Final Report Summary - TRANSTEARA (Determining the role of gamete small RNAs in propagating the trans-generational epigenetic silencing of transposable elements) 1. ResultsSummary of the project objectives:Both plant and animal genomes must be protected from the mutagenic effects of transposable element (TE) transposition. Gamete cells, both right before and after fertilization, are the battleground for the evolutionary arms race between TE activity and host-cell suppression of TEs. The protection of the germ cells and the very young embryo of the progeny are key steps of genome integrity that require trans-generational epigenetic suppression of TEs. The conceptual base of this project, is that TE derived siRNAs accumulating in the pollen sperm cells (SCs) are part of a trans-generational genome safeguard mechanism that protects TE expression and transposition in the SCs and after fertilization in the embryo. This project will determine the movement of TE derived sRNAs from the pollen SCs to the embryo and their involvement in genome stability by specifically investigating:Aim 1: the movement of pollen TE easiRNAs to the SCs, embryo and the endosperm.Aim 2: the role of TE easiRNAs in a siRNA amplification step initiation in the embryoAim 3: the role of TE easiRNAs in the protection of the embryo’s genome from the transposition of female active TEs in the endosperm or/and the embryo.Aim 4: the role of TE SC siRNAs genomic imprinting. Work performed:During this period the Fellow has performed the analysis of anti-TE RNA silencing in the different nuclei of the male and female gametophytes. A series of transgenic constructs expressing specific GFP in the vegetative nucleus (VN), SCs, egg cell (EC) and embryo/endosperm containing target sites for TEs have been successfully analyzed through microscopy. This analysis has determined that TE-derived siRNAs are active in the pollen grain VN, SCs, and in the post-fertilization tissues (endosperm and embryo). The fellow has also studied if an amplification step of TE-derived sRNAs could take place in the pollen grain. A series of transgenes inducing the production of secondary sRNAs in the VN have been analyzed through sRNA high-throughput sequencing and microscopy, leading to the conclusion that, indeed, TE-derived secondary siRNAs are mobile between the VN and the SCs. These mobile secondary siRNAs are active inducing the silencing of active TEs in the SCs. Further experiments have been performed in order to interfere with the physical movement of sRNAs between both nuclei through the use of viral silencing suppressor proteins (VSSs). These proteins, which are able to sequester sRNAs, have been specifically expressed in the pollen grain VN in order to avoid the movement of sRNAs between the VN and the SCs. The analysis of their effect, through their expression in the VN, against anti-TE RNA silencing in the SCs has revealed the mobile nature of anti-TE secondary siRNAs. These transgenic lines able to sequester VN-derived siRNAs would be further tested in the return phase partner laboratory in order to study the potential role of these male gametophyte-derived secondary siRNAs in the female gametophyte. Moreover the Fellow has dissected which are the potential components of the different RNA silencing pathways involved in the silencing of TEs in the VN. Several components have been identified and a specific RNA silencing pathway active in the VN has been uncovered. These results have been successfully published in the prestigious journal Nature Plants (http://www.nature.com/articles/nplants201630).Moreover, during the return phase of the fellowship, the Fellow has studied the potential role of pollen derived TE siRNAs in the development of the seed and influence on gene imprinting. Several sRNA and cytosine methylation genome-wide analysis have been performed both in the pollen grain and in seeds. The results of these experiments are being analyzed at this moment for future publication on a high impact factor journal and are expected to make a huge impact on the scientific community.ConclusionsThe main results obtained by the Fellow during this period can be summarized in: 1) TE silencing takes place in the VN, SCs and postfertilization tissues.2) Anti-TE secondary siRNAs are able to move from the VN to the SCs3) Male-derived TE siRNAs influence the development of the seed.4) Male-derived TE siRNAs influence genomic imprinting associated with TEs.These results enhance the knowledge of the trans-generational epigenetic regulation of plant genomes. Our results point to TE derived siRNAs accumulating in the pollen grain as part of a trans-generational genome safeguard mechanism that protects TE expression and transposition in the seed. The results obtained during this fellowship period contribute to answer how TEs are epigenetically silenced trans-generationally and the extent of their trans-generational role in hybrid incompatibility and gene imprinting. 