A new technical article in The Plant Journal describes a method developed by the authors to ascertain the number of transgenes inserted into a variety of important crops.
Knowing the number of genes successfully inserted into an organism as a result of a transformation event is useful information. Although there are simpler methods to determine whether a plant has been successfully transformed, being able to determine whether there is one or more transgenes present assists understanding the effect of one gene being inserted on plant phenotype and its segregation through following generations. It also allows researchers to eliminate the possibility of multiple copies of the gene being inserted and resulting in silencing of the gene.
The researchers outline the current problem with attempting to determine the number of genes inserted into an organism. Southern blot analysis is used to confirm the insertion of target genes, but even the most skillful analysis can struggle to identify when multiple transformations have occurred in the same segment of the dissected genome. Further, the analysis is expensive, labour intensive and requires the use of radioactive materials, in turn requiring special permits and handling procedures.
Quantitative Polymerase Chain Reaction, although rapid, isn’t able to provide the accurate measurement data obtained from Southern blot analysis and distinguishing between one and two transgenes is difficult even with extensive optimisation.
A More Accurate, Faster Method
The researchers made use of a newer PCR method, droplet digital PCR. Here is a short video covering some of the basics, but there are more detailed videos covering the technology:
For further reading, here is a 2012 Nature review of the technology that explains the process and its advantages really well as well as pointing out the differences with qPCR, and here is another from the IDT website.
The importance of this method lies in the use of the small droplets into which the reaction mixture is divided and in which the PCR amplification takes place. The division of the fractionated genome into the droplets results in either one or no DNA segments being contained within any particular droplet. With each droplet containing primers for the gene of interest for PCR amplification and a labeled probe for identification, subsequent analysis of the amplified droplets can detect the number of droplets containing the sequence of interest.
Figure from Nature review of dPCR illustrating the division of DNA between droplets and subsequent amplification.
Using the number of positive and negative reactions combined with the volume of the sample, the number droplets used and a Poisson statistical analysis to determine the likelihood of more than one segment of DNA being contained within a single droplet, a precise determination of the number of times the gene of interest is present in the genome can be performed. It is even possible to determine hemizygous or homozygous presence of the gene.
In order to determine the number of transgenes inserted into a genome, a known single-copy gene within the same genome must be selected for identification and comparison.
Making it Work for Plants
The purpose of the research was to optimise the use of droplet digital PCR for use in a number important crops, particularly rice, citrus, potato, tomato, maize and wheat, and to test the results of their protocols against Southern blot analysis results of the same plants for validation. The result, they hope, is to provide a cheaper, more efficient method to determine transgene number in plants that can then be used confidently in further studies.
To perform the study, the researchers picked common transgenic target sequences and single copy genes in each of the subjects to serve as a reference and ran a duplex analysis of each of the transformed food crops.
For each of the 6 subjects, the paper identifies the both the transgene and the reference gene used, whether the analysis was conducted in the transformed adult or progeny plant and how successful the analysis was in determining transgene copy number when compared with the Southern blot analysis.
Even with the varying sizes of the genomes tested, the results of the digital droplet PCR in each crop were comparable to the Southern blot analysis, providing confidence that the method could be used in transgenic research.
Some things to consider
Although the assessments were successful, the researchers note a few observations they made to assist anyone else using the technique, including:
- there is the possibility of obtaining ‘rainy’ droplets, where the droplet was found to be positive for the gene of interest but for which the signal wasn’t as strong as it was for the majority of the positive droplets. Despite the observation, the ‘rainy’ droplets didn’t appear to affect the result;
- plants with larger genomes require a larger quantity of genomic DNA for analysis in order to obtain an accurate analysis;
- sing either T0 plants or plants of known zygosity is important for an accurate analysis of transgene copy number using a single reaction;
- quality, accurately measured DNA must be used for analysis to avoid poor quality droplets;
- there is a range of optimal amounts of genomic DNA to be used dependent on the genome size in order that the confidence levels derived from the Poisson statistics are high enough that the results can be relied upon;
- fractionation methods chosen for use are important. Complete digestion to allow for random segregation in the droplets of fragments is required;
- if too few droplets are created the sample has been inadequately partitioned and results should be looked at closely; and
- copy numbers that are midway between integers indicate a problem with the reaction.
The paper outlines a method that other researchers can use to cheaply and accurately determine the number of transformation events in crop plants and should contribute to the efficiency of research in transgenic laboratories. The amount of reaction mixture, the primers and reporters used and the plant populations that the DNA are cultivated from can all be adjusted from the methods used in the paper and will hopefully lead to usable protocols in numerous plants.
* Feature image credit: Integrated DNA Technologies – http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/10/21/digital-pcr-(dpcr)-what-is-it-and-why-use-it-
Lately we’ve written a few posts about how crop resistance may be increased in the future. Our last post covered the development of a material able to pass RNAi protection to crops, and the one prior to that covered the multitude of sustainable new methods that may play a role in disease management.
This month, the Plant Biotechnology Journal (which is open – thank you Wiley) published an article looking at a possible way of reducing the evolutionary pressure caused by transgenic crops with one specific method of protection (Bt cotton in this study) as well as improving protection against pests that have developed resistance.
Bt cotton, a transgenic crop which contains a gene for a toxin (Cry toxin) found in Bacillus thuringiensis, provides protection against a range of insects. Prior to this gene being inserted into cotton, corn and a number of other crops, the Baccilus thuringiensis bacteria would be sprayed onto the crops to provide the same protection.
As successful as Bt crops have been, the use of a singular method of pest control puts pressure on the pests to evolve a method of overcoming the effects of the control mechanism. In the case of Bt cotton crops, farmers have begun using lines of transgenic cotton containing a number of different Bt toxins designed to kill the same pest. However, these pyramids of multiple Bt toxin genes are not entirely effective due to the toxins interfering with each other or causing cross-resistance within the pests.
Pyramids of Bt toxins and RNAi
The research tested the possibility of constructing a cotton plant containing both a Bt toxin gene and one of two genes for a double stranded RNA aimed at interfering with two particular genes within the pest Helicoverpa armigera, a moth which develops by feeding on important crops such as cotton and corn.
