Increasing Plant Pest Resistance with Bt + RNAi Pyramids

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.

bt and rnai pyramid figure 1

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.

Bt plus RNAi Fig 2

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.

Conclusion

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.

 

 

 

 

Using Clay Nanosheets to Give Plants Sustained RNAi-based Protection from Viruses

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.

bioclay-fig-3

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.

bioclay-fig-4

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.

Conclusion

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.

 

Genetic Engineering and Sustainable Plant Disease Management

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

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.

A suggested method of overcoming the serious threat to the citrus industry in Florida caused by citrus greening is to have the plant produce defensins from genes derived from spinach.

RNAi

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.

Conclusion

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.

Photoprotection and Crop Productivity

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.

Background

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.

The Biochemistry

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.

photoprotection

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 Study

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.

npq

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.

Discussion

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.

Conclusion

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.

Quantifying synthetic gene transcription in plants

The article we write about today, “Quantitative Characterization of Genetic Parts and Circuits for Plant Synthetic Biology“, was published online in Nature Methods about a year ago. But its importance is such that we still thought it worth describing and to point out to readers other sites that have also provided excellent overviews of this paper (see The New Leaf, the GARNet Community Blog and Science Daily‘s write ups).

The Study

The researchers behind the study were looking to address one of the biggest problems in plant synthetic biology (and synthetic biology generally), being the ability to design gene circuits with a solid understanding of the rate of transcription of each of the genes within the circuit. Being able to predict and ‘tune’ the amount that a gene is transcribed will be a great step forward in allowing researchers and biological engineers to design and test circuits on computers, saving the time and expense of having to synthesise and test each component in the wet lab.

But biology, particularly multicellular biology, is messy and noisy and affected by so many different factors that we are unlikely to know and control each and every one of them. This study showed as much, but also showed that with careful testing we can develop the underlying knowledge required to develop general rules that will assist higher throughput development and research.

Using protoplasts derived from Arabidopsis cells to express the synthetic constructs, the researchers developed three small circuits. One circuit was an inducible promoter which, when induced by an externally applied inducer to begin transcribing the circuit, would result in the production of the protein firefly luciferase (F-luc), a fluorescent protein that can be detected. The level of fluorescence detected is a function of how much the gene is being transcribed. The second circuit was also controlled by an inducible promoter which, when induced by the same inducer, would result in the transcription of a protein with DNA-binding domains which would bind to specific DNA sequences, in turn repressing the transcription of those genes. The idea behind the two circuits using the same inducer is that the amount of fluorescence from the first would act as a proxy reporter for the amount of repressor being transcribed.

The third circuit synthesised contained a gene for Renilla luciferase (R-luc), another protein which fluoresces but does so at a shorter wavelength than the firefly luciferase and is therefore distinguishable. This gene was linked to a constitutive (constantly active) promoter that contained a number of DNA sequences that could be bound by the repressor. The repressible promoter contained DNA binding sites at different points to test the ability to fine tune repression rates with different repressor/binding site combinations.

nature-methods-fig-1-protoplasts-inducers

Figure 1 from article. (a) is the repressible promoter with DNA binding sites at different points. (b) is the three

The result were protoplasts that (at least theoretically) would fluoresce at the shorter, R-luc wavelength when there was no inducer added to the cell as the gene circuit containing the repressor would not have been induced for transcription. When the inducer is added and increased, the R-luc fluorescence would reduce proportional to the amount of inducer added (as more and more of the repressor is transcribed) and simultaneously the amount of F-luc florescence would increase.

In a less messy environment, the input-output response would predictable somewhat like an electronic circuit – an increase in the input would result in an equivalent (whether that be linear, exponential, logarithmic etc) change in the output. The aim of the research was to quantify the input-output ratio and see if a mathematical formula could be applied to allow predictions of input and outputs to be made.

The Results

But biology is messy and the input to output ratio over a number of reproductions varied considerably even though the generally expected increase in F-luc and reduction in R-luc as more inducer was added was observed.

So the researchers looked at where the variability could be coming from and how it may be accounted for so that predictable quantification of the transcription rates could be derived. To test this they examined the amount of F-luc fluorescence in the protoplasts when no inducer was added which theoretically should result in no fluorescence or, if induced by something other than the external inducer, should be fluorescing at the same level. What they found was that there was an unexpected variability in the fluorescence levels even when there was not external inducer added. When sorted by different batches of protoplasts which were prepared on different days, it was apparent that some variation in the preparation of each batch (which they weren’t able to explicitly identify) was causing a variation in the F-luc levels that was not controlled for (Figure 2(b) below).

noise-in-protoplast-data

Figure 2 from article showing noise in the protoplast fluorescence data grouped by batch and inducer type.

