Synbio in Space!

The challenges of surviving in space (with or without hostile enemies intent on dominating the universe) has made and will continue for a long time to make for a good sci-fi story. The resourcefulness required to survive in the vast emptiness between (and on) planets also forces us think deeply about better ways of propelling, feeding, housing and medicating ourselves. The cost of launching extra weight into space plus the limited lifespan of necessities such as food and medicine leaves us with complex questions about how to sustain ourselves as we look to explore more of our solar system and, hopefully one day, beyond it.

A 2015 review in the Journal of the Royal Society Interface considered a number of space missions and analysed how synthetic biology may assist those missions in the four resource areas mentioned above; propellant, food, habitat and medication. Of most interest, given the significance of the challenges presented, is a six-person, 916 day return voyage to Mars which would include 420 days of traveling there and back plus a 496 day stay on the red planet.

What are we going to have to do to be real-life Martians?

Food

Although one of the shorter sections of the paper, how we will advance food production being the focus of this blog means that we will put the section of food production in space up-front and centre.

Based on astronauts living on the International Space Station, living in space requires 1.83kg of food per crew member per day. As a result, the six person Mars mission requires around 10 tonnes of food that needs to be shipped in order to sustain a mission if all of it was shipped from Earth although more accurate assessments of food amounts required to be produced whilst on mission have a lower estimate of 4.5 to 5 tonnes.

So the ability to produce food using available elements and compounds whilst away from the Earth is a significant problem. And the most accessible food production option is to use photosynthesizing bacteria and plants. Producing food using bacteria has the most significant weight savings, with Spirulina (a nutritional biomass made from either Arthrospira platensis or Arthrospira maximum) being promoted in earlier papers as a suitable option to producing food in space compared to attempting to grow and harvest plants. According to those previous papers, it is possible to produce 5.271kg of Spirulina per day using three 2,000 litre bioreactors. This rate of production would sustain the crew for the entirety of their stay on Mars and for the return to Earth and save a significant amount of launch mass.

Spirulina

Spirulina powder.

So how can synthetic biology help if Spirulina is already producible in adequate amounts to sustain the majority of a Mars trip? If you can imagine eating the same substance every meal for 706 days straight, one problem is easy to identify. It is suggested in the paper that synbio efforts could be directed to diversifying the textures and the tastes produced by the bacteria so that a variety of ‘foods’ can be consumed by those on the Mars mission.

Increasing the rate that the bacteria is able to reproduce will also aid the weight reductions at lift off and increase the redundancy built into the rations of food for the trip. There are some reported increases in production rates reported in the paper, but, for example, whether increased photosynthetic efficiency can be engineered into the bacteria to increase production is a question common to current research efforts in earth-bound food production.

Finally, being able to increase the nutrient content in the food produced to aid general health and space-specific health problems will increase the chances of a successful mission.

Pharmaceuticals

The shelf-life of pharmaceuticals are shortened in space travel and at present the issues caused by this are solved by  sending more supply missions. Given the time and distance involved in a trip to Mars, such a solution has little practical use.

Synthetic biology has the ability to play a role in readily producing pharmaceutical substances during a mission to Mars. For example, acetaminophen (paracetamol) can be produced using a modified chromisate pathway in E. coli. Unfortunately, space travel doesn’t suit E.coli as the compounds it requires to survive are not readily available. But if an autotroph can be manipulated to contain the same pathway and produce the same substance, we may have a new way to produce the pharmaceutical with the restricted inputs. The cyanobacterium Synechocystis sp. PCC 6803 has been identified as a possible candidate that will survive on the inputs available in space and could host the appropriate pathway to produce acetaminophen.

F8.large

Figure 8 from article demonstrating the proposed pathway to create acetaminophen from Synechocystis sp. PCC 6803.

It can be imagined that with enough research we should be able to produce a range of pharmaceuticals or at least their components from the limited inputs available in space.

Fuel

The further we go and the more weight we carry, the greater the fuel required. Complicating the matter further, the more fuel we carry into space, the more fuel we require to lift the extra fuel off the ground. The problem of needing more fuel to lift even more fuel to be able to propel more weight into space is one that permeates through the problems of supplying enough food and equipment to astronauts traveling for long periods of time.

