Progress 01/15/21 to 01/14/24
Outputs
Target Audience: The target audience for this research is the scientific community, especially those in the plant molecular biology community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training for graduate student Cecilia Chow, who completed her PhD in spring 2023 and is currently a postdoctoral scientist in the bay area. Cecilia learned how toanalyzehigh throughput sequence data, especially epigenomics data such as sRNA-seq and whole genome bisulfite sequencing. She also became adept at developing her own bioinformatic analysis pipelines and data visualization in R. The project also provided some genomics training to graduate student Tania Chakraborty and an opportunity for undergraduate student Kathryn Panferov to learn about plant breeding. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the final year of funding, we focused on arole for CLASSY (CLSY) proteins, which are essential in the initiation of siRNA production, duringseed development.We have identified mutations in fourof the five CLSY proteins in B. rapa (CLSY1, CLSY2, CLSY3,and CLSY4a). These lines are from a heavily mutagenized TILLING population and have a number of secondary mutations causing growth phenotypes. We are currently backcrossing each mutant to create clean lines for analysis.Preliminary data indicates that clsy3 mutants have reduced seed production, supporting our hypothesis that siren siRNAs rather than canonical 24-nt siRNAs are associated with seed development.
Publications
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Meyers B, Zhan J, Shevella D, Bologna N, and Mosher R. ⿿Plant Argonautes: Protein Effectors of Small RNAs. Agrisera Educational Poster 7, 2023. https://doi.org/10.6084/m9.figshare.24006819.v1
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Eckhardt NA, Axtell MJ, Barta A, Chen X, Gregory BD, Guo H, Manavella PA, Mosher RA, Meyers BC, ⿿Focus on RNA Biology The Plant Cell, 35:1617-1618, 2023. https://doi.org/10.1093/plcell/koad082
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The target audience for this research is the scientific community. I presented research progress at the following venues: * May, 2022 23rd Penn State Symposium in Plant Biology: RNA Biology; College Park, PA, USA. RNA-directed DNA Methylation: a maternal influence during seed development. * May, 2022 Department of Biology, Oxford University; Oxford, UK. RNA-directed DNA Methylation: a maternal influence during seed development. * April, 2022 The John Innes Centre; Norwich, UK. RNA-directed DNA Methylation: a maternal influence during seed development. * Mar, 2022 Department of Life Sciences, Imperial College London; London, UK. RNA-directed DNA Methylation: a maternal influence during seed development. * Mar, 2022 Department of Genetics, Development, and Cell Biology, Iowa State University; Ames, IA, USA. RNA-directed DNA Methylation: a maternal influence during seed development. Research on small RNAs and seed development was also shared in several manuscripts (listed in next section). Preprints are currently undergoing revision following peer review. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training for PhD student Cecilia Chow, who has become an expert on handling and analyzing high throughput sequence data, especially epigenomics data such as sRNA-seq and whole genome bisulfite sequencing. She is also adept at developing her own bioinformatic analysis pipelines and data visualization in R. Finally, this project has allowed Cecilia to develop her mentorship skills by overseeing multiple undergraduate research assistants. I anticipate that work from this project will form the bulk of Cecilia's dissertation, which she will submit in Spring 2023. Students that Cecilia has mentored include Collin Eckhauser, Eli Shlimovitz, Jeff Clark, and Kathryn Panferov. Each student has learned skills in molecular breeding, including plant husbandry, crosses, and genotyping (DNA extraction, PCR amplification, and agarose gel electrophoresis). They have also been involved in phenotyping for seed production. How have the results been disseminated to communities of interest?Unfortunately, an opportunity to present this research at the Plant and Animal Genomes meeting in Jan 2022 was cancelled due to COVID. However, the research was also discussed at the 23rd Penn State Symposium on Plant Biology in May 2022 and in four departmental research seminars. It was also shared via two research publications, as listed in Products, and contributed to a research review that is in press at The Plant Cell. What do you plan to do during the next reporting period to accomplish the goals?In the final reporting period, we will be focused on 1) investigating the link between siren siRNA accumulation and seed production, particularly as this relates to breeding system; andunderstanding the role of CLSY proteins in siren biogenesis and seed development
Impacts
What was accomplished under these goals?