Given the noise wasn’t completely random, the researchers looked to mathematics to attempt a solution to their quest to help predict output levels despite the noise.

The Mathematics

It was assumed that the input to output relationship would function in accordance with the repressing Hill function. But analysing their data compared to that expected according to the Hill function showed that the output amount was the Hill function multiplied by some factor that was related to whatever was affecting each batch. What they hypothesised what that the ratio between average of fluorescence of all batches to that of each batch, with no inducer applied, should normalise the fluorescence levels and eliminate the batch effect observed. Calculating the normalisation factor in this way resulted in the reduction in noise between batches.

normalisation

Figure 4 from article showing the effect of normalising the input-output data using the calculated normalisation factor.

Reproducibility and Usability

To test the circuits and normalisation of output quantification in a monocot, sorghum protoplasts were transiently transformed with the same result – normalisation of the fluorescence values reduced or eliminated the batch effects.

Further testing was done to compare the use of transient expression in protoplasts to expression of the same circuits in stably transformed plants to determine whether one is reliably indicative of the other. Again, the purpose of confirming this comparison is to allow faster testing of circuits using transient expression with confidence that similar results will flow from the same circuits being installed stably into transgenic lines. Comparing the two in Arabidopsis protoplasts, the researchers found that luciferase expression was lower in the stably transformed protoplasts compared to the transient expression but the difference could again be normalised and fitted with the repressing Hill function.

Thus, it was suggested by the authors of the paper that by using these normalisation techniques, transient expression of gene constructs in protoplasts could be reliably used to predict the expression levels of the same constructs in stably transformed plant cells.

Conclusion

The paper addresses the importance of predictable, reproducible quantification of genetic parts in multicellular organisms that produce a lot of noise when trying to quantify results. It demonstrates the issue that will consistently arise as we attempt to address food security and environment concerns with technology aimed to produce larger yields with lower input and land usage. But it gives an insight into the ability we have to overcome the hurdle and begin designing and testing circuits with larger throughput and greater reproducibility.

And was clearly worth writing about again!

Can we synthetically engineer C4 photosynthesis?

“Photosynthesis as the engine for life on earth has high engineering potential, which has not yet been fully exploited…By step-wise identification of all the components needed for engineering, it will eventually become possible to employ this powerful machinery to increase yields for the future.”

Schuler, ML, Mantegazza, O & Weber, APM, 2016, ‘Engineering C4 photosynthesis into C3 chassis in the synthetic biology age’. The Plant Journal, vol. 87, pp. 62

These lines from the conclusion of the review we write about here are indicative of why so much effort is being put into understanding the more productive C4 photosynthetic system and working to increase important crop yields with it.

Schuler, Mantegazza and Weber’s article in the special issue of The Plant Journal on plant synthetic biology provides an excellent overview of the current status, significant hurdles and possible solutions to those problems of the current research aimed at bolstering rice yield by converting it from the common C3 photosynthesis system to the more efficient C4 system. We’ve previously written about C4 photosynthesis here and here.

C4 photosynthesis

C4 photosynthesis has evolved independently at least 66 times and is likely linked to a sudden drop in atmospheric CO2 levels sometime in the past. It is characterised by the concentration of CO2 around Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), the carbon-assimilating enzyme, reducing the competition that CO2 has with O2 to interact with the enzyme. More CO2 means greater growth and reduced photorespiration, an energy requiring process that is used to remove the O2 reaction products.

The concentration of CO2 in C4 photosynthesis is usually caused by a two-celled (but one-celled is possible) distribution of the process of fixing carbon and the process of reducing it. The two-celled system combines mesophyll (M) cells, which take up the CO2 from the leaf air space, and the bundle sheath (BS) cells, where the Rubisco enzymes reside, the final destination of CO2 for fixation and entry into the Calvin-Benson cycle. These two cells are arranged in concentric layers (called ‘Kranz Anatomy’) around leaf veins, maximising the contact between the two types of cells and increasing the transport of the molecules between them.

2000px-C4_photosynthesis_is_less_complicated.svg

M cells convert CO2 to bicarbonate and then into the 4 carbon compound oxaloacetate via an enzyme that doesn’t react with oxygen. The modified compound is then passed to the BS cells where it is reformed into CO2 and fixed by Rubisco to enter the Calvin-Benson cycle.