To test whether the pyramids improved crop defence against H. armigera, cotton plants containing one of the two dsRNAs, the Bt toxin gene, a pyramid of Bt plus one of either of the RNAi constructs or a control plant were challenged with either a Bt susceptible or Bt resistant pest. Two essential genes which have previously been shown to effect pupation of H. armigera were the targets of the dsRNA constructs. The pest fed with an artificial diet containing either of the dsRNAs was shown to result in an increase in pest mortality and those pests that did survive had a lower weight compared to those feeding on the control plant.
Cotton plants were then transformed via Agrobacterium tumefacnians-mediated transformation to create lines of cotton with either of the dsRNAs or a Green Fluorescence Protein as a control and then selfed the resulting lines to create lines homozygous for each of the genes. Resulting lines were shown through Southern blot analysis to contain only one of the inserted genes.
Susceptible pests fed either of the dsRNA transformed plants showed lower rates of transcription of the target genes.
Having created lines of cotton containing dsRNA able to effect pest growth, the researchers crossed the dsRNA lines with Bt lines in order create crops containing both traits and developed those lines into homozygous cotton plants with the same number of copies of each of the genes.
Did the Pyramids make any difference?
Before testing the pyramided crops to compare their effect on Bt resistant pests to that of Bt-only crops, resistant H. armigera were fed Bt, transgenic GFP or non-transgenic cotton with no significant difference in their mortality or growth rates. Susceptible H. armigera pests fed the same diets showed the expected increased mortality when fed the Bt cotton diet.
When the resistant pests were fed the RNAi cotton crops, mortality rates were similar to that in susceptible pests, with both lines of pest having a mortality rate close to that of Bt susceptible pests when fed transgenic Bt plants. Of the two RNAi crops, there was no real difference in mortality rates and development between the two genes targeted by the RNAi constructs. Nor was there any significant difference between the effects of RNAi crops on resistant pest mortality and crops containing the Bt + RNAi construct, demonstrating that the pyramid did not have any effect on the pest save for the presence of the RNAi component of the pyramid.
Bt susceptible pests grown on the pyramid cotton crops did show an increase in mortality and days to development when compared to the RNAi crops alone. Using the index of multiplicative survival (“IMS” – comparing the mortality rates of the pyramid to the expected mortality rate of the pyramid which is calculated by multiplying the mortality rates of the pests when raised on crops containing only one or the other the pyramided genes) to determine whether the Bt and RNAi components were acting separately or in concert to cause the effect seen. Using this method of analysis it was thought that the two genes contained in the pyramid act independently against the susceptible pest.
Overall, the cotton crops containing the pyramids showed increased protection against both the susceptible and the resistant lines of pest.
Figure 3b from article. Comparative mortality rates between susceptible and resistant H. armigera on wild type (W0), Green Fluorescence Protein transformed (GFP), Bt, two RNAi transformed crops (JHA and JHB) and Bt + RNAi pyramids. Astrix indicates statistically significant differences between the two lines of pest.
Just in case there is a possibility that Bt resistance resulted in a fitness cost to the H. armigera that may interfere with the analysis, resistant lines were fed on wild type cotton and the transgenic GFP cotton and their development rates monitored. Interestingly, resistance to Bt does come with a cost to development time, resistant pests having a 15 to 16% increase in development time compared to their susceptible cousins. Mortality between the two lines however did not show any difference.
What effect may use of the construct have in reducing resistance evolution?
Computer simulations were used to demonstrate what effect the use of pyramid cotton will have on resistance evolution in a number of scenarios with parameters taken from common growing conditions in northern China and varying levels of pest fitness cost of resistance and time for resistance development.
The amount of refuge land used in the scenarios had a significant impact on resistance development times. Using a refuge percentage of 50%, it was found that adding RNAi defence either in succession with Bt crops or in a pyramid crop increased the time to development of resistance against the defence when compared to Bt crops alone. Using pessimistic parameters for the development of resistance and fitness cost (faster evolution and little to no fitness cost associated with resistance development) and a refuge percentage of 50, it was demonstrated that the time to resistance increased by 5 years when RNAi was used in tandom with Bt crops while time resistance increased to 10 used when the two methods were used consecutively in a pyramid.
Figure 5 from article. Simulations predicting years to resistance under a) realistic scenario, b) Optimistic scenario and c) Pessimistic Scenario with differing percentages of refuge area.
This may be the first time we have discussed a paper which experimented on something other than an important food crop. But Bt transformed food crops are in widespread use and the reliance on only one method of pest control results in the types of problems we are seeing today with the evolution of resistance. Therefore, developing the ability to provide more sustainable, longer term protection to crops could be fast-tracked using a technology like this where the gene targeted by the RNAi can be designed for a specific pest with minimal side effects on related species of insect.
The accuracy of the computer simulations is a little difficult to make out without a better knowledge of the underlying data but could be the basis of field tests and more sophisticated simulations.
The development of RNAi technology, from examples like this to the creation of crop protection technologies like BioClay, is impressive and seems likely to play significant role in the future protection of food production.
We have previously written about the possibility of using RNAi-based technologies to provide plants more sustainable and greater protection against viruses. RNAi, or RNA interference, is the protective process used in many eukaryotic cells against viruses which uses double stranded RNA (“dsRNA”) sequences complementary to that of a pathogen to silence the translation of that foreign RNA into proteins. It was recognised in a recent review article as one of the genetic technologies that could be used to provide sustainable crop protection in the future.
An article in January’s Nature Plants (sorry, the full-text article is behind a paywall) looked for a way to give RNAi the ability to withstand field conditions when topically applied to crop surfaces.
BioClay as a Delivery Mechanism
The researchers investigated the possibility of connecting the dsRNA to clay nanosheets (“LDH”) to form a substance, which the researchers called “BioClay”, that can be applied to crops and provide longer lasting protection than applying naked dsRNA.
BioClay nanosheets were created with an average diameter of 45nm. Loaded onto the nanosheets were dsRNA sequences complementary to segments of the pepper mild mottle virus (“PPMoV”) or the cucumber mosaic virus (“CMV”).
To check for successful loading, the dsRNA-LDH substances were subjected to electrophoresis. The fact that the dsRNA-LDH complexes didn’t migrate from the well at all was taken as evidence that the dsRNA had been successfully loaded onto the LDH. Sequences up to 1.8kbp were shown to be attachable to the LDH to form BioClay.
Transmission Electron Microscopy used to view the BioClay formed showed that the dsRNA chain is either adsorbed on the LDH surface or thread within a number of LDH particles.
