H2020 GeminiDECODER Project: Isolation and characterization of sRNA/target mobile pairs in plants using a viral protein as a probe
- Type Project
- Status Filled
- Execution 2020 -2023
- Assigned Budget 237.065,28 €
- Scope Europeo
- Autonomous community Andalucía
- Main source of financing Horizon 2020
- Project website https://doi.org/10.3030/896910
Small regulatory RNAs (sRNAs) are ubiquitous in plants. They play a critical role in the "spread" throughout a plant of RNA interference (RNAi), a genetic regulatory system that acts to silence the activity of specific genes. Given the diversity of RNAs and their targets, combined with their mobility, it has been challenging to fully identify and characterize mobile RNA pairs and their targets. The EU-funded GeminiDECODER project is using a novel probe to do so with unprecedented resolution: a protein from a plant virus recently shown to affect cell-to-cell RNAi spread. It promises to help scientists isolate mobile RNAs and their targets under different conditions in different plants, with potential application in regulating crop productivity. This project has enabled the isolation and characterization of mobile sRNAs and their potential targets, on a massive scale and with unprecedented resolution, using a completely new approach that overcame traditional limitations such as lack of specificity, lethality when using mutant plants, and sample contamination.
This work presents a comparative analysis of the efficiencies of some of these VSRs when used as viral probes, and evidence points to TYLCV C4 as the most efficient viral probe. Using C4, proteins involved in regulating the cell-to-cell movement of RNAs have been demonstrated, and their involvement in key biological processes, such as root xylem patterning, has been characterized. Lists of candidate mobile RNA/target pairs have been generated, opening avenues for the selection of putative pairs for further biological characterization. Furthermore, experiments with the C4 mutants generated in this project have shown that the symptoms induced during infection could represent a strategy deployed by viruses to attract the insect vector, thus increasing viral dispersal among plants: this factor should be taken into account for pest management. - Broader societal impact and implications. Because RNAi underlies most plant regulatory mechanisms, such as those involved in plant growth and defense responses, research on this topic may open new avenues for designing more productive and resilient crops, simply by harnessing the plant's endogenous regulatory mechanisms, without introducing foreign elements or using transgenesis. This topic is paramount for a constantly growing global population, where crop production must be optimized and carried out in an environmentally friendly and sustainable manner.
RSVs are viral proteins that naturally evolved to outperform RNAi at several levels, including sRNA movement. In recent years, a novel RSV, the C4 protein of Tomato yellow leaf curl virus (TYLCV), was shown to specifically interfere with the cell-to-cell movement of RNAi. Transgenic plants that artificially accumulate the C4 protein, i.e., C4-expressing plants, were generated by transgenesis. Because C4 interferes with the movement of sRNAs, its presence in C4-expressing plants will interfere with the movement of endogenous mobile RNAs.
This means that mobile RNAs will no longer reach their usual locations within these plants (they cannot move properly), and the RNAs they mark for degradation will no longer be degraded. This also means that if the accumulation of specific RNAs in C4-expressing plants is found to be increased compared to that in wild-type plants, these RNA species can be considered putative mobile RNAs. Following this experimental approach, the accumulation levels of different RNA species were analyzed by massive RNA sequencing in both C4-expressing and wild-type plants: these RNA sequencing analyses provide information on both the accumulation and the identity (sequence) of the specific RNA. Notably, RNAi constitutes the main defense mechanism that plants deploy against pathogens such as viruses, where complementary RNAs target virus-derived RNAs for degradation/repression.
In order to isolate these mobile sRNA species, analyses have been carried out combining different approaches, such as grafting and complementation combinations with transgenic tomatoes, involving the use of mutant TYLCV viruses. -Overview of results, exploitation and dissemination: By using C4 as a probe, the proteins responsible for controlling the cell-to-cell movement of sRNAs, BARELY ANY MERSITEM 1/2 (BAM1/2), were exposed for the first time. BAM1/2 are proteins located in the plasma membrane, which can be found associated with structures known as plasmodesmata (channels delimited by the plasma membrane that communicate one cell with another, creating a plasma continuum). From this location, BAM1/2 controls the cell-to-cell trafficking of mobile RNAs, and C4 interferes with their movement by directly interacting with BAM1/2. The manipulation of these BAM1/2 then emerges as a powerful technique to manipulate the movement of these mobile species.