Basically, by assimilating CO2 away from Rubisco, the plant reduces the ability of Rubisco to interact with O2 and instead it is steadily fed with CO2 from the M cells.

Of course, this description of the process is simplified and although most of the process and main enzymes that carry out the process are known, there are still gaps in our knowledge.

Recent Advances

The gathering of increasing amounts of genomic, trascriptomic and metabolimic data continue to improve our knowledge of C4 photosynthesis, how it evolved and how we might transition C3 crops to use the more efficient carbon fixation method.

Important C4 crop species have had their genomes sequenced and quantitative analysis of transcriptomes have begun to unravel the mystery behind the genes upregulated and downregulated, and the stage of development that these regulatory differences occur, that lead the formation of the Kranz anatomy. What we are finding is that many of the genes involved in C4 photosynthesis exist in C3 plants but are differently regulated at early stages to differentiate the BS and M cells, enable high throughput of metabolites between the cells and to increase the size of vascular tissue to support the increased activity.

Engineering C4 photosynthesis

Our initial attempts to engineer C4 photosynthesis relied on over-expressing one or more enzymes in C3 plants. However, given the enzymes involved in the C4 system are used in the C3 system in multiple alternative pathways, the effects of over-expression were multiple, varied and didn’t have the desire result. The compartmentalisation of reactions, whether in the single or two-celled reactions that make up the distinctive photosystem, is complex.

The notion of being able to engineer C4 photosynthesis is comforted by a number of factors:

  1. The main enzymes are already present in C3 photosynthesis;
  2. Characteristics such as the passing of metabolites between cells is seen in C3 species such as tobacco plants; and
  3. Nature has done it herself in the past on multiple, independent occasions.

But the authors of the paper also note a number of engineering steps that need to be accomplished if we are re-enact evolution ourselves;

  1. Higher order veins need to be initiated in plants (it previously being shown that such physical properties were already evolved in plants that subsequently evolved the Kranz anatomy);
  2. The ratio of BS to M cells must be increased, ideally in a similar concentric organisation to Kranz anatomy;
  3. Enlarging and enriching BS cells with additional chloroplasts;
  4. Increasing the connection between M and BS cells;
  5. Engineering the different morphologies of the chloroplasts to mimic the morphologies of chloroplasts found in M and BS cells;
  6. Mirror the differing roles that M and BS cells take on in C4 photosynthesis so Rubisco reduction of CO2 occurs only in the BS cells with M cells feeding CO2 to the BS cells and excluding the oxidation of O2.

The tools we need

If we are to achieve success we still have some tools to develop and refine.

Chief among this list is a model plant that can be engineered and tested easily with speedy regeneration without requiring too much growing room. The authors point out that rice crops have some limitations in these criteria but identify Brachypodium distachyon as a model C3 plant with a small, annotated genome with quick flowering time, low growing space requirements and an efficient transformation protocol. A model such as this could hasten the engineering, testing and data gathering on conversion which can then be tested on important crop species.

A C4 model plant with similar characteristics is also required. Setaria viridis has previously been suggested as a possible model plant, as has the Fast Flowering Mini Maize.

The ability to drive and control expression of a transgene is also required. Cis-regulatory modules that promote gene expression are still under development in the wider plant synthetic biology area. This leaves a chasm between the tools we have to hand and the possibility that a large number of genes need to be differentially expressed in order to convert C3 photosynthesis to C4 photosynthesis.

Huge strides are being made with genetic manipulation, particularly with the discovery and modification of the CRISPR/Cas 9 system. But, according to the article, the maximum number of genes successfully introduced into a plant, at present, is 9. To induce C4 photosynthesis in a C3 plant, we may need the ability to stably transform a far larger number of genes plus regulatory elements, and do so without disrupting the remainder of the genome or the phenotype characteristics of our food crops.

Even when we do have these tools at the ready, we are still missing some vital information about the genes and regulatory elements that compose C4 photosynthesis. Increasing our knowledge of minutia of genetic composition and regulation of C4 systems compared to C3 systems is still a top priority. Identifying genera with the underlying predisposition that have allowed species within it to evolve from C3 to C4 for comparative analysis, particularly species displaying characteristics of a C3-C4 intermediate with sister taxa displaying C3 and C4 phenotypes, would be idyllic in assisting the study of the evolution. The authors highlight Morandia  and Parthenium generas as possible true intermediates between C3 and C4 plants. Programs such as the Grass Phylogeny Working Group and the 1KP (1000 plants) project will greatly assist identifying and genotyping suitable candidates for understanding the genetics behind enhancing crop photosynthesis.