The mechanism of delivering dsRNA to the plant relies on the LDH degrading into a residue when exposed to CO2 and moisture. This process and the ability for BioClay to delivery dsRNA to the plant surface was tested by suspending the BioClay on the leaves of tobacco plants and incubating under atmospheric-like conditions for 7 days. The residue left after 7 days showed decreases in aluminium and magnesium, the conclusion being drawn that the LDH had degraded. The process was also tested by incubating test plants with CMV-loaded BioClay and collecting the residue after a week, finding that the amount of loaded BioClay had been reduced, indicating that the BioClay is releasing the loaded dsRNA.
How topically applied dsRNA provides protection to the subject plant is still a matter for further research. To test whether the dsRNA was being taken up by the plant after being released from the degraded BioClay nanosheets, the researchers attached a Cy3 fluorophore to LDH alone, to a dsRNA alone and to a dsRNA-LDH compound and tested all three by applying them Arabidopsis thaliana. 48 hours after application, the leaves were examined with confocal microscopy to determine whether any fluorophores and therefore, presumably, either the LDH, dsRNA or dsRNA-LDH complexes, had been taken up by the plant. The researchers observed the fluorophore within xylem of the leaves treated with Cy3 attached to dsRNA and dsRNA-LDH complexes, but was not internalised in treatments that did not contain dsRNA. Further, in the dsRNA-Cy3-LDH treatment showed flurophore uptake in the spongy mesophyll.
Not only was the fluorophore shown to enter the plant when attached to dsRNA, but was also seen to be transported to new apical meristem leaves that had not been directly treated.
Further testing of the uptake of dsRNA was undertaken on transgenic Arabidopsis that contained a β-glucuronidase reporter, the aim being to test whether a dsRNA directed towards the reporter gene interfered with its expression. Interference was measured with a fluorometric assay and plants treated with dsRNA-GUS complexes (with or without LDH) showed decreased β-glucuronidase activity, indicating that RNA interference was being induced by the treatments.
Did the BioClay Persist Longer?
The first few tests showed the dsRNA was being taken up by the plants and causing RNAi, but does the use of the LDH nanosheets to deliver the dsRNA result in greater protection?
The researchers tested the usefulness of the LDH nanosheets in a number of ways. First, they again labeled the dsRNA complexes with Cy3 and applied them to Arabidopsis leaves. After leaving them on the leaves for 24 hours half of the leaves in each treatment group were rinsed and the fluorescence levels measured. Complexes that contained LDH displayed residual flourescence while non-LDH treatments had little-to-no fluorescence after rinsing.
The LDH complexes were next tested with an RNase to test the ability of the different complexes to withstand degradation. dsRNA and dsRNA-LDH were treated with RNase. After treating, the dsRNA was released from the LDH in that treatment group and the two sets of dsRNA subjected to northern blot analysis. It was shown that the dsRNA originally attached to LDH had been degraded to a lesser extent than the naked dsRNA.
Figure 3 from article – Figures a – d show the microscopy images of the 4 treatment types to detect remains of treatments after washing. Figure e is the northern blot result showing the levels of degradation of the dsRNA by RNase when attached to LDH or naked. Figure f compares the dsRNA present on leaves at different time points after being sprayed with either the naked dsRNA or the dsRNA-LDH complexes.
Similarly, when the dsRNA and dsRNA-LDH were applied to leaves and their continuing presence on the leaves detected after application, the non-LDH attached dsRNA was barely detected after 20 days while the LDH connect dsRNA was detected 30 days after the treatment.
Similar to the findings about the translocation of the dsRNA into untreated leaves, the researchers used northern blot analysis on purposefully untreated leaves to test for the presence of the dsRNA 20 days after the spray was applied, finding that where the dsRNA was attached to LDH, the dsRNA was still detectable.
But Does it Afford Protection Against Viruses?
Showing that the BioClay can caused directed RNAi in plants and persist longer on plants is all well and good, but it must also provide the plants with additional protection against viruses.
Using nectrotic lesions caused by CMV as a marker for virus resistance, the study showed a significant reduction in the number of lesions in leaves treated with dsRNA and BioClay. A similar test used a PMMoV challenge to test the number of lesions formed. The leaves were challenged with the virus 20 days after being treated with dsRNA complexes and the researchers found that only the BioClay complex provided significant protection at this time point, demonstrating a longer period of protection.
Similarly, when a double-antibody ELIZA was used to test for the presence of CMV in leaves 20-days post challenge, the percentage of leaves positive for CMV was significantly less in leaves treated with BioClay compared to those treated with LDH alone and the dsRNA alone.
Figure 4 from article. Fig 4a and 4b visualise the lesion number of lesions per leaf resulting from being challenged at different times after being sprayed with the various treatments. The most significant result was the significant reduction in lesion numbers in BioClay treated leaves when challenged 20 days after the treatment when compared to the number of lesions formed after the other treatments.
Protection afforded to the non-treated leaves was tested by taking leaves that emerged 20 days after treatment and using the same double-antibody ELIZA to detect the level of infection. The researchers found a reduced level of infection in the new leaves when the plant had been treated with BioClay.
Finally, the researchers used RNA-seq testing on leaves challenged with CMV, some of which had been treated with BioClay or dsRNA, seeking out viral RNA. Leaves treated with the dsRNA or BioClay showed virus specific RNA was at least 10 times less abundant than in non-treated leaves.
The researchers have demonstrated through a series of steps that LDH nanosheets have the ability to deliver dsRNA to plants, be subsequently taken up by the plant and seemingly distributed throughout the plant, to provide useful protection against viruses. Most importantly, the LDH nanosheets were demonstrated to provide better protection to the dsRNA from being washed off the plant or from being degraded.
Field trials are the next obvious steps for a technology that seemingly has the ability to provide significant protection in a sustainable manner. The ability of the BioClay to withstand field stress, UV radiation for example, would further cement this technology as one that may alter agricultural practices and improve food security. Whether the RNAi protection can be extended to other pests is even more exciting.
A great piece of research which gives hope that this biological phenomenon can be used to assist crop protection and food production.
Some of the earliest and most prominent uses of genetic modification technology in crops have related to disease management. The insertion of a Bacillus thuringiensis gene into crops such as corn resulted in protection against damage caused by certain insects, eliminating the need for pesticides against those particular pests is one example. Another example, the ability of crops to thrive despite the application of glyphosate, was brought about by modifying crops so that the pathway affected by the chemical to cause plant death is cycled more regularly, helping the crop to survive.
