Periodic Reporting for period 1 - GREEN-SPECIALISTS (Specialised plant cell types via developmentally-controlled gene knockouts: a key step towards fully customised plants)
Reporting period: 2018-03-01 to 2020-02-29
Anthropogenic climate change is altering the environment faster than plants can adapt. Sensitivity to increasing temperatures, unpredictable rainfall and increased threats from insects and microbial pathogens are all substantial risks to crop security and there are limits to how well these risks can be managed through improved farming practices.
In the last century most crop plants have been improved by randomly mutating their seeds with radiation or chemicals, and then selecting new mutant plants with desirable properties like increased yields. The idea of modifying plants to climate-proof our food supply is being examined by scientists and government agencies worldwide. At this stage it is not clear whether the random mutagenesis approaches used in the past will be sufficient. It appears increasingly likely that we will need the ability to edit a plant’s genetic plan in a very precise and planned way. The last decade has heralded the rise of CRISPR-Cas9-based methods of genome modification. This new technology allows scientists to precisely remove or edit specific parts of a plant’s DNA.
Usually when the genome of an organism is edited, that modification exists permanently in every cell and is passed on to every cell in subsequent generations. But plants are highly sophisticated organisms with many different specialised cell types that each contribute particular roles to the overall function of the plant. Plants have some cells that are responsible for producing food or chemicals, and these are supported by cells responsible for other vital functions like photosynthesis, nutrient supply, and providing the plant with physical structure.
The overall objective of this study was to improve our technical ability to modify plants, in the hope that this will contribute to improving global food security. To engineer plants in a sophisticated way, we need to be able to edit specific cell types rather than applying modifications uniformly across the whole organism. This project aimed to investigate ways to modify particular cells in the leaves of plants without affecting the rest of the plant. The study focused on guard cells: these cells control the movement of water vapour and gases (like carbon dioxide) in and out of the leaf. Guard cells are very important for regulating a plant’s response to increased CO2 concentrations and changes in rainfall and humidity.
In the last century most crop plants have been improved by randomly mutating their seeds with radiation or chemicals, and then selecting new mutant plants with desirable properties like increased yields. The idea of modifying plants to climate-proof our food supply is being examined by scientists and government agencies worldwide. At this stage it is not clear whether the random mutagenesis approaches used in the past will be sufficient. It appears increasingly likely that we will need the ability to edit a plant’s genetic plan in a very precise and planned way. The last decade has heralded the rise of CRISPR-Cas9-based methods of genome modification. This new technology allows scientists to precisely remove or edit specific parts of a plant’s DNA.
Usually when the genome of an organism is edited, that modification exists permanently in every cell and is passed on to every cell in subsequent generations. But plants are highly sophisticated organisms with many different specialised cell types that each contribute particular roles to the overall function of the plant. Plants have some cells that are responsible for producing food or chemicals, and these are supported by cells responsible for other vital functions like photosynthesis, nutrient supply, and providing the plant with physical structure.
The overall objective of this study was to improve our technical ability to modify plants, in the hope that this will contribute to improving global food security. To engineer plants in a sophisticated way, we need to be able to edit specific cell types rather than applying modifications uniformly across the whole organism. This project aimed to investigate ways to modify particular cells in the leaves of plants without affecting the rest of the plant. The study focused on guard cells: these cells control the movement of water vapour and gases (like carbon dioxide) in and out of the leaf. Guard cells are very important for regulating a plant’s response to increased CO2 concentrations and changes in rainfall and humidity.
I made a two-stage plan to tackle this project.
1. Examine previously-characterised mechanisms for controlling gene expression in guard cells. Sections of DNA known as “promoters” are responsible for activating gene expression in different cell types, and any mechanism for controlling gene editing in guard cells would rely on promoters that are active only in that cell type. Examples of guard cell-specific promoters can be found in the scientific literature, but some of these studies are almost thirty years old and the conclusions sometimes depend on very different experimental approaches taken by different laboratories. I examined several promoters in parallel with a unified method that would make the experimental results clear and directly comparable.
2. Demonstrate DNA editing in guard cells without affecting the rest of the plant. Because of the importance of having a clear and unambiguous result for a proof-of-concept study like this, I first developed an Arabidopsis thaliana plant that produces a bright green fluorescent protein in all of its cells.
I discovered that current methods for producing engineered plants were going to be inadequate for this study. An important but laborious step when producing engineered plants is sterilising seeds prior to testing whether the preceding engineering step was successful. This study was going to require thousands of plants, meaning that the seed sterilisation stage would become extremely time consuming. In response I developed a new method that allows seeds to be screened without sterilization. This method will save hundreds of hours of tedious labour for plant scientists everywhere. This work was published as an open-access article in the plant-focused journal, Physiologia Plantarum (http://dx.doi.org/10.1111/ppl.13079). Additionally, five plasmids (transferrable DNA elements) produced in the course of this work have been made publicly available via addgene.org (https://www.addgene.org/plasmids/articles/28206747/).
I also designed a new method to assess promoters in a way that would be comparable across different plants, and even between different laboratories. Promoters are paired with fluorescent protein genes and measuring how much fluorescent protein is produced (expressed) as a result of the promoter activity. This was achieved by including an internal control: in addition to the promoter being examined, a known promoter was included that would drive expression of another fluorescent protein. Thereby, between different plants there is always an internal marker against which to measure promoter activity. I have provided the 21 genetic designs that I produced for this work for public distribution via addgene.org.