And some suggested means of pushing the research…

It is great to see that not only have the authors elucidated quite extensively the current knowledge and gaps within the field of C4 photosynthesis engineering, but have also suggested a couple of ways of advancing the research.

The first idea they suggested is synthetically replicating a simplified C4 photosynthetic system using known genetic components. The system replicates the targeting of specific enzymes to create a two-celled photosynthesis construct, limiting Rubisco to the BS cells using RNAi to interfere with its transcription in M cells. The article highlights specific transporters that can be used to transport the metabolites between the two cells.

A second suggested idea is using brute force to direct a speedy evolution of a C3 or C3-C4 intermediate species into a C4 plant. Identifying the minimum genetic requirements of a C4 plant in a candidate crop would then be followed by the repetitive growth under the selective pressure of a low CO2 atmosphere. By repeating genomic and transcription analysis of the evolving plant (if successful), a ‘mud-map’ of the road from C3 to C4 plants can be generated and be of enormous use to research seeking to synthetically install the same machinery.

Conclusion

Although its behind a pay-wall, get your hands on this article. Whether it be for a background in C4 photosynthesis or as a springboard for your own research, it is an area of immense potential that should be worthy of an X prize.

Could the Star Trek Food Replicator be the Future of Food?

It seems an age since we wrote about what the future of food may look like.

Many modern pieces of technology have drawn inspiration from Star Trek. Whether we think of mobile phones, tablet computers, translators and voice-base queries (hi Siri and Cortana), early versions of the technology can be found in Gene Roddenberry‘s imagination.

So, can scientists with a hint of Trekkie in them influence the way we feed ourselves?

The Food Replicator

On board the Enterprise are a number of voice-command activated food replicator terminals. By simply naming your desired meal it would materialise in just a few seconds – even tea, earl grey, hot –

Or, not….

So how could we possibly know how the food replicator worked? Look at the manual of course!

The food replicator works in a similar manner to the transporter save that it doesn’t start with a fully formed object to be encoded, transmitted, decoded and reformed at the desired location, nor is its fidelity as high as the transporter (the food replicator contains some single bit errors, of not much concern with food but a big problem in people). Instead, the food replicator contains a database of the molecular matrix of, reportedly, over 4,500 different types of food. When anyone on board the Enterprise orders a meal, the food replicator draws upon the database to obtain the required constituents parts of the meal and where each of those constituents parts must be placed in a three-dimensional map (called a quantum geometry transformation matrix).

But the parts aren’t like your usual ingredient list. Instead, the ship has a supply of organic particulate suspension made up of long-chain molecules (think of the atomic make up of lipids).

Common_lipids_lmaps

The molecules are dematerialised into energy (E=mc² you know) which travel to the required terminal and are then reformed down to the molecular level according the map of the meal ordered.

The ship’s supply of the suspension is restocked at Star Command but, given the length of time some of these journey’s must take, there must be a better, more ‘Star Trek’ way of keeping the crew fueled. According to the manual, osmotic and electrolytic fracturing of waste water (you know what that is) can lead to the reclamation and reuse of up to 82% of the food created.  Given the dilemma of food security we face, this sounds like something we should researching.

So, are we any closer to a food replicator ourselves?

Real life food replicators

We’re not quite there yet, but not for lack of trying.

There are a few recently released appliances that are inspired, at least according to the journalists writing the stories, by the Star Trek food replicator.

The Genie is a relatively new machine that looks a bit like a coffee machine but is in fact a quick-fire baking machine that takes in a pod of dehydrated ingredients (according to the meal that you want to cook) and, at a touch of a button on your connected phone, will mix the ingredients and add the required liquids, cooking it at the same time and, soon enough, out comes a ready meal.

A different method of automating the creation of a meal is that of a 3D printer which exudes the constituent proteins etc in the required 3D matrix that, at the end, will resemble your order. NASA has previously funded a project that aimed to 3D print a pizza for space crew (the patent specifically talks about it being for a space crew, which is a bit cool).

What we like the most about these inventions are the people behind them. The creators of the 3D printer for space pizza envisage the use of proteins derived from more sustainable sources as algae, insects and grass, while the creators of the Genie foresee the matching of meals to your microbiome and are very sure that the future of food will contain the possibility of getting your meal in a pill.