The possibility of producing fuel in space is limited, according to this paper, to using methanogens to create methane. The type of methanogen used in bioreactors effects the quality of methane gas created and the rate of production. Further, a number of methanogens are able to use the compounds, particularly CO2, created by the crew of a space mission.

Synthetic biology uses to address the problem of fuel production in space will likely focus on the production of methane by methanogens. Particularly, the ability to produce methane using the least amount of inputs and with the greatest nutrient recycling efficiencies will be key. Similarly, modification of cyanobacterial production of ethylene, an alternative propellant, for greater production efficiency is another possible use of synthetic biology to aid space flight.

Habitat Construction

Lastly, the ability of erect a habitat on Mars will require either the supply of material from Earth and/or the use of material available on Mars with equipment supplied from Earth. The proposed use of 3D printing to construct habitat allows the use of finer materials in an additive manufacturing process. But the possibility of biologically produced materials (biopolymers) provides a method of producing the base materials using inputs available away from the Earth and significantly reducing the launch mass of materials dedicated to habitat construction.

F7.large

Figure 7 from article demonstrating the shipping mass saved for habitat construction in missions to Mars (left) and the Moon (right).

Synthetic biology efforts focused on increasing the synthesis of biopolymer material per unit of bacteria will result in less equipment in the form of bioreactors being required to travel with the crew with the time for construction of the habitat being reduced as well.

Synbio in Space

At its most basic level, the quest to man a mission to Mars creates problems that must solved using the most efficient means possible. As a result, the use of synthetic biology to solve problems in the four areas listed above looks little further than what inputs are available in space and how they can be used to solve the problems spaceflight presents.

However, advances in modifying organisms to meet these challenges will likely lead to flow-on effects in the same areas here on a better-resourced planet. For example, should it be possible to create different textures and tastes in Spirulina in the confines of space then one would expect that it will be possible to use different inputs and different genetic circuits to create a variety of foods. Further, food produced should be more efficient, sustainable and economical than traditionally generated food.

Genetic Underpinnings of Heat Resistant Potatoes

Identifying the genetic basis of a particular trait is the building block on which molecular and transgenic breeding strategies build upon. How we connect the dots between a genotype and trait is the basis of a past post “How do we find genes related to traits? A review of bulked sample analysis”.

Despite only being behind rice and wheat as the most important crop worldwide, quantitative trait loci (QTLs) for heat tolerance in potatoes have not, until recently, been identified.

Potatoes are particularly sensitive to heat, with temperatures above the ideal maximum of 22°C resulting in significant decreases in growth and yield. The higher temperatures have a number of effects:

  1. Strongly suppresses tuberisation;
  2. Reduces the proportion of carbon assimilated for tuber starch;
  3. Reduces chlorophyll levels and CO2 fixation rates;
  4. Halts tuber dormancy resulting in early sprouting and secondary growths.

A recent study in the Plant Biotechnology Journal sought to identify QTLs for heat tolerance by comparing differences in gene expression and tuber yield between progeny of a biparental diploid potato (06H1) when grown at 22°C and 28°C.

Results

Temperature response analysis and gene identification

Single node cuttings were used as a method to quickly induce tuberisation of subject potato plants, allowing a faster assessment of tuber yield (see this article discussing its use – paywalled unfortunately). 170 genotypes from the 06H1 population were analysed using 6 nodal cuttings from each to assess tuber yield under 22°C and another 6 for each genotype assessed at 28°C. On average, tuber yield at the lower temperature (1.017 grams) was greater than for nodes grown at the higher temperature (0.285 grams). The difference in performance was used in combination with single nucleotide polymorphism (SNP) maps of the 06H1 genome to identify genetic markers which may be connected to the genes which alter expression rates at the two temperatures.

fresh weight tuber yield nodal cutting assay

Figure 1 from article. Comparison of tuber fresh weight yield when grown at 22°C and 28°C.

Statistical analysis of the differences in expression levels at each marker found 3 markers which may be connected to the difference in phenotype. Using the Potato Genome Browser the researchers browsed the list of genes connected to the markers for suitable gene candidates for further research. Of the three markers, one marker contained a possible gene candidate, that encoding Heat Shock Cognate 70, a gene that has been suggested as involved in the heat tolerance in cabbage and potato.