A recent review penned by Paul Vincelli in the journal Sustainability overviewed the possible targets of genetic modification to increase pest control, how and what types of modifications can increase immunity and the possible risks that must be addressed if engineering resistance is to be sustainable.
How can genetic engineering enhance disease management?
Engineering Pathogen-Associated Molecular Patterns (PAMPs) recognition
A common feature of the immune system of many eukaryotes is the ability to recognise particular patterns on pathogens (“PAMPs”). The patterns are conserved across species of pathogens and, once recognised by immune cells as they survey the cells present in their host they trigger an immune response.
Whilst all plants will have the ability to recognise a range of PAMPs, they wont recognise all of them. Therefore, if one species of plant has developed the ability to recognise a particular pathogen and defend against it, identifying the requisite gene and transplanting it into another plant that is struggling to defend against the same pathogen will quickly enable it to muster its own immune defence against it.
Resistance genes, or ‘R genes’, allow a plant to overcome effector molecules used by pathogens to increase their chances of successfully invading a host. In a never-ending arms race, a pathogen will develop an effector molecule to enhance susceptibility of the host to infection, while the host will in develop the ability to recognise the effector and induce the immune reaction again. In response, the pathogen may develop a new effector molecule, and the plant must again develop the ability to recognise the effector and respond when it is present.
The DNA encoding these new proteins developed by plants to detect new effectors are termed R genes, and the ongoing battle means that there are a multitude of R genes relating to a multitude of pathogens throughout the plant kingdom. Therefore, transferring R genes from a resistant plant to a susceptible plant will transfer resistance.
Transferring R genes can be done via conventional breeding, although some plants are easier and less time-consuming to cross-breed than others. Engineering the transfer of R genes will be quicker, more effective to use in difficult-to-cross crops and will enable more precise insertion of the genes, reducing the inheritance of unwanted genes along with the R genes.
Such a technique was used to transfer a gene from peppers which conferred resistance to bacterial leaf into tomato.
The obvious downside to conferring resistance this way is that the pathogen will again develop a new virulence method which will again need to be addressed, resulting in only a temporary resistance. However, helping crops quickly adapt to a new infection will help ensure short term yields while also allowing the engineering of specific resistance to specific pathogens as new effectors and R gene couples are discovered.
Giving Defence Responses a Boost
As well as increasing pathogen and effector recognition to allow immune responses to be initiated, increasing the size of those responses can also help combat specific pathogens.
An example provided in the paper is the use of a constitutive promoter from wheat to increase the expression of native immune gene which helped rice crops combat a number fungal pathogens including rice blast.
Changing DNA Sequences that Result in Increased Susceptibility
Some pathogens have developed the ability to exploit some required host protein to give itself a route of infection. The susceptibility genes encoding these proteins are problematic to deal with given the necessity of the gene product. However, modification to the gene, either natural or synthetic, which alters the protein enough to reduce their ability to be exploited by the pathogen but not so great as to render the protein useless in its required role, has been shown effective in increasing resistance.
Plants producing their own Antimicrobials
Like the Bt toxin producing corn crops that now protect themselves using the same chemistry used by conventional pesticides, crops producing their own antimicrobials provides a further potential basis for sustainable protection.
The discovery that double-stranded RNA results in the silencing of genes with a complementary sequence has led the ability insert genes into an organism coding for a double-stranded RNA which will silence a specific gene or, of use to us in plant immunity, will silence genes within a parasite to reduce or remove their pathogenicity. Such was the case with the papaya ringspot virus in Hawaii, which was overcome by engineering the papaya to produce a dsRNA complementary to a coat protein gene of the virus, removing a virulence factor relied upon by the virus and saving the industry.
Removing Host Virulence Factors
Similar to removing or modifying susceptibility genes to remove a target route of infection used by pathogens, removing or modifying host virulence factors such as a particular protein which allows strong binding of the pathogen is a potential method of reducing infection rates.
Detoxifying the toxins
Many pathogens produce toxins which will attack particular targets of plant cells to allow easier invasion. Being able to render the toxin ineffective will in turn reduce infection rates of many pathogens, and having the plant produce these detoxifying compounds is a possible means of sustainably reducing crop destruction from disease.
Using CRISPR/Cas 9
CRISPR and its ability to make target endonuclease activity to specific parts of a DNA sequence has seemingly limitless uses, including as a disease management tool. By targeting an endonuclease to DNA inserted into a plant, such as replicating DNA of Geminiviruses, will disrupt the replication and infection rates of numerous pathogens and holds the possibility of being an adaptable method of crop production.
Balancing Crop Protection and Resistance Selective Pressure
Although there are number of methods of increasing resistance through targeted means, evolution doesn’t allow us to simply pick a single method to eliminate a pathogen; resistance is an ongoing concern.
However, if the ability to use multiple methods of increased pest management is developed along with the ability to rotate what methods are used, we may manage to increase our crop protection and reduce the selective pressure we put on pests.
Stacking multiple genes into plants is a promising method of achieving this end and Plant Artificial Chromosomes may be the scaffold on which these methods of disease management can be simultaneously used in crops.
Concerns using Genetic Engineering
The usual concerns are raised and, although noted as risks that must be managed where there is a dearth of evidence confirming the size of the risk, those concerns are largely dismissed.
The health risks of genetically engineered crops are an oft-raised topic in public forums, but the science on the lack of risk is largely settled.
Flow of recombinant DNA into related plant species is discussed but is again largely dismissed save where further research should be conducted to quantify such risks. Given the context of such genes being already present in wild-type plants and therefore already available for gene flow, the lack of evidence of microorganisms being transformed by transgenes plus the ability to design synthetic gene components so as to be not usable by prokaryotic microorganisms, the use of genetic modification use has little chance of increasing the risk.
The control of genetic engineering by large companies and the promotion of monocultures are also raised as potential concerns related to using this technology in disease management programs. Again, the concerns raised fail to look at the current state of agriculture where large companies already hold the majority of patents for non-GMO technologies and where monoculture farming is a symptom of economic pressure unrelated to the use or disuse of genetic modification technology.
The review provides a great basis for further researching possible methods of sustainable disease management, pointing out the multiple paths that may be taken to research and develop protections against any particular plant pathogen.