So, although the media certainly likes to add ‘Star Trek’ to the title of any article featuring these new products, none of them use a quantum geometry transformation matrix to ‘print’ a three dimensional replication of a piece of fish made up of parts reclaimed from your bowel movement. But, that is not to say that the parts and knowledge required to make up such a replicator aren’t completely beyond us.

What may the future hold?

We can reclaim water from poop, we can use algae to produce a myriad of basic food components, we can can teleport quantum information and we may one day have the ability to easily store the amount of data needed to keep the instructions for the molecular-scale replication of food.  Although the use of quantum transformation matrices to reconstruct food via teleported components may be a fair way from being realised given our current quantum teleportation abilities, it is foreseeable that farming practices may pivot in the future to the production of the basic proteins, carbohydrates, lipids and essential elements for later combination to form the ingredients of a meal.

There would certainly seem to be many environmental advantages to having algae, bacteria or yeast produce required ingredients, such as significant control over growing systems and conditions, reduced input energy for production and reduced environmental effects from land clearing, eutrophication of waterways from excess nutrient run off and reduced food miles.

Who knows, perhaps one day your visit to the farmers market will see you face-to-face with a microbiologist who has same enthusiasm for his or her wares as the local micro-brewer does today.

Live long and prosper!

Using CRISPR to Enhance BLAST Resistance in Rice

Clustered Regular Interspaced Short Palindromic Repeats (CRISPR) specific nucleases have quickly become one of the most promising methods of making specifically placed double-stranded breaks in DNA. By expressing a guide RNA with a Cas9 nuclease within an organism, DNA can be completely restricted (amongst other uses), lending itself to the ability to rejoin the DNA with a new piece of DNA inserted or, as in this particular study, simply disrupting a specific gene by rejoining the DNA but with one or more nucleotides missing from the genetic code via non-homologous end joining (see our collaborative piece with PLoS SynBio for a description).

Rice_blast_Magnaporthe_grisea

Rice Blast lesion.

A research article in PLoS One recently (have we mentioned we love open science) tackled the issue of increasing resistance to rice blast (Magnaporthe oryzae) using this new tool. Rice, feeding nearly 50% of the world population, is an important staple crop and rice blast, one of the most destructive diseases in rice production, makes the desire to reduce yield loss caused by this fungus one of significant effect should it be realised.

The researchers identified previous studies which had found that a particular gene, OsERF922, is a negative regulator of BLAST resistance. The previous study used a host-expressed RNAi construct to knock-out this gene in rice and, when the rice plants were challenged with the fungus, the transgenic plants showed greater resistance.

The problem with using a RNAi-expressing transgenic plant is that the modification, being an insertion of DNA into the genome, comes under transgenic regulations throughout most of the world, making its adoption for use much more difficult. Traditional breeding to achieve a similar knockout can take nearly a decade to produce. The purpose of this study, therefore, was to use CRISPR to mutate and knockout the gene without leaving any foreign DNA in the genome.

The experiment

The researchers created a DNA construct that would express the guide RNA and direct the restriction of this gene 7 base pairs away from the initiation codon. To test whether the construct worked it was tested by transforming it into rice protoplasts and then amplifying the target section of DNA. 3 mutant protoplasts were recovered, one with a single base substitution, one with a five base deletion and one with a 30 base insertion, demonstrating that the CRISPR designed worked in the cell.

With the activity of the CRISPR confirmed, rice plants were transformed by using Agrobaterium tumefaciens to deliver the DNA construct which resulted in 50 first generation transgenic plants, of which 21 were sequenced. More than half of the transgenic plants analysed contained base pair deletions (about one third of which were deletions of less than 10 base pairs) while about a quarter had a single base pair insertion and about one-tenth has simultaneous insertions and deletions. Importantly, 16 of the 21 transgenic plants had bi-allelic mutations (mutated on both copies of the gene, although mutated in different ways) and three were homozygous for the mutation.

Having ascertained that mutations could be made in the rice chromosomes, 6 of the analysed rice plants were self pollinated and then genotyped at the target gene. The 120 plants produced all carried the mutations (the progeny of the bi-allelic parents segregated according to the Mendellian ratio of 1:2:1).

Using three of the homozygous, one bi-allelic and one heterozygous plants, a second generation was bred. The homozygous parents produced progeny all of whom had stably transferred the mutation. The bi-allelic and heterozygous plants again followed Mendellian genetic segregation.