With a candidate gene identified, the gene was isolated at amplified for analysis of allelic differences within the gene, of which 4 were identified and named A1, A2, A3 and A4. Nodes of potatoes containing each the four alleles were assessed for tuber yield, with allele A2 having the greatest fresh weight at both 22°C and 28°C and combined A2A3 genotypes having the highest yield. Gene transcript levels were assessed in plants grown at both temperatures which showed that the HSc70 gene was upregulated in A2A3 plants but not in the A1A4 plants, while the HSc70 allele up-regulated in plants grown at the warmer temperature were all from the A2 allele.

Analysis of the amino acids of each of the alleles themselves found that the A2 and A3 proteins contained an additional C-terminal sequence of KIEEVD (thought to be involved in interactions with co-chaperones), while no other variations were unique in a single allele.

What was unique to the A2 allele was a difference in the promoter sequence where a run of ten TA repeats was found as opposed to a run of four TA repeats in the promoters of the other repeats. The researchers later hypothesize that the greater number of TA repeats may result in enhanced expression levels, a hypothesis based on previous research in yeast, although the specific effect of the repeat TA sequence is not known.

Confirming the effects of the HSc70 A2 allele

To test the A2 allele and its promoter sequence, the gene and upstream component was transferred into Nicotiana benthamiana plants and subjected to 45°C heat in a growth chamber. After 24 hours the expression of the HSc70 gene was 7 times greater than in the control plants and cell membrane injury in the transgenic plants were significantly lower.

The unique promoter was then targeted for further analysis. The A2 allele was put downstream of a series of promoters with TA repeats varying between four and ten times. After being exposed to the higher temperatures the level of HSc70 transcription was measured. Alleles with eight to ten TA repeats in the promoter showed statistically significant elevated transcript levels compared to alleles with four to six TA repeats in the promoter.

HSc70 expression levels

Figure 6 from article. a) HSc70 expression levels for promoters containing 4, 6, 8 and 10 TA repeats and b) cell membrane injury percentage for plants containing the same numbers of TA repeats.

A2 allele-containing plants with significantly increased expression under 28°C heat were selected for analysis under 40°C conditions for 24 hours in comparison to wild-type plants. Extracts of plants after 4 hours at this temperature showed HSc70 expression being enhanced by up to 50 times more than in control plants. However, after 24 hours the transcript levels fell back to that seen prior to being subjected to the extreme heat. The A2 allele-containing lines has significantly less cell membrane damage compared to the wild-type.

Finally, the ability of the A2 allele to protect tuber yield at elevated temperatures was tested. Using the same nodal cutting tuber yield assay as before, A2 allele containing plants were compared with wild Desiree potato plants when grown at 20°C showed little difference in tuber yield. However, at 28°C the Desiree plants reduced yield by 75% compared to yield at 22°C. By comparison, transgenic lines had significantly lower yield reductions, with some lines have twice the fresh and dry weight yield as the wild type.

Conclusion

The identification of the heat shock cognate protein 70 within the potato genome and the quantification of the effects of the specific A2 allele demonstrates the significance of quality genome research, genome annotation and the ability of powerful molecular tools to seek out expression variances that allow us to pinpoint the basis of variances in phenotype. Coupled with well prepared and executed experiments, the development of these tools will continue to enable us to extract information that will assist us to assist crops and food production.

Whether it is through transgenic technology or molecular breeding, the identification of genes such as the one discovered in this study will assist food production meet the challenges of a changing climate.

Identifying the specific allele of the HSc70 protein that contributes to its resistance to the negative effects of elevated temperature is surely a big step into future-proofing the security of this important crop.

C4 Photosynthesis: Discovery, Understanding and Future Development

“The C4 pathway is one of the most complicated biochemical pathways likely to be placed in front of a university undergraduate, and attempts at C4 engineering have only served to reveal additional uncertainties about how the pathway operates, how it is regulated at the level of gene expression and post-translationally, and some of the physical properties of the anatomical specialization of Kranz C4 leaves.”