The new technologies and any risks that come with them must be subject to rigorous risk analysis and the pros and cons weighed before implementation. Combining the use of multiple types of technology and managing the evolutionary pressure that could be caused if only one type of technology or only one target is used could create methods more sustainable and more targeted than those used today.
Being able to turn genes on and off as required is a significant goal in genetic manipulation. Synthetically constructed gene circuits which turn on and off in response to specific activators have previously been demonstrated and the ability knock out specific genes with such technology as CRISPR/Cas 9 has made gene manipulation easier and cheaper.
But the purpose of this particular article in the Plant Biolotechnology Journal (open-access = awesome) was focused on manipulating the mechanics of gene transcription repression to turn off gene repressors (and activate expression) and then reverse the process in subsequent generations as required.
Transcription factors, the proteins that form part of the machinery that transcribes DNA sequences into RNA, contain motifs that allow for them to be activated and deactivated. Regulation of gene transcription using the activation and deactivation of transcription factors is a necessity in all cells, allowing the expression of genes to be limited to particular moments within a cycle or at different stages of development.
The examples used in this study when demonstrating the novel bit of tech the researchers developed to force the repression and activation of different genes illustrate the need for this function well. One gene, MYB80, codes a transcription factor necessary for pollen development and the programmed cell death (PCD) of the tapetal in plant anthers. If allowed to transcribe one of its targeted genes unregulated, PCD occurs too early and the male flowers are rendered sterile, meaning the transcription must be regulated if the plant is to develop properly. This consequence was discovered in an earlier study when a mutant of the transcription factor was created which deleted part of the encoding gene which resulted in the loss of the regulatory motifs, likely meaning the the transcription factor was unhindered in transcribing its target genes.
Another transcription factor studied by the researchers was that encoded by WUSCHEL (WUS), required for the development of the organising centre beneath the stem cells in the meristem. Regulation of WUS activity is required to create distinct cell boundaries and a lack of regulation leads to disorganised growth and male sterility. Similar to MYB80, the transcription factor has a number of regulatory motifs at the C-terminal end of the protein which interact with corepressors.
Manipulating the function of transcription factors to regulate gene transcription is therefore another potential method of turning genes on and off to test the knock-on effects and potentially alter and improve plant characteristics.
Synthetically Regulating the Transcription Factors
How these regulatory motifs inhibit gene transcription, and how we might be able to cause the same inhibition, was the focus of this research. Using the MYB80 and WUS as targets, the researchers created what they dubbed “Conserved Sequence-guided Repressor Inhibition”, or “CoSRI”.
The name of the protein gives away how it works. This study and previous studies have found that within specific repressors are regions that are conserved across many species. Studies that have removed these conserved regions have resulted in the types of defects discussed above due to the unrestrained transcription of the genes targeted by the subject transcription factors. When the conserved sequence is reinstated, repression of transcription is reinstated and phenotypes return to match the wild-type plants.
Fig 1a from the article. The figure demonstrates how the CoSRI is targeted to the conserved section of the repressor gene and the section of the CoSRI interfering with the binding of the corepressor to the repressor.
So the researchers targeted these conserved sequences as being the sites to direct their newly constructed proteins, thereby building in some specificity to which repressors are targeted. Conjugated to this new protein is a repression-motif interacting region, a section of protein which interacts with the DNA binding section of the transcription factor, stopping the corepressors from interacting with the DNA binding domain of the transcript factor.
Testing the Constructs
Genes for the CoSRI proteins designed specific to the MYB80 and WUS transcription factors were transformed into Arabidopsis thaliana separately.
When the MYB80-specific CoSRI was introduced, the same silique abortion and male sterility were observed. Sterile lines analysed showed that the transcript levels of the CoSRI were at the same levels of greater than the levels of the endogenous transcription factors. Further, transcripts of the genes targeted by the endogenous genes were reduced in sterile lines.
Similarly, CoSRI genes specifically targeting the WUS transcription factor was transformed into A. thaliana, again resulting in altered phenotypes, specifically defective shoot meristems resulting in poor growth and failure of silique elongation leading to loss of fertility.
In short, the researchers had developed a synthetic construct that competed with corepressors for the ability to bind to the repression-binding motif of the transcription factor and blocked the active repression of gene transcription normally mediated by the transcription factors.
Restarting the Repression
Being able to stop the repression of gene transcription is great, but what about reversing it? The example of such a use given in the paper is forcing male sterility in, say, a hybrid plant species, but then being able to reverse the sterility later.
To achieve this, the researchers created what they termed a restorer construct which combined the first 188 amino acids at the N-terminus of the corepressor (the part required to repress transcription by the transcription factor) with a conserved region of the target transcript factor. The result is a protein that is directed to, in one example, the MYB80 transcription factor that carries with it the necessary part of the corepressor to repress the workings of the target transcription factor and restoring the wild-type phenotype.
Figure 1b from the article. The figure depicts the interaction of the CoSRI with the repressor to block corepressors from the repression binding site of the transcription factor, and the use of the restoring CoSRI to reverse the sterility caused by the first CoSRI.
The researchers tested whether the new construct interfered with the transcription levels of any other genes or the transcript levels of the MBY80 targets in wild-type A. thaliana that had not been previously transformed with a CoSRI, finding no interference.
In lines that had both the CoSRI and the restorer construct, sterility was reversed when the transcript levels of the restorer were similar to that of the CoSRI. Fertility was only partially restored or not restored at all when the restorer construct levels were significantly lower than their competition.
This new technique appears to provide an indirect and reversible method of altering the transcription of specific genes. Being able to stop and restart transcription factors for specific genes could be invaluable to plant (and other) research and the technique could find its way in the the toolbox of genetic engineering technology that is exploding at the moment.
The technology could also open up the possibility of being able to alter plant characteristics to suit seasonal requirements, particularly if we develop the ability to not just knockdown and re-initiate gene transcription but also to carefully calibrate gene transcript levels.
Last October, the journal Developmental Cell published this article about tissue growth control in Arabidopsis thaliana, examining the effects of mutating a particular gene thought to influence plant growth.
The title of the article buries the lead a little though, as the method they developed to assess the effects of modifying this particular gene could potentially be put to good use in systems engineering studies.
The article starts with the researchers noting the importance of the apical meristems in plants, being the source of stem cells which differentiate to allow plant growth at both the root and shoot ends of the plant. Root apical meristems are well studied and while the effects of different treatments on shoot apical meristems and consequent growth of the stem, leaves and flowers have been studied, how these tissues are initiated within the apical shoot apical meristem is still a bit of mystery.