2. Societal implicationsThe data obtained in the period covered by this fellowship has helped to provide the European Community and its research environment with tools that could be helpful to improve crops for genome instability, hybrid compatibility and TE transposition. In addition, our results are helping to increase the basic knowledge of TE epigenetic regulation that could also improve the tools to control their carcinogen effect in humans. The applications to plant molecular biology obtained from our results have just opened new research fields and are enhancing the scientific excellence of the European Union.The fellow has presented part of the results exposed here at the Keystone Symposia on “RNA Silencing in Plants” that took place in Colorado (USA) in February 2015 and in the Keystone Symposia on “Plant Epigenetics: From Genotype to Phenotype” that took place in New Mexico (USA) in February 2016. These two biannual meeting concentrate the best researchers in the fields of plant RNA silencing, plant epigenetics and plant molecular biology. On both meetings, the results submitted by the Fellow were selected for a talk and, both at the oral and poster sessions, the interest in the results presented were evident supporting the importance of the research carried out during this period. Moreover, the results published in Nature Plants have had an extensive repercussion in the social media, being the 15th article, in repercussion terms, of the articles of the same age in Nature Plants (http://www.nature.com/articles/nplants201630/metrics).During the return phase period, the Fellow have had the opportunity to carry different dissemination activities directed to young researchers through his participation in the “Marie Curie Individual Fellowships Practical Workshop” that took place at La Fe Hospital (Valencia, Spain) on June 2015. This activity helped to disseminate the research of this project, the importance of the Marie Curie Fellowship programs and the European Research Council activities. The workshops lead to an actual increase in the number of proposals presented by young researchers from the center. Together with this, several activities of dissemination directed to the scientific community and general public will take place once the last results from the project are published. Due to the importance of those results, they will be presented in the best plant biology meetings both at the European and global scientific community levels. At the same time, the results will be implemented for the general public. 1. ResultsSummary of the project objectives:Both plant and animal genomes must be protected from the mutagenic effects of transposable element (TE) transposition. Gamete cells, both right before and after fertilization, are the battleground for the evolutionary arms race between TE activity and host-cell suppression of TEs. The protection of the germ cells and the very young embryo of the progeny are key steps of genome integrity that require trans-generational epigenetic suppression of TEs. The conceptual base of this project, is that TE derived siRNAs accumulating in the pollen sperm cells (SCs) are part of a trans-generational genome safeguard mechanism that protects TE expression and transposition in the SCs and after fertilization in the embryo. This project will determine the movement of TE derived sRNAs from the pollen SCs to the embryo and their involvement in genome stability by specifically investigating:Aim 1: the movement of pollen TE easiRNAs to the SCs, embryo and the endosperm.Aim 2: the role of TE easiRNAs in a siRNA amplification step initiation in the embryoAim 3: the role of TE easiRNAs in the protection of the embryo’s genome from the transposition of female active TEs in the endosperm or/and the embryo.Aim 4: the role of TE SC siRNAs genomic imprinting. Work performed:During this period the Fellow has performed the analysis of anti-TE RNA silencing in the different nuclei of the male and female gametophytes. A series of transgenic constructs expressing specific GFP in the vegetative nucleus (VN), SCs, egg cell (EC) and embryo/endosperm containing target sites for TEs have been successfully analyzed through microscopy. This analysis has determined that TE-derived siRNAs are active in the pollen grain VN, SCs, and in the post-fertilization tissues (endosperm and embryo). The fellow has also studied if an amplification step of TE-derived sRNAs could take place in the pollen grain. A series of transgenes inducing the production of secondary sRNAs in the VN have been analyzed through sRNA high-throughput sequencing and microscopy, leading to the conclusion that, indeed, TE-derived secondary siRNAs are mobile between the VN and the SCs. These mobile secondary siRNAs are active inducing the silencing of active TEs in the SCs. Further experiments have been performed in order to interfere with the physical movement of sRNAs between both nuclei through the