C4 has also been greatly improved as a viral probe. One drawback of the original C4 is that, when expressed transgenically in plants, it induces strong developmental phenotypes, which interferes with the isolation of potential mobile RNAs. Different C4 mutants were generated that are still capable of interfering with RNA movement but unable to induce developmental phenotypes. With the generation of these C4 mutant forms, not only new biotechnological tools were made available, but key aspects of the molecular interaction between TYLCV and tomato were also uncovered, namely:
- Symptom induction is based on C4.
- Viral symptoms have been shown to serve as attractants for the insect vector of TYLCV, the whitefly Bemisia tabaci, probably contributing to virus dispersal.
Both aspects will contribute to the efficient design of pest control strategies. Although the research is still ongoing, these findings have been made publicly available in international journals, such as PNAS (Fan, Aguilar et al., 2021), New Phytologist (Aguilar and Lozano-Duran, 2022), and Stress Biology (Aguilar and Lozano-Duran, 2022), which can be found in the EU public repository Zenodo.
The regulation of gene expression is key in living organisms, enabling responses to both internal and external stimuli, modulating growth, development, and defense against attacking pathogens. By controlling gene expression, the living organism alters the accumulation of specific proteins in the cell, promoting some functions and inhibiting others, thereby maintaining equilibrium (homeostasis) free from potential disturbances.
Since protein expression from a gene depends on an RNA molecule acting as an intermediary, targeting this intermediate RNA for degradation is an important way to achieve gene expression in a highly sophisticated and specific manner: this mechanism is known as RNA silencing (RNAi). In RNAi, a small RNA (sRNA) acts as the central element of this mechanism, targeting another complementary RNA molecule (the "intermediary" RNA) for degradation. In plants, these RNAs have been characterized as mobile molecules capable of spreading RNAi from cell to cell, beyond the initiation sites, thus acting as a cell-non-autonomous process.
Despite their central importance for plant biology, the number of fully characterized mobile RNA/target RNA pairs is scarce, due to their elusive nature and the low sensitivity of traditional approaches followed so far. The overall objective of this research project was the isolation and biological characterization of mobile RNAs and their potential target RNAs, following a completely novel approach based on the use of viral suppressors of RNA silencing (VSRs) as probes.
Conclusions of the action: A viral protein, the C4 protein of Tomato yellow leaf curl virus (TYLCV), has been characterized as the most suitable probe for isolating mobile sRNAs in bulk and with improved resolution. This provides new mobile RNA species for biological characterization, and the genetic elements responsible for regulating their movement have been exposed. These findings open up the possibility of increasing crop resilience and production solely through the exploitation of the plant's endogenous regulatory mechanisms.
In plants, RNA interference (RNAi) is involved in processes ranging from development to stress responses. The central element of this regulatory mechanism, small RNA (sRNA), has been characterized as a mobile molecule capable of propagating RNAi beyond initiation sites, acting as a cell-non-autonomous process. Examples of mobile RNAs are: mobile interfering RNAs (siRNAs), derived from viruses that spread throughout the plant and are responsible for activating viral silencing in uninfected tissues; other RNAs, along with microRNAs (miRNAs), have been described to translocate through different cell layers, acting as morphogens; finally, some miRNAs have been shown to translocate through the vasculature under stress conditions, activating protective responses.