The cause of the mystery is not a result of under-appreciation of the importance of this part of the plant, but due to the complexity of the structure and growth characteristics plus the difficulty of studying the inaccessible region of the plant in real time.
Within the introduction the researchers underpin the importance of the shoot apical meristem with a description of its structure. It has distinct zones with distinct functions, including a peripheral zone from which come leaves and flowers and a central zone which replenishes the peripheral zone cells. Underlying these zones is the rib zone from which stem growth originates. The rib is named as it is due to it being made up of a cells which divide in a different orientation to the overlying layers.
Fig 1 from article. Figure 1F (bottom left) shows the locations of the peripheral (PZ), central (CZ) and rib zones (RZ) within the stem apical meristem.
As can be made out from the coloured lines indicating the orientation of cell division in figure 1G above, cells within the peripheral and central zones divide horizontally (or perpendicular to the surface of the meristem), while cells in the rib zone divide so that the daughter cells are orientated vertically, causing upward stem growth.
These cell divisions are controlled and vary depending on the stage of plant growth. During the transition to the vegetative stage of growth the outer zones are active while the rib zone is restrained from cell division. During the flowering stage the rib zone is active and stem elongation rates are increased.
Therefore, knowing how the rates of cell division are controlled could lead to advances in crop growth and production, and the method of visualising the effects of manipulating genes could be invaluable to food production.
The 3D imaging method
Imaging the division of the cells within the shoot apical meristem in real time is something that has not previously been achieved but is a technical advance required to understand the underlying basis of plant growth. So, these clever researchers at the Norwich Research Park invented a way to do it.
Based on the fact that the cell wall is extended perpendicular to the mitotic spindle during cell division, the researchers thought it likely that the new wall will be thinner than the existing walls. To utilise this characteristic of cell wall extension and the information it provides about the orientation of cell division, the researchers cross-linked polysaccharides in the cell wall with fluorescing propidium iodide. The thinner, newer cell wall should fluoresce at a lower intensity compared the thicker, existing wall and therefore the orientation of cell division within the meristem can be discovered and imaged.
It was using this method that the great images shown in figure 1 were obtained.
Using the new imaging technique to test the effects of gene modifications
The researchers examined the role of REPLUMLESS (RPL), a gene which transcribes a transcription factor known to regulate stem growth. The method by which RPL controls stem elongation and the transitions between different growth stages is unknown however. Therefore, using the new imaging method, the study looked at the differences in cell division between a wild-type A. thaliana and an rpl mutant.
Imaging showed that the RZ of the wild-type was well-defined when the orientations of the cell divisions were analysed while cell divisions in the rpl mutant were less well organised, indicating that the differentiation between the different boundaries may be controlled by the gene.
Testing these findings, the researchers labeled cells with a Green Fluorescence Protein to track cells as they divided and the directions of tissue growth, finding that the outer regions of the shoot apical meristem grew laterally in relation to the stem. Conversely, the central region of the meristem divided and grew vertically and this was different between the wild-type and rpl mutant.
Delving further in the function of RPL, chromatin immunoprecipitation was used to detect regions of the genome bound by the transcription factor, finding that RPL interacts with a large number of genes that control stem development. Particularly, differential gene expression of a gene LSH4, a gene that controls organ boundary development, was detected.
Therefore, using their new technique, the researchers found the RPL affects the organised division of cells in the shoot apical meristem which in turn affects the control of growth during the various stages of the growth cycle. The result of mutating the RPL gene on plant growth can be seen in figure 6G and 6H from the article.
Figure 6 from article. 6G is the wild-type and 6H is the rpl mutant, demonstrating the effect of the disorientated meristem divisions when RPL was not controlling stem elongation. Growth rates are shown in figure 6K while the orientation of cell divisions are demonstrated in the top left of the figure.
The researchers have developed a method of imaging cell divisions within the stem apical meristem which was used to study the method of growth control exerted by a gene known to play a role in stem morphogenesis.
Critically, the method described can be used by other researchers to research and test genetic modifications that could help increase crop productivity or adapt crops for better or more efficient growth in differing conditions.
This recent research article in Science has received a decent amount of attention for good reason given the possible impact it could have on crop productivity through increasing photosynthetic efficiency. However, the approach to increasing efficiency in this paper varies considerably from the efforts to transport C4 photosynthesis into C3 crops more regularly seen.
We have a good understanding of the working of photosynthesis and its use of photons and excited electrons to fix carbon. But built into this system is a protection mechanism that kicks in when the intensity of light is too great for the CO2 fixation capacity of the photosystems, a damaging state for the plant to be in. When the excitation energy is too great, the energy is dissipated as heat, a process called nonphotochemical quenching of chlorophyll fluorescence (NPQ).
At high light intensity, NPQ is a useful process. However, NPQ at light intensities lower than that which could cause damage to the delicate photosynthetic components results in a reduction in CO2 fixation. When a leaf goes from high light intensity to low light intensity, NPQ reduces accordingly. However, the transition of NPQ lags behind the transition of the leaf from high to low light intensity, resulting in a temporary reduction in CO2 fixation and, therefore, plant growth. Earlier research indicated that these losses were in the range of 7.5 to 30% of fixation rates.
These researchers therefore tested the possibility of increasing the speed with which the transition from photoprotection to full resumption of carbon fixation could occur.
NPQ results from a conformational change to the photosystem II antennae from an unquenched to quenched state, which results in the excess excitation energy being dissipated. NPQ levels correlate with the amount of photosystem II subunit S (PsbS) and the flux of xanthophyll cycle. While over-expression of PsbS in plants can enhance NPQ and photoprotection as well as increase the rate of change between quenched and unquenched states, overexpression can also reduce CO2 fixation rates when light intensity is at a level lower than that which could cause damage. Further, the lag between transition from quenched to unquenched states due to the effects of PsbS on NPQ is minimal (10 to 90 seconds) compared to the to the same transition when NPQ is triggered by zeaxanthin (10 to 15 minutes), a product of the xanthophyll cycle.
Figure 1 from article showing the factors affecting NPQ under different light intensity.
Therefore, the researchers looked at whether adjusting the xanthophyll cycle could assist the transition rate of NPQ. Specifically, they hypothesised whether accelerating the cycle and simultaneously increasing PsbS would result in a faster reduction of NPQ when leaves transition from high to low light intensity.