 

Now that the researchers have identified progeny with stably transferred mutations, they searched for the possible transfer of the CRISPR DNA or the transfer DNA from the Agrobacterium tumefacians anywhere in the rice genome of the 120 first generation transgenic rice plants. They generated primers for the Cas9 gene and subjected the primers and the DNA to PCR amplification. The result was the generation of no amplicons for these primers. The story was a little different in the two progeny generations where only 10% of the plants in the second generation didn’t have any detectable remnants of the transfer DNA construct. However, all 30 T2 progeny of one homozygous T1 parent contained no amplified remnant of the CRISPR or transfer DNA, demonstrating that it is possible to make a stably inherited homozygous mutation without incorporating the DNA construct.

T-DNA electrophoresis image

Amplified PCR products of T-DNA within mutagenic rice crops. All 30 progeny of T1 plant KS2-45-6 (column 6 of the second row from the top) showed no detectable trace of the T-DNA construct.

Interlude..

So, the story so far…..

We have piece of DNA which can cause mutations in this specific gene in rice protoplasts and rice plants, and the mutation can be passed from generation to generation and can do so without incorporating the CRISPR or vector constructs.

The experiment (cont.)

Now that the ability to mutate the gene has been demonstrated, 6 homozygous T2 transgenic rice plants with differing types of mutation (deletion, addition, substitution) were inoculated with the rice blast fungus at the seedling stage of development. Leaves of the wild type control plant nearly died from the inoculation but the transgenic plants showed significantly decreased lesion areas and lengths, demonstrating that the loss-of-function mutation of OsERF9222 resulted in enhanced resistance to rice blast.

journal.pone.0154027.g003

Results from study: Figure A showed sequenced wild-type and the 6 mutated genes of the homozygous mutated crops. Figures B – E show the effects of challenging each of the crops with Magnaporthe oryzae.

Rice blast resistance isn’t worth anything if the mutation also negatively affects the agronomic traits of the crop. Therefore, the 6 homozygous mutants were assessed and compared to the wild type plants in relation to their:

  • plant height;
  • flag leaf length and width;
  • number of productive panicles;
  • panicle length;
  • number of grains per panicle;
  • seed setting rate; and
  • thousand seed weight.

All 6 plants (and importantly, the plant which produced all devoid of the CRISPR and vector DNA) showed no significant difference in each of the criteria compared to a health wild type plant.

And a little extra…

Just to check the frequency of producing mutagenic crops from directing mutations to a specific gene, the researchers checked whether the frequency could be be increased by targeting multiple sites in that gene with the one CRISPR construct. They created one construct which targeted two sites in the gene, and another which attacked three sites in the gene. The result was an increased frequency of mutations as the number of target sites increased and a corresponding increase in the percentage of mutants with homozygous sequence changes.

Conclusion

This research demonstrates the ability to induce a specific, advantageous change with this recently discovered, highly adaptable tool of genetic modification, and do so without adding any extraneous DNA to genome. The result is creating a resistant crop in a much shorter amount of time than traditional breeding will allow using a tool that can be adjusted quickly to pivot should evolution of the pest occur. Importantly, the changes in this particular gene didn’t affect the agronomic traits of the rice crop, resulting in a consumer acceptable staple food and decreased yield loss, a significant development in our ability to feed more people with the same amount of land and resources and potentially less fungicides.

 

The quest for the ultimate on-farm stress sensor

Very recently, Dr Lee Hickey of the University of Queensland told a short story about a farmer in the future being alerted to a disease outbreak on his farm via his phone. He swiftly commands one of his drones to spray an RNAi specific to the disease on the infected field and averts the crises. Instead of worrying about the problem and the potential effect on his crop’s yield for the year, he goes to the footy with mates without a worry.

The idea of remotely sensing and specifically addressing a crop under stress is a current area of technology under development. There are some commercial sensors available to detect a small array of stressors and the use of drones in agriculture is increasing, but the ability to monitor and address a number of potential in-crop problems at the same time is a technology still confined to a distant world in an Isaac Asimov novel.

But that doesn’t mean scientists aren’t researching how the technology might work.

A recent study in the journal Agriculture took three commercial sensors, two being spectrometers and one a fluorometer. The spectrometers can use a variety of indices to give an output which relates the amount of light hitting a plant and the amount of light being reflected to, for example, how green the plant is. Fluorescence sensors measure the fluorescence of the cells of the plant and can adjust to measure such spectra as red fluorescence, blue green fluorescence and infra-red fluorescence. From these measurements certain conclusions can be made about the health of the crop being monitored.

