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.