use of viral silencing suppressor proteins (VSSs). These proteins, which are able to sequester sRNAs, have been specifically expressed in the pollen grain VN in order to avoid the movement of sRNAs between the VN and the SCs. The analysis of their effect, through their expression in the VN, against anti-TE RNA silencing in the SCs has revealed the mobile nature of anti-TE secondary siRNAs. These transgenic lines able to sequester VN-derived siRNAs would be further tested in the return phase partner laboratory in order to study the potential role of these male gametophyte-derived secondary siRNAs in the female gametophyte. Moreover the Fellow has dissected which are the potential components of the different RNA silencing pathways involved in the silencing of TEs in the VN. Several components have been identified and a specific RNA silencing pathway active in the VN has been uncovered. These results have been successfully published in the prestigious journal Nature Plants (http://www.nature.com/articles/nplants201630).Moreover, during the return phase of the fellowship, the Fellow has studied the potential role of pollen derived TE siRNAs in the development of the seed and influence on gene imprinting. Several sRNA and cytosine methylation genome-wide analysis have been performed both in the pollen grain and in seeds. The results of these experiments are being analyzed at this moment for future publication on a high impact factor journal and are expected to make a huge impact on the scientific community.ConclusionsThe main results obtained by the Fellow during this period can be summarized in: 1) TE silencing takes place in the VN, SCs and postfertilization tissues.2) Anti-TE secondary siRNAs are able to move from the VN to the SCs3) Male-derived TE siRNAs influence the development of the seed.4) Male-derived TE siRNAs influence genomic imprinting associated with TEs.These results enhance the knowledge of the trans-generational epigenetic regulation of plant genomes. Our results point to TE derived siRNAs accumulating in the pollen grain as part of a trans-generational genome safeguard mechanism that protects TE expression and transposition in the seed. The results obtained during this fellowship period contribute to answer how TEs are epigenetically silenced trans-generationally and the extent of their trans-generational role in hybrid incompatibility and gene imprinting. 2. Societal implicationsThe data obtained in the period covered by this fellowship has helped to provide the European Community and its research environment with tools that could be helpful to improve crops for genome instability, hybrid compatibility and TE transposition. In addition, our results are helping to increase the basic knowledge of TE epigenetic regulation that could also improve the tools to control their carcinogen effect in humans. The applications to plant molecular biology obtained from our results have just opened new research fields and are enhancing the scientific excellence of the European Union.The fellow has presented part of the results exposed here at the Keystone Symposia on “RNA Silencing in Plants” that took place in Colorado (USA) in February 2015 and in the Keystone Symposia on “Plant Epigenetics: From Genotype to Phenotype” that took place in New Mexico (USA) in February 2016. These two biannual meeting concentrate the best researchers in the fields of plant RNA silencing, plant epigenetics and plant molecular biology. On both meetings, the results submitted by the Fellow were selected for a talk and, both at the oral and poster sessions, the interest in the results presented were evident supporting the importance of the research carried out during this period. Moreover, the results published in Nature Plants have had an extensive repercussion in the social media, being the 15th article, in repercussion terms, of the articles of the same age in Nature Plants (http://www.nature.com/articles/nplants201630/metrics).During the return phase period, the Fellow have had the opportunity to carry different dissemination activities directed to young researchers through his participation in the “Marie Curie Individual Fellowships Practical Workshop” that took place at La Fe Hospital (Valencia, Spain) on June 2015. This activity helped to disseminate the research of this project, the importance of the Marie Curie Fellowship programs and the European Research Council activities. The workshops lead to an actual increase in the number of proposals presented by young researchers from the center. Together with this, several activities of dissemination directed to the scientific community and general public will take place once the last results from the project are published. Due to the importance of those results, they will be presented in the best plant biology meetings both at the European and global scientific community levels. At the same time, the results will be implemented for the general public.