Promoter activity in different cell types was measured with a confocal microscope by recording the amount of fluorescent protein produced. The fluorescence was compared between different cell types in absolute terms and with comparison to the fluorescence from the control promoter-protein pair. These experiments clarified discrepancies observed with some previously-reported promoters, and provided insight into the roles of some previously uncharacterised promoters. The data are still being processed to prepare a manuscript for public communication of the results.
During the course of this project, studies were published by two other university groups demonstrating the cell-type-specific CRISPR-Cas9 engineering concept that I proposed in this fellowship. The data published included demonstration of CRISPR-Cas9 activity targeted to guard cells, among other specific cell types (https://doi.org/10.1105/tpc.19.00454 https://doi.org/10.1101/793240). It was gratifying to see that the concept and the approach were valid. The Arabidopsis thaliana plant that I produced that expresses green fluorescent protein in all cell types and will be useful for validating the published methods for kncoking out genes in other specific cell types.
1. Examine previously-characterised mechanisms for controlling gene expression in guard cells. Sections of DNA known as “promoters” are responsible for activating gene expression in different cell types, and any mechanism for controlling gene editing in guard cells would rely on promoters that are active only in that cell type. Examples of guard cell-specific promoters can be found in the scientific literature, but some of these studies are almost thirty years old and the conclusions sometimes depend on very different experimental approaches taken by different laboratories. I examined several promoters in parallel with a unified method that would make the experimental results clear and directly comparable.
2. Demonstrate DNA editing in guard cells without affecting the rest of the plant. Because of the importance of having a clear and unambiguous result for a proof-of-concept study like this, I first developed an Arabidopsis thaliana plant that produces a bright green fluorescent protein in all of its cells.
I discovered that current methods for producing engineered plants were going to be inadequate for this study. An important but laborious step when producing engineered plants is sterilising seeds prior to testing whether the preceding engineering step was successful. This study was going to require thousands of plants, meaning that the seed sterilisation stage would become extremely time consuming. In response I developed a new method that allows seeds to be screened without sterilization. This method will save hundreds of hours of tedious labour for plant scientists everywhere. This work was published as an open-access article in the plant-focused journal, Physiologia Plantarum (http://dx.doi.org/10.1111/ppl.13079). Additionally, five plasmids (transferrable DNA elements) produced in the course of this work have been made publicly available via addgene.org (https://www.addgene.org/plasmids/articles/28206747/).
I also designed a new method to assess promoters in a way that would be comparable across different plants, and even between different laboratories. Promoters are paired with fluorescent protein genes and measuring how much fluorescent protein is produced (expressed) as a result of the promoter activity. This was achieved by including an internal control: in addition to the promoter being examined, a known promoter was included that would drive expression of another fluorescent protein. Thereby, between different plants there is always an internal marker against which to measure promoter activity. I have provided the 21 genetic designs that I produced for this work for public distribution via addgene.org.
Promoter activity in different cell types was measured with a confocal microscope by recording the amount of fluorescent protein produced. The fluorescence was compared between different cell types in absolute terms and with comparison to the fluorescence from the control promoter-protein pair. These experiments clarified discrepancies observed with some previously-reported promoters, and provided insight into the roles of some previously uncharacterised promoters. The data are still being processed to prepare a manuscript for public communication of the results.
During the course of this project, studies were published by two other university groups demonstrating the cell-type-specific CRISPR-Cas9 engineering concept that I proposed in this fellowship. The data published included demonstration of CRISPR-Cas9 activity targeted to guard cells, among other specific cell types (https://doi.org/10.1105/tpc.19.00454 https://doi.org/10.1101/793240). It was gratifying to see that the concept and the approach were valid. The Arabidopsis thaliana plant that I produced that expresses green fluorescent protein in all cell types and will be useful for validating the published methods for kncoking out genes in other specific cell types.
My work introduces a new way to analyse promoters in plants. I anticipate that when published, this new approach will improve the accuracy and reproducibility with which these important genetic control elements are studied. This will increase the value of scientific data generated, making it easier for scientists to adapt published data to their own work without having to spend as much time repeating studies.
The development of the antimicrobial seed screening method will likely have a larger and more direct impact on plant scientists, encouraging them to plan more comprehensive and ambitious synthetic biology experiments by alleviating a major limitation in their experimental throughput.
Although I was not the first to publish a successful implementation of cell type-specific CRISPR-Cas9 activity in plants, it is great to see that this approach has been successful. These results open up new and exciting opportunities for designing sophisticated genetic programs to better manage plant responses to heat and drought.
The development of the antimicrobial seed screening method will likely have a larger and more direct impact on plant scientists, encouraging them to plan more comprehensive and ambitious synthetic biology experiments by alleviating a major limitation in their experimental throughput.
Although I was not the first to publish a successful implementation of cell type-specific CRISPR-Cas9 activity in plants, it is great to see that this approach has been successful. These results open up new and exciting opportunities for designing sophisticated genetic programs to better manage plant responses to heat and drought.