A team of researchers from the Spanish National Research Council, an entity attached to the Spanish Ministry of Science, Innovation, and Universities, has made a significant advance in plant biotechnology by developing a new method for silencing genes. The novel technique uses ultra-short ribonucleic acid (RNA) sequences carried by genetically modified viruses to achieve genetic silencing, allowing the customization of plant traits. The work, published in the Plant Biotechnology Journal, opens up new avenues for crop improvement, functional genomics, and sustainable agriculture.

Viral vector technology involves modifying viruses, removing the genetic material that causes disease, to turn them into vehicles that carry the RNA sequence to be introduced into an organism. This technique, when applied to plants, has already proven effective under experimental conditions in inducing flowering and accelerating the development of improved crop varieties, modifying plant architecture to facilitate adaptation to mechanization, improving drought tolerance, and producing metabolites beneficial to human health, among other applications.

Now, the method developed by the CSIC, together with the Valencian University Institute for Research on the Conservation and Improvement of Agrodiversity (COMAV) and the Italian Department of Applications and Innovation in Supercomputing (Cineca), represents an optimization of technological platforms to accelerate the development and validation of agricultural applications based on viral vectors.

“We have implemented synthetic biology approaches compatible with future industrial-scale production,” says Fabio Pasin, a Ramón y Cajal researcher at the Margarita Salas Center for Biological Research (CIB-CSIC), who led the study.

The new technique, called virus-mediated short RNA insertions (vsRNAi), represents a breakthrough in the field that explores the use of viral vectors to improve the agronomic characteristics of crops. By using a benign plant virus, short RNA molecules are transported to plants, triggering a process known as RNA interference (RNAi) to specifically silence genes, preventing the information in a gene from being translated into a protein. This is a new approach that improves the efficiency of reducing the expression of target plant genes.

Virus-delivered short RNA inserts (vsRNAi) trigger gene silencing

(a) JoinTRV vector assembly and delivery of 32-, 28-, 24- and 20-nt vsRNAi targeting the two N. benthamiana CHLI homeologues (vCHLI, vCHLI-28, vCHLI-24 and vCHLI-20). Leaf phenotypes and chlorophyll levels (mean ± SD, n = 3) are shown; CTRL—control. Different letters indicate significant differences (P < 0.05), by one-way ANOVA and Tukey’s honestly significant difference (HSD) test. (b) Transcriptomics of vCHLI and CTRL samples (n = 3) detects a significant downregulation of CHLI expression (FDR <0.05). (c) In vCHLI samples, small RNA sequencing shows predominance of 21- and 22-nt sRNAs mapping to CHLI, and with a read depth peak (mean, n = 3) localized to the vsRNAi-targeted region. (d) Leaf phenotypes and chlorophyll levels (mean ± SD, n = 3) are shown for vCHLI-b and vPDS, targeting CHLI and PDS homeologues, respectively; ***, P < 0.001, Student’s t-test. (e) vsRNAi portability to scarlet eggplant (Solanum aethiopicum). Phylogeny and fruits are shown, alongside leaf phenotypes and chlorophyll levels (mean ± SD, n = 3) of ‘Rossa di Rotonda’ plants treated with vCHLI-DK, a pTRV1 + pTRV2 derivative targeting CHLI; ***, P < 0.001, Student’s t-test.

The researchers have used a combination of comparative genomics and transcriptomics to design vsRNAi targeting specific genes in plants, demonstrating that the insertion of such short RNA sequences, consisting of 24 nucleotides (the basic structural units of ribonucleic acid), can effectively silence genes in plants. These are ultra-short sequences, as viral vector technology typically uses sequences of around 300 nucleotides.

“This innovation dramatically reduces the size and complexity of traditional virus-induced gene silencing constructs, enabling faster, cheaper, and more scalable applications,” Pasin notes.

To achieve this, the research team focused on the CHLI gene, which is essential for chlorophyll biosynthesis, and designed viral vectors that carried insertions of between 20 and 32 nucleotides, which were introduced into a model plant. The treated specimens showed visible yellowing of the leaves and significant reductions in chlorophyll levels, confirming robust gene silencing.

“Small RNA sequencing revealed that the vsRNAi approach triggers the production of small RNAs, 21 and 22 nucleotides long, which correlates with effective negative regulation, a process by which gene expression is reduced or stopped, of transcription,” adds the CIB-CSIC researcher.

An effective technique for boosting agriculture

The work involved applying this new approach to the model plant Nicotiana benthamiana, demonstrating its effectiveness in producing the desired phenotypic changes in crops of the Solanaceae botanical family, one of the most important worldwide, as it includes vegetables and staple crops for human consumption, such as potatoes. Within this family, the technique was used on tomato and scarlet eggplant (Solanum aethiopicum) crops, an underutilized species with great potential for cultivation beyond its current areas in Africa and Brazil, and which could even be extended to Europe, where it has niche production and local ecotypes such as the Italian “Rossa di Rotonda.”

Among the advantages of the new method over existing RNAi techniques are its simplicity, specificity, and cost-effectiveness, as well as the absence of stable modifications in plant genomes.

“This is a major advance in plant biotechnology, and we are excited about its potential applications,” says Pasin. “We believe that the technique could be a revolutionary change for basic research, especially for non-model plants with limited availability of genetic resources and biotechnological tools, but also for agriculture, as it allows for on-demand alteration of crop traits and selective control of pests and diseases

The results have significant implications for agriculture, as they could be utilized to temporarily modify crop traits to achieve specific phenotypes that enhance yield, disease resistance, and nutritional content. In addition, the portability of vsRNAi between species highlights its potential for high-throughput functional genomics and the modulation of specific traits in both model crops and underutilized crops.

Source – Spanish National Research Council