The Experiment

Although the sensors mentioned above are normally used to monitor an individual stress such a water deficiency, nitrogen deficiency, fungal infection or weed competition, this paper used a combination of stress of factors to determine whether a single band or index could be used to detect and identify a single stress whilst one or more other stressors were present.

The experiment was set up so that every combination of the four types of stress and a control was tested using each index available on each device. 12 days after planting, the water stress testing commenced with water reduced to 30% of the pot water holding capacity while non-stressed plants were watered at 70% of the water holding capacity. For nitrogen stressed plants no nitrogen fertiliser was used throughout the experiment while the non-stressed wheat received fertiliser at predetermined amounts on predetermined days. Weed plants and fungal infections were added at the same stage of the experiment.

Sensors1

Table from article. Combinations of stress treatments.

Each treatment combination was measured every third day after two leaves had appeared on the spring wheat that was being tested. For each index to be measured, five readings were taken on each reading day, and the five readings averaged.

Results

Each stress resulted in a significant effect on the growth of the spring wheat either by the amount of biomass or the ratio of root to shoot growth. Many indices provided significantly different spectral ratios and fluorescence levels between control plants and plants tested with one type of stress. The tables of average values for each index tested for each device and whether the difference between stressed and non-stressed values were statistically significant are provided below.
Sensor2

sensor3sensor4

For a given index, combining the average values of more than one stress made it more difficult to ascertain whether a plant was stressed and, if so, which stress was being applied.

Without pulling the entirety of the article here, an example of this difficulty is given. Where two different types of stress resulted in an increase of the index value for one stress and a decrease in the index value for the other stress, determining whether one or the other stress is present is easy – if the value goes down compared to the control, then the individual stressor that causes the index to be lower is present, and if the value is higher than in the control then obviously the stressor that increases the value is present. However, if both stressors are present then the value given will be somewhere in between the two extremes and can be difficult to distinguish from the control. A graph of the values of the FLAV index from the Multiplex sensor reading nitrogen deficiency and water shortage is given as an example of this difficulty.

sensor5

From Figure 7 of article (“-” = not stressed, “+” = stressed). Using the FLAV index for water and nitrogen monitoring, the unstressed plant gives the lowest reading, the water stress only treatment gives the highest reading, the nitrogen stress only treatment gives a reading only slightly higher than the unstressed treatment, and the treatment with both types of stress gives a reading roughly half way in between the unstressed and the water stress only treatment.

The difficulty caused by this is that, pretending you were the farmer in Dr Hickey’s story, it may be difficult for the app on his phone to differentiate between a nitrogen stressed crop, an unstressed crop and a crop with both nitrogen and water stress. The lack of precise diagnosis results in the lack of precise management.

What was also clear from the data obtained was that an index value for an unstressed plant at one particular stage of development can be identical with the value for a stressed plant at a different stage of development. The result is an added complexity in differentiating between a stressed and unstressed crop as a function of growing time compared to a stress that can be differentiated from a control plant at the same stage of growth.

Conclusion

The experiment shows that there are a number of current indexing methods of differentiating between a plant faced with a particular stress to an unstressed plant. However, for some indices the stressed and unstressed plants need to be at the same growth stage in order for the results to be of use. Further, while some indices are able to show the presence of one type of stress when other types of stress are also present, the interplay between the effect of different stressors can make detecting a stress or identifying whether one ore more particular stressors are present difficult.

For the time being, developing a set of standard readings over a range of indices for unstressed and differently stressed species at different growth stages is required. The standards can be incorporated into sensors that may use either complex algorithms to determine whether a plant is stressed and, based on the deviation of the reading to known standards for a range of types of stress, evaluate the likely type or types of stress present. Alternatively, a sensor using a variety of indicies which can each individually distinguish a different type of stress and deviation from a set standard may result in more accurate and dynamic sensing system.

We are still quite a way from Dr Hickey’s vision, but the technology and the methods to distinguish different types of crop stress already exists. Its not hard to envision a breakthrough piece of technology becoming available soon, and harder not to imagine that this type of technology will soon be incorporated into automated precision agriculture such as the Farmbot and indoor agricultural systems.

Plant Artificial Chromosomes

If you leave to one side the public acceptance of genetically modified crops as a means to overcome constraints on agriculture, a significant problem with genetically engineered crops is that few genes have been successfully transferred and expressed in commercial crops and most only contain one gene addressing one particular issue eg herbicide resistance.

