Periodic Reporting for period 2 - GeminiDECODER (Isolation and characterization of mobile sRNA/target pairs in plants by using a viral protein as a probe)
Periodo di rendicontazione: 2022-06-01 al 2023-05-31
Regulation of gene expression is key in living organisms, allowing responses to both internal and external stimuli, modulating growth, development or defence responses against attacking pathogens. By controlling gene expression, the living organism alters accumulation of specific proteins in the cell, promoting some functions while inhibiting others, in such a way that balance (homeostasis) is kept away from potential disturbances. Since the expression of one protein from one gene relies on one RNA molecule acting as intermediary, targeting this intermediate RNA for degradation constitutes a major way by which gene expression can be achieved in a very sophisticated and specific way: this mechanism is the so-called RNA silencing (RNAi). In the RNAi, a small RNA (sRNA) acts as the core element of this mechanism, targeting for degradation another complementary molecule of RNA (the “intermediary” RNA). In plants, these sRNAs have been characterized as mobile molecules able to spread the RNAi cell to cell, beyond the initiation sites, so acting as a cell non-autonomous process. Despite their capital importance for plant biology, the number of mobile sRNA/target RNA pairs fully characterized is scarce, because of their elusive nature and the low sensitivity of traditional approaches followed so far. The overall aim of this research project has been the isolation and biological characterization of mobile sRNAs and their potential target RNAs, following a completely new approach based on the use of viral suppressors of RNA silencing (VSRs) as probes.
-Conclusions of the action: One viral protein, the C4 protein from tomato yellow leaf curl virus (TYLCV), has been characterized as the most suitable probe for the isolation of mobile sRNAs, in a massive way and with improved resolution. By doing so, new mobile sRNA species are now available for biological characterization, and genetic elements responsible for regulating their movement have been exposed. These findings open the possibility of increasing crop resilience and production just through the exploitation of the plant endogenous regulatory mechanisms.
-Conclusions of the action: One viral protein, the C4 protein from tomato yellow leaf curl virus (TYLCV), has been characterized as the most suitable probe for the isolation of mobile sRNAs, in a massive way and with improved resolution. By doing so, new mobile sRNA species are now available for biological characterization, and genetic elements responsible for regulating their movement have been exposed. These findings open the possibility of increasing crop resilience and production just through the exploitation of the plant endogenous regulatory mechanisms.
VSRs are viral proteins naturally evolved to overcame RNAi at different levels, including sRNA movement. In recent years, a new VSR, the C4 protein from tomato yellow leaf curl virus (TYLCV), was shown to interfere specifically with the cell-to-cell movement of RNAi. Transgenic plants artificially accumulating the C4 protein, that is 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 sRNAs. That means mobile sRNAs will no longer reach their usual localizations inside these plants (they cannot move properly), and the RNAs they mark for degradation will no longer be degraded. This also means that, if accumulation of specific RNAs in C4-expressing plants is found as increased when compared to that of wild-type plants, these RNA species can be considered as putative mobile RNAs. By following this experimental approach, levels of accumulation of different RNA species were analyzed by massive RNA sequencing in both C4-expressing plants and wild-type plants: these RNA sequencing provides information of both the accumulation and identity (sequence) of the specific RNA. Of note, RNAi constitutes the main defence mechanism plants deploy against pathogens such as viruses, where complementary sRNAs target for degradation/repression virus-derived RNAs. In order to isolate these mobile sRNA species, analysis have been carried out combining different approaches, such as combinations of grafting and complementation with transgenic tomatoes, involving the use of mutant TYLCV viruses.
-Overview of results, exploitation and dissemination: By using C4 as probe, 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 localized at plasma membrane, which can be found associated with structures known as plasmodesmata (plasma membrane-delimited channels that communicate one cell to each other, creating a plasma continuum). From this localization, BAM1/2 controls the cell-to-cell trafficking of mobile sRNAs, and C4 interferes with their movement by interacting directly with BAM1/2. Manipulation of these BAM1/2 emerges then as a powerful technique to manipulate the movement of these mobile species.
