein from Flock house virus has been reported to be used for detecting viral-derived long dsRNA in plants efficiently. Nicotiana benthamiana is widely used as a model plant for plant-microbe interactions owing to its susceptibility to diverse plant diseases, especially viral diseases. Here, we describe the use of Nicotiana benthamiana stably expressing GFP-tagged dsRNA binding protein (B2: GFP) to pull down dsRNA and associated host and viral proteins from turnip mosaic virus-infected plants. The obtained protein complexes are compatible with functional assays, Western blotting, and mass spectrometry. This system provides a valuable and robust tool to study VRC proteome in N. benthamiana upon plant viral infections.

Analysis of Plant Virus-Induced Immunity by Using Viral-Derived Double-Stranded RNA in Arabidopsis thaliana.

Pattern-triggered immunity is the first line of defense against infection by pathogens such as bacteria and fungi in plants, and this mechanism remains poorly defined in plant viruses. Double-stranded RNA (dsRNA) is an intermediate in the replication of plant RNA viruses, and is considered to be a conserved structure of plant viruses similar to pathogen-associated molecular pattern. Whether dsRNA is the elicitor that activates plant immunity in response to virus infection remains obscure. In this method, we use the cDNA of turnip mosaic virus genome as the template to in vitro synthesis of viral dsRNA and examine whether viral dsRNA could activate plant immunity in Arabidopsis thaliana, including MAPK kinase cascade and reactive oxygen burst. In order to provide some references for researchers studying dsRNA in terms of research methodology and experimental methods, we use western blot to measure MAPK kinase cascade and luminol-based assay to measure ROS burst in Arabidopsis thaliana treated by viral dsRNA.

Intrinsic disorder in the dynamic evolution of structure, stability, and flexibility of potyviral VLP assemblies: A computational study.

An all-atom Molecular Dynamics (MD) study was applied to three viral nanoparticles (VLPs) of Turnip mosaic virus (TuMV), a potyvirus: the particles genetically functionalized with two peptides, VIP (human vasoactive intestinal peptide) and VEGFR (peptide derived from the human receptor 3 of the vascular endothelial growth factor), and the non-functionalized VLP. Previous experimental results showed that VIP-VLP was the only construct of the three that was not viable. VLPs subjected to our MD study were modeled by four complete turns of the particle involving 35 subunits of the coat protein (CP). The MD simulations showed differences in structures and interaction energies associated to the crucial contribution of the disordered N-terminal arms of CP to the global stability of the particle. These differences suggested an overall stability greater in VEGFR-VLP and smaller in VIP-VLP as compared to the unfunctionalized VLP. Our novel MD study of potyviral VLPs revealed essential clues about structure and interactions of these assembled protein particles and suggests that the computational prediction of the viability of VLPs can be a valuable contribution in the field of viral nanobiotechnology.

Plant and animal positive-sense single-stranded RNA viruses encode small proteins important for viral infection in their negative-sense strand.

Positive-sense single-stranded RNA (+ssRNA) viruses, the most abundant viruses of eukaryotes in nature, require the synthesis of negative-sense RNA (-RNA) using their genomic (positive-sense) RNA (+RNA) as a template for replication. Based on current evidence, viral proteins are translated via viral +RNAs, whereas -RNA is considered to be a viral replication intermediate without coding capacity. Here, we report that plant and animal +ssRNA viruses contain small open reading frames (ORFs) in their -RNA (reverse ORFs [rORFs]). Using turnip mosaic virus (TuMV) as a model for plant +ssRNA viruses, we demonstrate that small proteins encoded by rORFs display specific subcellular localizations, and confirm the presence of rORF2 in infected cells through mass spectrometry analysis. The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes. The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase, and mutation of rORF2 completely abolishes virus infection, whereas ectopic expression of rORF2 rescues the mutant virus. Furthermore, we show that several rORFs in the -RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have the ability to suppress type I interferon production and facilitate the infection of vesicular stomatitis virus. In addition, we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs. Taken together, these findings indicate that the -RNA of +ssRNA viruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection, revealing a viral proteome larger than previously thought.

Aphids on Aphid-Susceptible Cultivars Have Easy Access to Turnip Mosaic Virus, and Effective Inoculation on Aphid-Resistant Cultivars of Oilseed Rape (Brassica napus).