The researchers transformed tobacco plants with Arabidopsis-derived sequences for violaxanthin de-epoxidase, zeaxanthin epoxidase and PsbS and promoters for their expression in leaves. Transcript and protein levels of the three sequences were shown to be increased in the transformed plants compared to the wild type controls. Leaves of transformed and wild type plants were then subjected to fluctuating intensities of light. The NPQ relaxation rate due to the altered xanthophyll cycle increased significantly compared to the wild type, having an average relaxation time of 753 seconds compared to 2684 seconds in the wild type while relaxation due to the additional PsbS expression decreased from 21 to 15 seconds on average.
The recovery of CO2 assimilation was also analysed under the same fluctuating light. After transitioning from high to low light the CO2 assimilation decreased and was at a minimum 30 seconds after the transition before increasing again as the photoprotection relaxed. The rate of CO2 fixation increased faster in the transgenic lines with 9% higher fixation rates compared to the wild type tobacco plants.
Further testing the effects of the overexpressed genes on CO2 fixation, the researchers looked at the variation in CO2 fixation rates in response to variations in light. Two tests were performed: vary light intensity leaving enough time at each intensity to allow the fluorescence and gas exchange to reach a steady state, and varying light intensity every 4 minutes.
During the steady state experiment, the maximum CO2 fixation didn’t vary between the transgenic and wild type plants (both averaging 0.092 CO2 per absorbed photon), indicating that the overexpression didn’t effect the photosynthetic capacity generally as was seen in previous experiments.
Figure 4 from article showing CO2 fixation per photon, quantum yield per photon and NPQ levels in the wild type and the three transgenic lines.
Under the alternating light experiment the CO2 fixed per absorbed photon was decreased compared to the steady state experiment but was greater in the transgenic lines (0.066CO2/photon) than the wild type (0.058CO2/photon), an 11.3% increase. Similar findings were observed in relation to quantum yield of whole-chain electron transport.
Plants grown under field conditions showed the same differences in fixation rates. Further, a randomised block design consisting of 12 blocks was used to test the agronomic performance of experimental and control lines, with 14 to 20% greater dry weight observed in the transgenic lines compared to the wild type with noted increases in leaf, stem and root weights and leaf area.
Finally, whether the transgenic lines may suffer from altered photoprotection under high intensity was tested in seedlings. After 2 hours being exposed to excessive light the photoprotection appeared to be similar or higher in transgenic lines.
In the discussion section the researchers point out that under field conditions an individual chloroplast can be subjected to instantaneous and repetitive changes from high to low light conditions due to shading from other parts of the same plant or from nearby plants. The ability to reduce the response time of the photoprotective system will significantly assist crop productivity.
Further, stomatal conductance increases under high light conditions and remains so for minutes after transferring back to shade, resulting in excess water loss compared to when the leaves receive less than a harmful level of light. Speeding the relaxation of NPQ, the researchers point out, should also result in better water use efficiency.
This results of this research are really novel and demonstrate just how complex and evolved the photosynthesis machinery is. The xanthophyll cycle and PsbS are found in vascular plants, leaving open the possibility of transferring these faster transition rates to important crops.
We have previously reported on an article in Nature Scientific Reports (“the Rangan paper”) that indicated the possibility that C4 photosynthesis was being carried out in the grain of wheat, an important C3 crop that belongs to a clade with no evidence that C4 photosynthesis has ever evolved within it. As C4 photosynthesis is more efficient in fixing CO2 than C3 photosynthesis, the possibility of the pathway being active in such an crop would give hope that it could be manipulated to improve its growth properties and that other important food crops.
Shortly after we posted our article it was pointed out that, although the evidence gathered in that paper are a useful analysis of gene transcript data, the presence of C4 gene transcripts should not amount to the conclusion that there is an active C4 biochemical pathway in the wheat grain. In the last week, two articles have appeared rebutting the conclusions of the paper.
“Seeds of C4 photosynthesis”
This article by Julian Hibberd and Robert Furbank in Nature Plants points out that although what is presented in the Rangan paper provides an interesting in-depth assessment of the transcripts derived from the wheat grain, the transcript data by itself doesn’t evidence a functioning pathway.
Whether the transcribed proteins are active and to what extent they are active cannot be evidenced solely from transcript data. If they are active, whether they are linked appropriately to form the C4 pathway for metabolite flux also needs to be evidenced if we are to accept the possibility of a functioning pathway.
The Rangan paper also cited differences in particular cell types within the pericarp of wheat grain and combine these differences with the abundance of C4 gene transcripts to hypothesize the possible existence of the two-celled C4 system. There were two problems with this hypothesis:
- No cell specific transcript evidence is given to support the assertion, let alone the protein and metabolite data. Although there are a number of different types of cells in the pericarp, the transcript analysis does not distinguish between these types of cells which would help confirm or deny a difference in transcript abundance between the types of cells; and
- Both the Hibberd and Furbank paper and the paper discussed below cited a protein labelling experiment which showed the PEPC protein (an important protein in the C4 pathway) is not found in the pericarp but rather the aleurone layer.
The paper concludes by saying that the transcript data isn’t enough to form a conclusion that engineering C4 photosynthesis in wheat will be any easier in wheat than in any other crop.
“Poor Evidence for C4 Photosynthesis in the Wheat Grain”
This article in Plant Physiology make the same criticisms about the lack of biochemical evidence supporting the claim of C4 photosynthesis in the wheat grain, the lack of evidence showing which cells within the grain are the origin of the transcripts obtained and that previous immunolabeling studies have shown PEPC being localised in the aleurone layer and endosperm, not the pericarp as suggested by Rangan et al.
A further criticism of the Rangan paper was the assertion made that the increase in PEPC and decrease in RuBisco transcripts is evidence of C4 photosynthesis. Again, the lack of biochemical evidence supporting an active pathway was noted. However, the authors also note that C4 photosynthesis still requires the use of RuBisco to assimilate the CO2 concentrated by PEPC and that an approximate ratio of one-to-one is required. Therefore, an over-abundance of PEPC compared to RuBisco doesn’t result in the increased photosynthetic efficiency we relate with C4 photosynthesis.
And a bit more…
We contacted Professor Robert Furbank, co-author of the Nature Plants letter, who was kind enough give us some insight into the issue, its historical context and how big a discovery it would be if C4 photosynthesis existed in wheat grain:
“It has been known since Tom ap Rees’ work in the 1970’s that a suite of C4 enzymes are present in C3 seeds but the flux is into amino acid synthesis and gluconeogenesis.