As described in last year's report, genetic characterization of additional suppressor mutants has not identified strong candidate alleles in addition to BrCLV1, which we characterized in a recent publication.To continue our effort to understand the genetic underpinnings of seed production, we have refocused on the role of siren siRNAs in influencing gene expression during seed development and move on to a candidate gene approach investigating CLASSY proteins. A point mutation in the LRR domain of CLAVATA1 causes multilocularity in B. rapa We completed this work and published a manuscript describing our findings (in Plant Direct). This work will also form a thesis chapter for graduate student Cecilia Chow. Reproductive-specific "siren" siRNAs trans-methylate protein-coding genes In this reporting period, we focused on completing revisions to our manuscript describing methylation of protein-coding genes by highly-abundant siRNAs produced in the ovule and developing seed coat (called "siren" siRNAs). This work was published in The Plant Cell. Interestingly, the nrpd1a-2 mutant, which eliminates siren siRNAs while leaving other 24-nt siRNAs intact, has as strong a seed development defect as the rdr2 mutation that eliminates all 24-nt siRNAs, suggesting that siren siRNAs are the critical species influencing seed development. In a collaboration with Dr. Mark Beilstein, we have established that loss of 24-nt siRNAs has a stronger seed development phenotype in the outbreeder Capsella grandiflora than its closely-related inbreeder Capsella rubella. We have therefore begun investigating a link between breeding system and siren siRNA production. Preliminary evidence suggests that more loci are responsible for siren production in C. grandiflora, and we will be following this line of research through the next reporting period. A role for CLASSY (CLSY) proteins in seed development Although loss of 24-nt siRNAs does not result in seed production defects in Arabidopsis thaliana (another component of our breeding system hypothesis), Arabidopsis does produce siren siRNAs. Research from Julie Law's laboratory demonstrates that the putative SWI/SNF nucleosome remodeling protein CLSY3 is necessary for siren expression (Zhou et al 2022), and this is consistent with our observation that CLSY3 is highly expressed during seed production in B. rapa. To determine whether CLSY proteins are also required for siren expression in B. rapa, and whether disruption of these proteins impacts seed development, we have identified mutations in each of the five CLSY proteins in B. rapa (CLSY1, CLSY2, CLSY3, CLSY4a, and CLSY4b). We have confirmed nonsense alleles for all except CLSY3, for which we have multiple missense mutations. Preliminary data indicates that two of the clsy3 alleles have reduced seed production, supporting our hypothesis. However, these lines are from a heavily mutagenized TILLING population and will need a couple of generations of backcrossing before concrete conclusions can be reached. We are now working on those backcrosses, as well as molecular and developmental characterization of each CLSY mutation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Burgess D, Chow HT, Grover JW, Freeling M, and Mosher RA. Ovule siRNAs methylate protein-coding genes in trans The Plant Cell, 34:3647-3664, 2022. https://doi.org/10.1093/plcell/koac197
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Chow HT, Kendall T, and Mosher RA, A novel CLAVATA1 mutation causes multilocularity in Brassica rapa Plant Direct, 7:e476. http://dx.doi.org/10.1002/pld3.476
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Chow HT and Mosher RA, Small RNA mediated DNA methylation during plant reproduction The Plant Cell, accepted, 2023.
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The target audience for this research is the scientific community. Unfortunately, scientific conferences and seminars remained sharply constrained in 2021 due to the ongoing COVID-19 pandemic. Nevertheless, I presented research progress at the following venues: * Jul, 2021 UA/KAUST joint seminar series; virtual; Maternal small RNAs influence seed development in Brassica rapa * Sept, 2021 Molecular and Cellular Biology Department, University of Arizona; Tucson, AZ, USA. RNA-directed DNA Methylation: a maternal influence during seed development. Research on small RNAs and seed development was also shared in several manuscripts and preprints (listed in next section). Preprints are currently undergoing revision following peer review. Changes/Problems:As described above, genetic characterization of additional suppressor mutants has not identified other strong candidates. Although the suppressive effect is clearly heritable, we are unable to map additional mutations in crosses to a different variety. To continue our effort to understand the genetic underpinnings of seed production, we have refocused on the role of siren siRNAs in influencing gene expression during seed development (described above). Future work will consider both the mechanism (components and intercellular communication) and the downstream targets of this pathway. We continue to adapt our research program to the ongoing COVID-19 pandemic. Capacity in the laboratory has increased throughout the first year of this award, as members of the laboratory were able to be vaccinated, and we are building contingency plans in the face of rising Omicron cases. Global demand for molecular biology reagents, as well as disruption to global shipping, has occasionally delayed experiments as well. However, we are committed to pushing ahead. What opportunities for training and professional development has the project provided?This project has provided PhD Cecilia Chow an opportunity to develop her research skills, particularly with respect to handling and analyzing high throughput sequence data. Cecilia has learned to prepare a variety of Illumina sequencing libraries, both for DNA (BSA-seq) and RNA (RNA-seq). In addition to analysis of these libraries, she has developed her own analysis pipelines to identify genes that might be regulated by siRNAs in trans during seed development. Cecilia has also developed her communication skills by presenting her research in the School of Plant Sciences Research seminar. Finally, she is developing excellent leadership and mentorship skills by overseeing an undergraduate student. This project has provided an opportunity for undergraduate student Collin Eckerhauser to participate in laboratory research and learn more about genetics and molecular biology. Collin has learned to extract DNA, run PCRs, genotype mutants, and calculate segregation ratios. How have the results been disseminated to communities of interest?This research was discussed in two research seminars and multiple publications been published or posted on BioRxiv as preprints. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we anticipate: - finishing our characterization of Brclv1 and preparing it for publication - submitting revisions of our first paper on trans-methylation of protein-coding genes by RdDM - investigating potential intercellular and inter-generational activity the trans-methylation pathway - identifying loss-of-function alleles for upregulated trans-methylation target genes and beginning crosses to see if they suppress seed abortion in nrpd1.