Plant viruses improve transmission efficiency by directly and indirectly influencing vector behavior, but the impact of plant cultivars on these modifications is rarely studied. Using electropenetrography (EPG) technology, a comparative study of the effects of turnip mosaic virus (TuMV) infection on quantitative probing behaviors of the cabbage aphid (Brevicoryne brassicae) was conducted on two oilseed rape cultivars ('Deleyou6' and 'Zhongshuang11'). Compared to mock-inoculated plants, cabbage aphids on infected plants increased the frequency of brief probing, cell penetration, and salivation. Additionally, aphids on infected 'Deleyou6' prolonged cell penetration time and decreased ingestion, but not on infected 'Zhongshuang11', suggesting that aphids were more likely to acquire and vector TuMV on the aphid-susceptible cultivar 'Deleyou6' than on resistant cultivars. TuMV also affected aphid probing behavior directly. Viruliferous aphids reduced the pathway duration, secreted more saliva, and ingested less sap than non-viruliferous aphids. In comparison with non-viruliferous aphids, viruliferous aphids started the first probe earlier and increased brief probing and cell penetration frequencies on the aphid-resistant cultivar 'Zhongshuang11'. Based on these observations, viruliferous aphids can be inoculated with TuMV more efficiently on 'Zhongshuang11' than on 'Deleyou6'. Although aphid resistance and TuMV infection may influence aphid probing behavior, oilseed rape resistance to aphids does not impede TuMV transmission effectively.

P1 of turnip mosaic virus interacts with NOD19 for vigorous infection.

P1 protein, the most divergent protein of virus members in the genus Potyvirus of the family Potyviridae, is required for robust infection and host adaptation. However, how P1 affects viral proliferation is still largely elusive. In this work, a total number of eight potential P1-interacting Arabidopsis proteins were identified by the yeast-two-hybrid screening using the turnip mosaic virus (TuMV)-encoded P1 protein as the bait. Among which, the stress upregulated NODULIN 19 (NOD19) was selected for further characterization. The bimolecular fluorescent complementation assay confirmed the interaction between TuMV P1 and NOD19. Expression profile, structure, and subcellular localization analyses showed that NOD19 is a membrane-associated protein expressed mainly in plant aerial parts. Viral infectivity assay showed that the infection of turnip mosaic virus and soybean mosaic virus was attenuated in the null mutant of Arabidopsis NOD19 and NOD19-knockdown soybean seedlings, respectively. Together, these data indicate that NOD19 is a P1-interacting host factor required for robust infection.

P3 and NIa-Pro of Turnip Mosaic Virus Are Independent Elicitors of Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus.

Serological, biological and molecular characterization of viruses causing mosaic diseases on cabbage (Brassica sp L.) in Central Ethiopia.

The productivity of cabbage (Brassica oleracea var. capitata) in Ethiopia has been generally low due to several biotic and abiotic constraints among which are several viral diseases. There is a recent report indicating that this economically important vegetable is seriously affected in Ethiopia by cauliflower mosaic virus (CaMV) and turnip mosaic virus (TuMV). However, little information exists on the incidence and distribution of these viruses as the previous report is based on samples only from Addis Ababa. In this study, a total of 370 leaf samples were collected from 75 cabbage growing fields in Central Ethiopia in two rounds of survey. Two cabbage varieties locally known as "Habesha gomen" and "Tikur gomen" with virus-like symptoms were collected and tested with Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) using polyclonal antibodies specific to CaMV and TuMV. Results from serological diagnosis were confirmed with PCR and Sanger sequencing. The results indicated a high incidence and wide distribution of both viruses in Central Ethiopia with an average of 29.5% infection for CaMV and 40% for TuMV. Biological inoculation tests for CaMV or TuMV or both on healthy cabbage seedlings gave similar symptoms as those observed in the field. Symptom severity was higher with co-infection of CaMV and TuMV followed by TuMV single infection. BLAST analysis showed that TuMV and CaMV isolates from Ethiopia have nucleotide identity of 95-98% and 93-98%, respectively to previously reported isolates. Phylogenetic analysis revealed that CaMV isolates from Ethiopia are closely related to isolates from USA and Italy within Group II clade whereas TuMV isolates have close similarities with isolates from World B clade including isolates from Kenya, UK, Japan and the Netherlands. The identification of the causative agents of the mosaic disease observed on cabbage in Central Ethiopia may lay the foundation for future management studies.

Proteomics Identified UDP-Glycosyltransferase Family Members as Pro-Viral Factors for Turnip Mosaic Virus Infection in Nicotiana benthamiana.