Mobile RNAs are therefore of vital importance to plant biology, and various approaches have been followed to analyze them; however, the number of fully characterized mobile RNAs/target pairs is scarce. Recently, the C4 protein of Tomato yellow leaf curl virus (TYLCV) has been shown to specifically interfere with the intercellular movement of RNAi, without affecting the accumulation or activity of small RNAs. In this project, C4 will be used as a novel and powerful tool to isolate mobile small RNAs and their targets, under different conditions (basal vs. viral infection) and in different plants (Arabidopsis and tomato), on a large scale and with unprecedented resolution. By using C4-expressing plants, we expect to readily detect off-site mobile small RNAs, as well as increased levels of their unprocessed targets.
Thanks to this viral probe, a complete picture of the different mobile small RNA populations and their targets will be obtained, and the biological relevance of the most prominent candidates will be assessed. As a result, relevant new knowledge will be obtained about the regulation of key biological processes in plants, which could open new avenues for the design of more productive crops.
Small RNAs (opens in new window) (sRNAs) are molecules found within plants that are involved in virtually every regulatory process. They control responses to internal and external stimuli, for example, and modulate growth. They also play a critical role in the spread throughout a plant of RNA interference (opens in new window) (RNAi), a genetic regulatory system that acts to silence the activity of specific genes. “sRNAs are mobile molecules,” explains GeminiDECODER project coordinator Eduardo Bejarano of the Institute of Mediterranean and Subtropical Horticulture ‘La Mayora’ (opens in new window) in Spain. “Their mobility and diversity has made it difficult to fully identify and characterize them, as well as their potential RNA targets.”
These challenges have limited our understanding of several important plant processes; for example, how local plant responses are transformed into systemic ones. This process is key to activating plant defenses against nutrient or water deprivation, or disease. Protein probes derived from plant viruses were used in the GeminiDECODER project, which was supported by the Marie Skłodowska-Curie Actions program and coordinated by the University of Málaga.
In Spain, the project sought to develop new ways to isolate and characterize these mobile RNAs and their potential target RNAs. "To do this, we followed a completely new approach: using proteins derived from plant viruses as probes to detect RNAs with unprecedented resolution," explains Bejarano. This technique, Bejarano believes, could help scientists better isolate mobile RNAs and their targets in different plants, and could have potential applications in regulating crop productivity. The Role of Proteins in Developmental Processes To achieve these goals, the project genetically transferred plants so that they accumulated a viral protein called C4. The protein from Tomato yellow leaf curl virus (TYLCV, Geminivirus) was selected due to its ability to interfere with the movement of RNAs. "The presence of C4 in plants interferes with the movement of mobile RNAs," adds Bejarano. "This means that mobile RNAs can no longer reach their usual locations, and RNAs marked for degradation will no longer be silenced."
By analyzing abnormal RNA accumulation levels, Bejarano was able to identify potential RNA targets for mobile RNAs. He was also able to gather information on the identity of the specific mobile RNA in question. Once these target genes were identified, Bejarano was able to analyze their biological function. "As a proof of concept, we applied C4 to analyze the patterning of root tissue determined by mobile RNAs," says Bejarano. "This allowed us to expose the role that certain plant proteins play in promoting sRNA movement and controlling developmental processes in plants." Optimizing Sustainable Crop Production GeminiDECODER has made several important discoveries by using C4 as a potential mobile RNA probe. New putative mobile RNA/target pairs have been isolated and are awaiting confirmation and biological characterization. "RNAi is fundamental to most regulatory mechanisms in plants," notes Bejarano. "Therefore, this project could help open new avenues of research." On a practical level, identifying plants in nature that express higher or lower levels of a specific RNA could help explain why they might exhibit greater resistance to pathogens, drought, or nutrient starvation. "This issue is paramount for a constantly growing global population, when crop production must be optimized and carried out in an ecologically sound and sustainable manner," says Bejarano.
- UNIVERSIDAD DE MALAGA
- SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES
- CORDIS project factsheet (pdf)
- Web researcher
- BAM1 and BAM2 receptor-like kinases are required for root xylem patterning
- On the inhibition of RNA silencing movement by the tombusvirus P19 protein: dep…
- Plant viruses as probes for engineering abiotic stress tolerance in crops
- University of Malaga website
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