Impacts
What was accomplished under these goals?
Brclv1 causes multilocularity in B. rapa We successfully identified the mutation causing multicellularity and increased seed production in the RdDM-deficient nrpd1 background. This mutation is a single amino acid change in the extracellular LRR domain of Br CLAVATA1. While disruption of the Clavata signaling pathway is well-known to trigger multilocularity, we did not expect this change because the R-o-18 variety already carries a mutation in this pathway (in CLAVATA3, encoding the ligand for CLAVATA1). However, it is clear that our unique clv1 allele causes multilocularity independent of the genotype at CLV3. We have further demonstrated that this allele can trigger multilocularity in Arabidopsis when the native CLV1 allele is absent, indicating that it will work in multiple Brassicaceae species. We still have some loose-ends to tie up before preparing this work for publication, but we have submitted a provisional patent application based on our unique genetic material. Cloning of additional nrpd1 suppressor mutations We created mapping populations for three additional suppressor mutations, and performed Bulked Segregant Analysis sequencing. Surprisingly, no genomic regions showed linkage to the R-o-18 background in any of these three populations. We also phenotyped F1 individuals from crosses between suppressors. Although these mutations came from independent pools of EMS-treated seeds, they all complement, suggesting that either they are alleles of the same gene (unlikely) or that our phenotype is not genetically stable. An unstable phenotype might also explain the lack of linkage in the BSA-seq analysis. Given these challenges, as well as new findings described below, we are shifting our focus from suppressor mutations toward other molecular approaches to identify the mechanism behind RdDM's role during seed development. Reproductive-specific "siren" siRNAs trans-methylate protein-coding genes In collaboration with researchers at UC Berkeley, we have discovered that a class of highly-abundant, ovule-specific siRNAs can trigger DNA methylation at protein-coding genes, causing their transcriptional suppression. These siRNAs, produced from "siren" loci, account for >90% of the siRNAs in unfertilized ovules, and remain highly abundant in the developing seed coat (Grover et al, 2020 PNAS). Most siRNAs come from gene fragments embedded in siren loci, and production of these siRNAs is correlated with non-CG methylation and reduced mRNA accumulation at homologous protein-coding genes. Because RdDM generally function in cis at transposons and other repetitive sequences, our work reveals an exciting new mechanism of gene regulation through RdDM. We are currently investigating the possibility that this mechanism also functions intercellularly and intergenerationally, allowing the maternal soma to influence gene expression in the developing endosperm. We also have a list of genes regulated by this pathway, some of which are strong candidates for the seed abortion we detect when maternal RdDM is eliminating. Investigating these downstream genes is one of our goals for the next reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Chakraborty, T., Kendall, T., Grover, J.W. et al. Embryo CHH hypermethylation is mediated by RdDM and is autonomously directed in Brassica rapa. Genome Biol 22, 140 (2021). https://doi.org/10.1186/s13059-021-02358-3
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Schmitz, Robert J., et al. "Quality control and evaluation of plant epigenomics data." The Plant Cell (2021). https://doi.org/10.1093/plcell/koab255
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Mosher, Rebecca A. "Small RNAs on the move in male germ cells." Science 373.6550 (2021): 26-27. https://doi.org/10.1126/science.abj5020
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Chakraborty, Tania, et al. "A Null Allele of the Pol IV Second Subunit is Viable in Oryza sativa." bioRxiv (2021). https://doi.org/10.1101/2021.10.21.465363
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Sundar, Vivek H., et al. "Plant Polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains." bioRxiv (2021). https://doi.org/10.1101/2021.08.25.457601
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Burgess, Diane, et al. "Ovule siRNAs methylate and silence protein-coding genes in trans." bioRxiv (2021). https://doi.org/10.1101/2021.06.10.447945
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