Khalid Amari

Aphids are major agricultural pests due to their rapid reproduction, insecticide resistance, and role as vectors of plant pathogens. RNA interference (RNAi) has emerged as a promising strategy for species‐specific pest control. However, RNAi in aphids remains inconsistent, limited by dsRNA instability, inefficient uptake, and variable gene silencing. Here, we investigate key biological barriers limiting RNAi efficiency by evaluating the delivery, stability, and efficacy of double‐stranded (ds)RNA in the pea aphid Acyrthosiphon pisum . Feeding bioassays using food dyes and Cy3‐labeled dsRNA demonstrated ingestion and persistence of fluorescent signals within the aphid midgut for up to 120 h, confirming that the feeding system effectively delivers dsRNA to the insects. Ex vivo degradation assays revealed rapid dsRNA breakdown by aphid nucleases, with complete degradation within 2 h. Formulations improved stability: chitosan‐protected dsRNA resisted aphid nuclease activity, and layered double hydroxide (LDH) nanosheets induced elevated expression of RNAi‐associated genes. Functional assays targeting the vacuolar H ⁺ ‐ATPase 26 kDa‐like gene ( apvhal26 ) produced variable knockdown but consistently induced the expression of RNAi‐associated genes ( ago2, dicer1 ), suggesting activation of the RNAi pathway or recognition of exogenous dsRNA despite inconsistent gene silencing. Our results demonstrate that several key barriers to RNAi in aphids can be partially mitigated through improved delivery and nanoparticle formulations. However, rapid nuclease‐mediated degradation and inconsistent target gene silencing remain major obstacles to reliable RNAi efficacy. This study highlights both the potential and limitations of RNAi for aphid control and emphasizes the need for optimized formulations, target gene selection, and delivery methods to advance RNAi as a sustainable biopesticide strategy.

As obligate intracellular pathogens, plant viruses interact with host factors to achieve efficient replication and spread. Host factors also play important roles in virus detection and antiviral defence. Furoviruses cause substantial losses in cereals and efficient control measures are lacking. We therefore used Japanese soil-borne wheat mosaic furovirus movement protein (MP ^JSBWMV ) in immunoprecipitation experiments to identify host interaction partners in the model host plant Nicotiana benthamiana . We identified a P-type pentatricopeptide repeat-containing protein (NbPPR) as the interaction partner of the MP ^JSBWMV . Fluorescent protein-tagged NbPPR localized to chloroplasts and to the cytosol and plasmodesmata (PD) depending on the location of the fluorescent protein tag, suggesting that the protein may be dually localized to these cellular compartments. MP ^JSBWMV :RFP and GFP:NbPPR colocalized and interacted at PD as shown by fluorescence resonance energy transfer fluorescence lifetime imaging and co-immunoprecipitation. To functionally characterize the MP ^JSBWMV -interacting NbPPR, we immunoprecipitated GFP:NbPPR-interacting RNAs and identified a motif overrepresented in the GFP:NbPPR-bound RNAs. The motif is consistent with the PPR target sequence predicted using PPR code. While most of the RNA targets encode subunits of the organellar NADH-dehydrogenase complex, we also identified RNAs encoding cell wall proteins. Downregulation of NbPPR, using virus-induced gene silencing and subsequent infection with fluorescent protein-tagged viruses, resulted in increased numbers of infection sites for JSBWMV in plants silenced for NbPPR compared to controls. We propose a model to explain how virus infection may affect NbPPR function and how NbPPR may regulate the infection cycle. As PPRs are increasingly explored for the modulation of gene expression, virus-targeting PPRs may be developed into tools for virus control in future.

The use of double-stranded RNA (dsRNA) for plant protection shows great potential as a sustainable alternative to traditional pesticides. This review summarizes the current state of knowledge on using exogenous dsRNA in plant protection and includes the latest findings on the safety and efficiency of this strategy. The review also emphasizes the need for a cautious and comprehensive approach, considering safety considerations such as off-target effects and formulation challenges. The regulatory landscape in different regions is also discussed, underscoring the need for specific guidelines tailored to dsRNA-based pesticides. The review provides a crucial resource for researchers, regulators, and industry stakeholders, promoting a balanced approach incorporating innovation with thorough safety assessments. The continuous dialog emphasized in this review is essential for shaping the future of dsRNA-based plant protection. As the field advances, collaboration among scientists, regulators, and industry partners will play a vital role in establishing guidelines and ensuring the responsible, effective, and sustainable use of dsRNA in agriculture.