Although this improvement has resulted in considerable gains in productivity, yield, sustainable chemical use and the like, research into other genes that confer advantages like resistance to drought and salinity stress are making real the possibility that we could benefit from the ability to transfer multiple genes into a crop with confidence of inheritance and the ability to swap in and swap out different genes.

For multiple genes to be inserted into a chromosome, directing both genes to be located in close proximity to each other in order for the gene set to passed to subsequent generations is a tool still under development. Even so, the randomness of meiosis can disrupt our best work.

Could the development of Plant Artificial Chromosomes (“PACs”) be a means to overcome these issues?

Plant Artificial Chromosomes

A review article in Plant Biotechnology Journal suggests that recent advances in genetic engineering such as specific site-directed breaking of DNA with CRISPR/Cas9 systems and subsequent homologous recombination processes could be combined with PACs to construct synthetic chromosomes conferring a stack of advantageous genes into crops..

PACs, or ‘minichromosomes’, are described in the paper as “super vectors for foreign gene organisation, expression and manipulation”, with properties such as:

  1. they are small with few genes of their own, leaving them capable of accepting large gene inserts;
  2. they are separate chromosomes to the plant’s, resulting in minimal interference with and from the plant’s own genetic material;
  3. they are stable during mitosis and meiosis, leading to their stability through growth and into subsequent generations;
  4. they can be constructed to allow subsequent manipulation whilst in the plant.

Construction

There are two methods of constructing PACs, the first being to clone the essential components like a centromere, origin of replication and telomeres, assembly the gene outside the plant cell and then transfer them into the plant cell. However, this approach is yet to be demonstrated unequivocally and plant centromeric repeats are species specific, adding a further complexity to their use at all.

A second method called ‘telomere-mediated chromosomal truncation’ however has been demonstrated as a way of creating minichromosomes in crops such as maise and rice. The process works on past observations that if telomere repeat sequences, conserved throughout most plant species, is inserted in to a plant chromosome, the chromosome will truncate itself by seeding new telomeres at the site of transformation.

Telomere_mediated_minichromosome_production

Telomere-Mediated Chromosomal Truncation. Credit DWilliams4, Wikipedia.

Although this truncation process has been known for some time, this review talks-up the potential use of the tool in conjunction with site specific engineering tools that have recently been developed. These tools can direct sites of truncation with precision and create minichromosomes that can be easily re-engineered with stacks of genes covering a number of traits with a level of stability that cant be achieved if the stack were inserted directly into the genome. Further, unlike insertions into the genome, inserting an artificial genome into a crop avoids linkage drag, a detrimental process where the inserted gene is linked to a deleterious gene on the chromosome that, when expressed, harms the plant.

PACs and the recent advances in site specific genetic engineering tools

Producing these minichromosomes currently requires the generation and screening of numerous plants to find those successfully transformed. Tools such as CRISPR/Cas9 can improve our productivity given they can

  • direct the insertion of telomere sequences to specific sites on the chromosome to be transformed;
  • simplify the process of removing the selectable marker genes from the chromosome after the screening process (to free up more room on the chromosome, avoid antibiotic resistance genes being expressed in crop or avoid us having green fluorescing pea plants!); and
  • easily add, remove or change single genes or stacks of genes either after creation or whilst in the plant they are desired to be expressed in.

The article discusses previous research performed by the authors demonstrating this last point. The selectable marker gene (SMG) and the Gene of Interest (GOI) in a particular minichromosome were removed and replaced by those on a donor plasmid using specifically added restriction sites on the minichromsome and homologous recombination.

pbi12466-fig-0002

Site specific recombination swapping out SMG1 and GOI1 for SMG2 and GOI2 in a Plant Artificial Chromosome. Reproduced from the article.

Insertion in to recipient plant genomes

PACs, after being generated in plants, can be cloned in vitro and transferred back into plants of the same species they were generated in. A different method, fusing protoplasts of the recipient plant to a protoplast containing the PAC is suggested as way of overcoming this species specificity and allowing the insertion of the minichromosome into alien species.

Conclusion

Plant Artificial Chromosomes combined with the advanced genetic engineering tools could be a pathway to stable, inheritable stacks of traits into crops with the ability to change the required genes as needed. As we expand our catalogue of genes conferring advantages on crops through research, PACs may start being used in research to test combinations of genes and stable products specifically tailored to particular environments may see general application.