Viruses encounter numerous host factors that facilitate or suppress viral infection. Although some host factors manipulated by viruses were uncovered, we have limited knowledge of the pathways hijacked to promote viral replication and activate host defense responses. Turnip mosaic virus (TuMV) is one of the most prevalent viral pathogens in many regions of the world. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach to characterize cellular protein changes in the early stages of infection of Nicotiana benthamiana by wild type and replication-defective TuMV. A total of 225 differentially accumulated proteins (DAPs) were identified (182 increased and 43 decreased). Bioinformatics analysis showed that a few biological pathways were associated with TuMV infection. Four upregulated DAPs belonging to uridine diphosphate-glycosyltransferase (UGT) family members were validated by their mRNA expression profiles and their effects on TuMV infection. NbUGT91C1 or NbUGT74F1 knockdown impaired TuMV replication and increased reactive oxygen species production, whereas overexpression of either promoted TuMV replication. Overall, this comparative proteomics analysis delineates the cellular protein changes during early TuMV infection and provides new insights into the role of UGTs in the context of plant viral infection.

Environmental Conditions Modulate Plant Virus Vertical Transmission and Survival of Infected Seeds.

Seed transmission is a major mode for plant virus persistence and dispersal, as it allows for virus survival within the seed in unfavorable conditions and facilitates spread when they become more favorable. To access these benefits, viruses require infected seeds to remain viable and germinate in altered environmental conditions, which may also be advantageous for the plant. However, how environmental conditions and virus infection affect seed viability, and whether these effects modulate seed transmission rate and plant fitness, is unknown. To address these questions, we utilized turnip mosaic virus, cucumber mosaic virus, and Arabidopsis thaliana as model systems. Using seeds from plants infected by these viruses, we analyzed seed germination rates, as a proxy of seed viability, and virus seed transmission rate under standard and altered temperature, CO2, and light intensity. With these data, we developed and parameterized a mathematical epidemiological model to explore the consequences of the observed alterations on virus prevalence and persistence. Altered conditions generally reduced overall seed viability and increased virus transmission rate compared with standard conditions, which indicated that under environmental stress, infected seeds are more viable. Hence, virus presence may be beneficial for the host. Subsequent simulations predicted that enhanced viability of infected seeds and higher virus transmission rate may increase virus prevalence and persistence in the host population under altered conditions. This work provides novel information on the influence of the environment in plant virus epidemics. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Corrigendum: Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines.

[This corrects the article DOI: 10.3389/fimmu.2022.986823.].

Wild and weedy Hesperis matronalis hosts turnip mosaic virus across heterogeneous landscapes in upstate New York.

Turnip mosaic virus (TuMV) is a widespread and economically important pathogen in agricultural crops and has the widest known host range in the virus family Potyviridae. While management of the virus and its aphid vectors in agricultural fields decreases virus incidence, many alternative wild hosts for TuMV may serve as source populations for crop infection through spillover. Over thirty years ago, research demonstrated that the introduced brassica, Dame's Rocket (Hesperis matronalis) hosts several viruses, including TuMV. Here, we use both enzyme-linked immunosorbent assays (ELISA) and next generation sequencing to document the frequent infection by TuMV of Dame's Rocket, which is common and widespread in disturbed areas around crop fields in upstate New York. Deep sequencing of multiple tissue types of symptomatic hosts indicate that the infection is systemic and causes diagnostic, visible symptoms. In a common garden experiment using host populations from across upstate New York, we found evidence for genetic tolerance to TuMV infection in H. matronalis. Field surveys show that TuMV prevalence varies across populations, but is generally higher in agricultural areas. Examining disease dynamics in this and other common alternative hosts will enhance our understanding of TuMV epidemiology and, more broadly, virus distribution in wild plants.

Battle for control of anthocyanin biosynthesis in two Brassicaceae species infected with turnip mosaic virus.

It has previously been found that turnip mosaic virus (TuMV) greatly suppresses anthocyanin accumulation (AA) in Brassica rapa leaves, and that such leaves become infected whilst anthocyanin-enriched leaves on the same plants are rarely infected. To clarify whether AA is a defense against TuMV, in this study we examined tissue-level patterns of spontaneous AA in relation to the cellular localization of a TuMV strain that expresses a yellow fluorescent protein. We found that TuMV infection was significantly blocked by AA, suggesting that it functions as a chemical barrier against TuMV. We next analysed changes in expression of genes related to anthocyanin biosynthesis in TuMV-infected leaves of Arabidopsis. TuMV also suppressed AA that is induced by high light in Arabidopsis, and this this suppression was mainly due to inhibited expression of anthocyanin late-biosynthesis genes (LBGs). Most positive transcription factors of LBGs were also down-regulated, while the negative regulator SPL15 was highly up-regulated. Cucumber mosaic virus (CMV) also moderately suppressed AA in Arabidopsis, but in a different manner. Since it appeared that anthocyanin-enriched leaves of Arabidopsis were resistant to TuMV but not CMV, our results suggested that the anthocyanin-associated resistance that we observed was specific to TuMV.