The Arabidopsis thaliana genome encodes several genes that are known or predicted to participate in the formation of stress granules (SG). One family of genes encodes for Ras GTPase-activating protein–binding protein (G3BP)-like proteins. Seven genes were identified, of which one of the members was already shown to interact with plant virus proteins in a previous study. A phylogenetic and tissue-specific expression analysis, including laser-dissected phloem, by qRT-PCRs was performed and the sub-cellular localization of individual AtG3BP::EYFP fluorescent fusion proteins expressed in Nicotiana benthamiana epidermal cells was observed. Individual AtG3BP-protein interactions in planta were studied using the bimolecular fluorescence complementation approach in combination with confocal imaging in living cells. In addition, the early and late induction of G3BP-like expression upon Turnip mosaic virus infection was investigated by RNAseq and qRT-PCR. The results showed a high divergence of transcription frequency in the different plant tissues, promiscuous protein–protein interaction within the G3BP-like gene family, and a general induction by a viral infection with TuMV in A. thaliana. The information gained from these studies leads to a better understanding of stress granules, in particular their molecular mode of action in the plant and their role in plant virus infection.

Virus-induced plant diseases in cultivated plants cause important damages in yield. Although the mechanisms of virus infection are intensely studied at the cell biology level, only little is known about the molecular dialog between the invading virus and the host genome. Here we describe a combinatorial genome-wide approach to identify networks of sRNAs-guided post-transcriptional regulation within local Turnip mosaic virus (TuMV) infection sites in Brassica napus leaves. We show that the induction of host-encoded, virus-activated small interfering RNAs (vasiRNAs) observed in virus-infected tissues is accompanied by site-specific cleavage events on both viral and host RNAs that recalls the activity of small RNA-induced silencing complexes (RISC). Cleavage events also involve virus-derived siRNA (vsiRNA)–directed cleavage of target host transcripts as well as cleavage of viral RNA by both host vasiRNAs and vsiRNAs. Furthermore, certain coding genes act as virus-activated regulatory hubs to produce vasiRNAs for the targeting of other host genes. The observations draw an advanced model of plant-virus interactions and provide insights into the complex regulatory networking at the plant-virus interface within cells undergoing early stages of infection.

Cyclophilins (CYPs) are a group of ubiquitous proteins characterized by their ability to bind to the immunosuppressive drug cyclosporin A. The CYP family occurs in a wide range of organisms and contains a conserved peptidyl-prolyl cis/trans isomerase domain. In addition to fulfilling a basic role in protein folding, CYPs may also play diverse important roles, e.g. in protein degradation, mRNA processing, development, and stress responses. We performed a genome-wide database survey and identified a total of 94 CYP genes encoding 91 distinct proteins. Sequence alignment analysis of the putative BnCYP cyclophilin-like domains revealed highly conserved motifs. By using RNA-Seq, we could verify the presence of 77 BnCYP genes under control conditions. To identify phloem-specific BnCYP proteins in a complementary approach, we used LC-MS/MS to determine protein abundances in leaf and phloem extracts. We detected 26 BnCYPs in total with 12 being unique to phloem sap. Our analysis provides the basis for future studies concentrating on the functional characterization of individual members of this gene family in a plant of dual importance: as a crop and a model system for polyploidization and long-distance signalling.

Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection. Viruses are parasites that require the host cell machinery for their propagation within and between cells. Myosins are molecular motors involved in the trafficking of cargos along actin filaments. Plant viruses have evolved to borrow this transport mechanism to aid their infection and spread within the plant. However, little is known about which of the many plant myosins are essential and at which specific steps they act to support virus infection. Here we investigated the role of different N. benthamiana myosins during the infection by Tobacco mosaic virus (TMV). Our results show that class XI myosins play specific roles in the reproduction and intracellular movement of TMV in association with the dynamic endoplasmic reticulum network, whereas class VIII myosins support the specific targeting of the viral movement protein to plasmodesmata and thus the cell-to-cell movement of the virus. Together these results indicate that TMV interacts with distinct myosins during specific infection steps.

Plasmodesmata (PD) are dynamic cell wall microchannels that facilitate the intercellular trafficking of RNA and protein macromolecules playing cell nonautonomous roles in the orchestration of plant development, growth, and plant defense. The trafficking of macromolecules and organelles within cells depends on cytoskeletal components and their associated motor proteins. Plant viruses evolved to hijack this transport system to move their infectious genomes to PD. Current efforts concentrate on dissecting the role of specific myosin motors in transporting plant or viral proteins to the channels. Here we describe a method that addresses the role of specific myosins by expression of myosin tails that cause the repression of myosin activity in a dominant-negative manner. As an example, we explain the use of myosin tails from Nicotiana benthamiana to address the role of N. benthamiana myosins in the targeting of PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) to PD.