C4 has also been greatly improved as viral probe. One drawback the original C4 presents is that, when expressed transgenically in plants, induces strong developmental phenotypes, interfering with the isolation of potential mobile sRNAs. Different C4 mutans still able to interfere with the movement of sRNAs but unable to induce developmental phenotypes were generated. With the generation of these C4 mutant forms, not only new biotechnological tools were made available, but also key aspects of the molecular interaction between TYLCV-tomato were uncovered, namely i) symptom induction relies on C4, and ii) viral symptoms have proven to serve as attractants for the TYLCV insect vector, the whitefly Bemisia tabaci, probably contributing to virus dispersal. Both aspects will contribute to efficiently design strategies aim at controlling pests.
Although 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 public EU repository Zenodo.
-Overview of results, exploitation and dissemination: By using C4 as probe, 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 localized at plasma membrane, which can be found associated with structures known as plasmodesmata (plasma membrane-delimited channels that communicate one cell to each other, creating a plasma continuum). From this localization, BAM1/2 controls the cell-to-cell trafficking of mobile sRNAs, and C4 interferes with their movement by interacting directly with BAM1/2. Manipulation of these BAM1/2 emerges then as a powerful technique to manipulate the movement of these mobile species.
C4 has also been greatly improved as viral probe. One drawback the original C4 presents is that, when expressed transgenically in plants, induces strong developmental phenotypes, interfering with the isolation of potential mobile sRNAs. Different C4 mutans still able to interfere with the movement of sRNAs but unable to induce developmental phenotypes were generated. With the generation of these C4 mutant forms, not only new biotechnological tools were made available, but also key aspects of the molecular interaction between TYLCV-tomato were uncovered, namely i) symptom induction relies on C4, and ii) viral symptoms have proven to serve as attractants for the TYLCV insect vector, the whitefly Bemisia tabaci, probably contributing to virus dispersal. Both aspects will contribute to efficiently design strategies aim at controlling pests.
Although 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 public EU repository Zenodo.
This project has made it possible the isolation and characterization of mobile sRNAs and their potential targets, in a massive way and with unprecedented resolution, by using a completely new approach which overcame traditional limitations such as lack of specificity, lethality when mutant plants are used, or sample contamination. This work has presented a comparative analysis of efficiencies of some of these VSRs, when used as viral probes, and the evidence pointing out C4 from TYLCV as the most efficient viral probe. By using C4, proteins involved in regulating the cell-to-cell movement of sRNAs have been shown, and their involvement in key biological processes such as root xylem patterning has been characterized. Lists of candidate mobile sRNA/target pairs have been generated, opening avenues for the selection of putative pairs for further biological characterization. In addition, experiments with the C4 mutants generated in this project have shown that symptoms induced during infection could represent a strategy that viruses deploy to attract the insect vector, so increasing viral dispersal among plants: this factor needs to be considered for pest management.
-Impact and wider societal implications. Because RNAi relays at the bases of most regulatory mechanisms in plants, as those involved in plant growth and defence responses, research on this topic may open new avenues for the design of more productive and resilient crops, just by taking advantage of the endogenous regulatory mechanisms of the plant, without introducing foreign elements nor using transgenesis. This topic is paramount for an ever-growing world population, when crop production needs to be optimized and conducted in green, sustainable ways.
-Impact and wider societal implications. Because RNAi relays at the bases of most regulatory mechanisms in plants, as those involved in plant growth and defence responses, research on this topic may open new avenues for the design of more productive and resilient crops, just by taking advantage of the endogenous regulatory mechanisms of the plant, without introducing foreign elements nor using transgenesis. This topic is paramount for an ever-growing world population, when crop production needs to be optimized and conducted in green, sustainable ways.