Host Plants Shape the Codon Usage Pattern of Turnip Mosaic Virus.

Turnip mosaic virus (TuMV), an important pathogen that causes mosaic diseases in vegetable crops worldwide, belongs to the genus Potyvirus of the family Potyviridae. Previously, the areas of genetic variation, population structure, timescale, and migration of TuMV have been well studied. However, the codon usage pattern and host adaptation analysis of TuMV is unclear. Here, compositional bias and codon usage of TuMV were performed using 184 non-recombinant sequences. We found a relatively stable change existed in genomic composition and a slightly lower codon usage choice displayed in TuMV protein-coding sequences. Statistical analysis presented that the codon usage patterns of TuMV protein-coding sequences were mainly affected by natural selection and mutation pressure, and natural selection was the key influencing factor. The codon adaptation index (CAI) and relative codon deoptimization index (RCDI) revealed that TuMV genes were strongly adapted to Brassica oleracea from the present data. Similarity index (SiD) analysis also indicated that B. oleracea is potentially the preferred host of TuMV. Our study provides the first insights for assessing the codon usage bias of TuMV based on complete genomes and will provide better advice for future research on TuMV origins and evolution patterns.

Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines.

Virus-like particles (VLPs) have been gaining attention as potential platforms for delivery of cargos in nanomedicine. Although animal viruses are largely selected due to their immunostimulatory capacities, VLPs from plant viruses constitute a promising alternative to be considered. VLPs derived from Turnip mosaic virus (TuMV) have proven to present a tridimensional structure suited to display molecules of interest on their surface, making them interesting tools to be studied in theragnostic strategies. Here, we study their potential in the treatment of food allergy by genetically coupling TuMV-derived VLPs to Pru p 3, one of the most dominant allergens in Mediterranean climates. VLPs-Pru p 3 were generated by cloning a synthetic gene encoding the TuMV coat protein and Pru p 3, separated by a linker, into a transient high-expression vector, followed by agroinfiltration in Nicotiana benthamiana plants. The generated fusion protein self-assembled in planta to form the VLPs, which were purified by exclusion chromatography. Their elongated morphology was confirmed by electron microscopy and their size (~400 nm), and monodispersity was confirmed by dynamic light scattering. Initial in vitro characterization confirmed that they were able to induce proliferation of human immune cells. This proliferative capability was enhanced when coupled with the natural lipid ligand of Pru p 3. The resultant formulation, called VLP-Complex, was also able to be transported by intestinal epithelial cells, without affecting the monolayer integrity. In light of all these results, VLP-Complex was furtherly tested in a mouse model of food allergy. Sublingual administration of VLP-Complex could effectively reduce some serological markers associated with allergic responses in mice, such as anti-Pru p 3 sIgE and sIgG2a. Noteworthy, no associated macroscopic, nephritic, or hepatic toxicity was detected, as assessed by weight, blood urea nitrogen (BUN) and galectin-3 analyses, respectively. Our results highlight the standardized production of allergen-coated TuMV-VLPs in N. benthamiana plants. The resulting formula exerts notable immunomodulatory properties without the need for potentially hazardous adjuvants. Accordingly, no detectable toxicity associated to their administration was detected. As a result, we propose them as good candidates to be furtherly studied in the treatment of immune-based pathologies.

Turnip Mosaic Virus Infection Differentially Modifies Cabbage Aphid Probing Behavior in Spring and Winter Oilseed Rape (Brassica napus).

Direct and indirect effects of plant virus infection on vector behavior have been discovered to improve virus transmission efficiency, but the impact of plant cultivars in virus-vector-plant interactions has received little attention. Electropenetrography (EPG) allows real-time tracking and quantification of stylet penetration behaviors, pathogen transmission, and plant resistance mechanisms. Quantitative probing behaviors on a spring oilseed rape cultivar, 'Xinyou17', and a winter oilseed rape cultivar, 'Zheping4', were investigated using EPG to compare turnip mosaic virus (TuMV) regulation of cabbage aphid probing behavior. Results for indirect effects showed that compared to mock-inoculated plants, cabbage aphids on infected plants increased brief probing frequency, cell penetration frequency, intracellular probing time, and decreased time to first probe and pathway time, potentially promoting viral acquisition. TuMV also directly influences aphid probing behavior. Viruliferous aphids had reduced pathway time, increased cell penetration frequency, increased intracellular probing time, increased salivation frequency, and ingested less sap than non-viruliferous aphids, primed for viral infection. Although oilseed rape cultivars can also influence aphid behavior, the main effect of cultivars was not significant on TuMV-infected plants.