Instituto de Biología Molecular y Celular de Plantas

ABSTRACTPotato virus X (PVX), a filamentous, positive‐sense RNA plant virus, has been engineered into a molecular tool for diverse biotechnological applications, including cancer cell targeting. Here, we present the production and functional characterization of genetically‐encoded PVX‐derived nanoparticles decorated with nanobodies targeting two common receptors in human cancer cells, epidermal growth factor receptor (EGFR) and human epidermal growth factor 2 (HER2). We first generated a series of PVX‐derived nanoparticles displaying distinct nanobodies against EGFR and HER2 in Nicotiana benthamiana plants. Self‐assembly and structural integrity of the recombinant nanoparticles were confirmed by immunogold electron microscopy. We next characterized in vitro the cancer‐cell binding capacity of the different recombinant viral nanoparticles (VNPs) by flow cytometry and confocal microscopy. Select VNPs were further assayed in a pilot in vivo study using tumor‐bearing mice. Preliminary results showed that nanobody decoration can increase retention time and myeloid cell recruitment in the tumor microenvironment in HER2+ mouse tumor models in vivo. Nanobody‐displaying PVX‐derived nanoparticles may constitute a new plant‐produced biotechnological product for cancer immunotherapy.

Summary Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus ) comprises the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompass most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I ( CHLI ). Second, we developed a tobacco etch virus (genus Potyvirus )-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus , these findings must be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

SUMMARYMicroRNAs (miRNAs) are endogenous 21‐nucleotide small RNAs that direct sequence‐specific silencing of complementary messenger RNAs to regulate a wide range of biological processes. In plants, miRNA precursors are processed from imperfect foldback structures by the RNase III enzyme DICER‐LIKE1, in coordination with accessory proteins. While mismatches flanking the miRNA/miRNA* duplex in endogenous precursors can strongly influence miRNA accumulation, their impact has not been thoroughly examined in the context of artificial miRNAs (amiRNAs) used for targeted gene silencing in plants. Here, using silencing sensor systems in Nicotiana benthamiana, we systematically investigated how base pairing at or near DCL1 cleavage sites affects amiRNA production from the recently described minimal shc precursor. Independent pairing of naturally mismatched positions revealed that introducing a G–C pair immediately upstream of the mature amiRNA remarkably enhances amiRNA accumulation and silencing efficiency. This effect was further validated in Arabidopsis transgenic lines targeting endogenous genes and confirmed by deep sequencing, which revealed highly accurate processing and predominant release of the intended amiRNAs, supporting the specificity of the approach. Our findings show that a single structural modification in an amiRNA precursor can significantly enhance the efficacy of amiRNA‐mediated gene silencing. This optimized amiRNA platform is well suited for large‐scale functional genomics screens and should facilitate the development of next‐generation crops with enhanced resilience to environmental stresses.

The fast development of the DNA sequencing technologies has moved the bottleneck to produce high quality genomes to the HMW DNA extraction and to the genome annotation processes. The identification of gene models can be performed based on experimental evidence and/or ab-initio models. Many factors such as incomplete representation of the gene space, phylogenetic distance of the protein data to the target species genome, specificity of the ab-initio models, and transposon dynamics can impact negatively the quality of the genome structural annotation. GAQET2 is a tool designed to assess thee quality of the gene model annotation for non-model species, combining tools like AGAT, BUSCO, OMArk, PSAURON, and sequence homology searches by Diamond. It also has a module to detect TE miss-identified as genes called DeTEnGA.

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.Key features• Syn-tasiRNAs allow the simultaneous silencing of multiple genes with high specificity, as they are computationally designed to avoid off-target effects.• This protocol describes the design and obtention of syn-tasiRNAs for the simultaneous silencing of one or several endogenous genes in any plant species.• This protocol also describes a non-transgenic alternative for applying syn-tasiRNAs to plants using a viral vector to induce whole-plant gene silencing.• This protocol can also be applied to induce antiviral protection against pathogenic viruses, reducing viral mutational escapes when expressing multiple syn-tasiRNAs targeting different viral sites.

ARGONAUTE (AGO) proteins associate with small RNAs to form RNA-induced silencing complexes (RISCs). Arabidopsis AGO1 effects post-transcriptional silencing by microRNAs (miRNAs) and small interfering RNAs (siRNAs) and is necessary for siRNA amplification through conversion of RISC target RNAs into double-stranded RNA by the RNA-dependent RNA Polymerase RDR6 and its mandatory cofactors SGS3 and SDE5. Many AGO proteins harbor hydrophobic pockets that interact with tryptophan residues, often surrounded by glycine (GW/WG), in intrinsically disordered regions (IDRs) of RISC cofactors. Here, we show that GW/WG dipeptides in the IDR of SGS3 and the hydrophobic pockets in AGO1 are required for fully functional RDR6-dependent siRNA amplification. We also show that this mechanism requires AGO1-specific structural elements, including positively charged residues surrounding the binding pockets, and a conserved, negatively charged patch in the IDR of SGS3. Thus, the same, conserved protein-protein interaction site is used for different purposes in distinct eukaryotic AGO proteins: the GW/WG-mediated TNRC6-Ago2 interaction is crucial for miRNA-guided silencing in metazoans whereas the GW/WG-mediated SGS3-AGO1 interaction facilitates siRNA amplification via RDR6 in plants.

Abstract How animals repeatedly adapted to life on land is a central question in evolutionary biology. While terrestrialisation occurred independently across animal phyla, it remains unclear whether shared genomic mechanisms underlie these transitions. Here, we combined large-scale comparative genomics, machine learning, and multi-omics data, including proteomics and transcriptomics from stress experiments relevant to terrestrial environmental challenges in 17 species, to investigate the genomic basis of animal terrestrial adaptation. Gene co-expression networks revealed that genes relevant to stress were largely lineage-specific, yet converged in function through the co-option of gene families pre-dating terrestrialisation events. Phylogenomic and machine learning analyses supported a dominant role for early-evolving genes, enriched in stress-related functions, paired with a higher gene loss than gain at terrestrialisation nodes. Our findings support a model of lineage-specific genomic changes involving mostly conserved genes that converged at the functional level during the independent transitions to terrestrial life.