What I find interesting in the context of the Rangan paper is that the same mistakes were made which delayed the discovery of the C4 pathway by a decade. Careful measurements of photosynthetic flux which can separate a respiratory role of PEPC and other C4 enzymes from a photosynthetic one are required. To ensure that RNAseq does not become the emperor’s new clothes, we must combine biochemistry, physiology and our new next gen sequencing tools to build a cogent and robust story.
I would be very surprised in C4 photosynthesis existed in grains. The evolution of C4 was to concentrate CO2 around rubisco in a low CO2 atmosphere, which is certainly not the case in a developing cereal grain. Even in the glumes, there is hard evidence that the initial evidence for C4 photosynthesis was flawed.”
The Hibberd and Furbank response to the Rangan paper acknowledges that the transcript analysis performed and reported on provide interesting evidence regarding differences in gene expression between different plant tissue, but warn that the gap between gene transcription and functioning protein pathways is something that must be bridged before the type of conclusion made by Rangan et al. can be made with confidence.
We must also admit that, in our post on the Rangan paper, our enthusiasm for the conclusions presented was greater than our critical analysis of evidence supporting it. We thank Steven Burgess and Julian Hibberd for their tweets pointing out issue with the conclusions drawn and Robert Furbank for taking the time to talk to us. In future, we will improve on our analysis of new articles and will seek out comments from relevant experts when significant claims are being made.
The CRISPR/Cas9 endonuclease system is likely to be the restriction enzyme currently in most widespread use. Its ease of manipulation to target different nucleotide sequences has made it valuable to systems, transgenic and synthetic biology, allowing researchers the ability to observe the effects of manipulating specific genes by knocking them out, down or up regulating them or for adding new genes into genomes.
Related to our recent post about how we identify genes related to traits, many traits that we research and possibly seek to modify, particularly in crop species, are usually controlled by more than one gene. Therefore, studying the effects of altering a particular phenotype characteristic requires the ability to target multiple genes in the one organism.
A recent PLoS One research article looked at the use of CRISPR/Cas9 to target multiple sites in one transformation event in Arabidopsis thaliana. Particularly, they looked at its efficiency transforming a number of genes and also assessed the rate of off-target modifications made.
The researchers targeted 7 genes from the GOLVEN (GLV/RGF/CLEL) gene family. The proteins produced by genes belonging to this family are important regulators of root stem cell development. In the discussion section of the paper the researchers also mention that the targeting of these genes combined with their selection of guide RNAs resulted in a diversity of DNA sequences being targeted and a significant number of identified potential off-target sites that may also be effected by editing. Therefore, they believe that their results targeting members of this family of genes will be representative of multi-site targeting in Arabidopsis generally.
For each of the seven genes two restriction sites were targeted. gRNA units targeting these sites were synthesised into clusters of 4 and the clusters containing all 14 of the gRNAs were then assembled and cloned into a binary vector. Wild-type A. thaliana were transformed using Agrobacterium tumefacians via the floral dip method.
Six T1 plants generated from the transformed wild-type plants were screened for editing by sequencing one of the targeted genes (GLV1). Only one of the six plants screened showed a transformation at this location.
The leaves of 48 T2 plants generated from this one transformed T1 plant were sampled in a pool. The pooled DNA was resequenced and the reads mapped against the TAIR10 reference genome.
The criteria the researchers set in assessing whether the targeted genes had been edited was to see whether there were any insertion deletion (indel) events at or near three base pairs upstream of the PAM sequence, noting that while large deletions may not be mappable such large deletions are likely to be infrequent (based on their previous work and experience).
Using the above criteria and assessing the pooled reads of each of the 14 targets, two of the targets showed no transformation (and from the table below, appear to avoided resequencing altogether. The researchers opined that in their experience such events occur in a similar frequency in single restriction events). Efficiency of editing in the 12 successfully transformed targets ranged from 33% to 92%.
Table 1 from article.
Being able to target multiple genes linked to a specific trait for mutation is great, but study results can be effected by the endonuclease causing mutations at other sites in the plant DNA. To assess the risk of this occurring the researchers used CRISPRP, a tool that can be used to find highly specific CRISPR sites and allows researchers to assess likely off-target sites within a genome for a particular gRNA. Assessing each gRNA with the tool and limiting the results to the top 20 possible off-target sites for each gRNA sequence, 178 possible off-target sites were identified and were covered in the sequencing data.
Analysing the near 43,500 reads that covered the identified potential off-target sites, the researchers found no indel events attributable to the endonuclease (presumably using the same criteria used to assess whether targeted sites were edited).
Thus, it appears that stacking gRNAs and targeting multiple genes at one time is still highly specific in Arabidopsis.
To check for any other possible off-target events that were not covered in the identified sequences the researchers altered the filters applied to the possible indel sets and obtained a further 4 potential sites that had an allele frequency with with the gRNA of over 3%. Screening these sites in both the wild and the transgenic lines showed a number indel events in both lines, suggesting, say the researchers, that the identified locations are susceptible to deletion events.
Finally, the researchers looked for possible translocations resulting from multiple editing events occurring at once. To do this, they looked for reads of targeted editing events that were contiguous with other editing events (see figure below) and found two independent events, demonstrating that such events can happen but at a very low frequency in Arabidopsis.
Figure 2 from article. The sequences in part A of the figure are target sequences of three gRNA. Part B shows the sequence of the translocation of one of the sequences categorised by the concatenation of the target sequences.
The study suggests that using CRISPR to target multiple genes can result in a targeted and precise gene mutation. However, this is limited to Arabidopsis manipulation and these results do not necessarily extend to other plants. That said, Arabidopsis is widely used model plant for research and the findings here may assist researchers looking to manipulate arrays of genes at the same time.
The low transformation frequency of an admittedly small sample size is a little concerning when compared to high efficiencies of transformation generally seen when CRISPR is used. The researchers point out that this may be due to genes targeted being essential or late embryogensis related. But it would be great to see a replication of the study using other phenotype-related loci to confirm whether there is any transformation effect caused by the genes targeted.
Overall, the results of the study suggest that CRISPR/Cas9 editing continues to find new and useful application and may be of use to those tinkering with plant traits or synthesising new ones.