Robert Furbank (2016) “Walking the C4 pathway: past, present, and future”, Journal of Experimental Botany, vol. 67, no. 14,  pp. 4057 – 4066

Whilst writing our post titled “C4 Photosynthesis Evolution: Why some intermediates may not assist our understanding” we came across a 2016 paper written by Professor Furbank covering the discovery of C4 photosynthesis, the research and stories behind our understanding of this important photosynthetic trait, the gaps in our knowledge that remain to be filled and where the research may lead moving forward.

Readers of this blog may recall that Professor Furbank generously gave us the time to explain the problems in a research article proposing that C4 photosynthesis already existed in wheat which greatly assisted our updated post on the issue. He has also recently been a guest on the Australian Broadcasting Corporation’s “The Science Show” talking about C4 photosynthesis and the positive effects it could have on our food security (listen here).

Discovery of an important trait

Prior to the biochemical description of C4 photosynthesis detailed by Hal Hatch and Roger Slack in 1966, the special properties of particular plants that were later understood to use C4 photosynthesis had been documented since the turn of the 20th century. Professor Furbank’s review lists the important research articles which led the way to our current understanding, such as the discovery of Kranz anatomy, plants increased water use efficiency and increased photosynthetic rates compared other plants.

The separation between the collection and concentration of CO2 in mesophyll cells before pumping the CO2 to bundle sheath cells, where it is reduced and decarboxylated, is the most important and distinguishing trait used to differentiate C4 photosynthesis from other forms of photosynthesis. The concentration of CO2 allows Rubsico to operate at greater efficiency, reducing the competition for the active site of the enzyme that normally plays out between CO2 and O2 in non-C4 species, benefiting the growth rate and resource efficiency of the plant.

Furbank - C4 diagram

Figure from article demonstrating the Kranz anatomy (ring of purple bundle sheath (BS) cells and mesophyll cells (M) within the plant tissue. The numbers on the right of the figure denote; 1. carbonic anhydrase, 2. PEP carboxylase, 3. malate dehydrogenase, 4. NADP-malic enzyme, 5. pyruvate orthophosphate dikinase.

Unpublished data, criticisms of legitimate research findings and unnoticed publications of important research in obscure or foreign-language journals hampered early progress in discovering C4 photosynthesis until the seminal work of Hatch and Slack (who we are proud to learn are Australian scientists) in 1966.

Hatch and Slack’s 1966 research paper used 14C-labelled CO2 to study the phenomenon, applying a pulse of labeled CO2 and then illuminating the subject leaf to follow the progression of the labeled CO2 as it made its way through the photosynthetic cycle. This method is commonly known as a ‘pulse-chase’ analysis. The idea for the experiment came from similar earlier experiments conducted previously by other researchers but which had provided results that lacked certainty in the identification of the labeled compound. Whilst having chatting over a beer during the Australian Biochemical Society conference held in Hobart in 1965, Hatch and Slack decided to try their hand at replicating these earlier experiments in a manner that would reduce or remove the uncertainty contained within the earlier results and shed further light on the strange data that was being produced in these earlier works.

In something reminiscent of the oversight of Gregor Mendel’s early research on genetics in pea plants, it was discovered in the late 1960’s that a Russian researcher named Yuri Karpilov had reported similar findings from his research as that obtained by Hatch and Slack in an article he published in 1960 in a little known journal.

What wasn’t grasped at the time was that this newly discovered pathway resulted in a pool of CO2 concentrated in some cells while Rubsico, an enzyme difficult to extract, was present and operating in the separate bundle sheath cells. This discovery was slowly uncovered in a series of papers in the late 1960s and early 1970s, as was the importance of the concentration of CO2 in reducing photorespiration and its link to improved plant performance.

As our methods of isolating and probing at different cells have improved, so has our knowledge of the variety of biochemical pathways that exist within the C4 pathway. However, we are still grasping at which of these pathways provides the greatest flux of metabolites. When we acquire this understanding it will focus our attention to those pathways likely to result in the greatest improvements in plant performance and harness our attempts to engineer C4 photosynthesis in important crops.

Recent research

Since the significant increase in our knowledge of this important photosynthetic pathway around the time of Hatch and Slack’s 1966 paper, genetic transformation of plants and the increase in our ability to extract and subject plant data to bioinformatics techniques has continued to push research forward. The use of green foxtail millet as a C4 model grass by the biofuels community has allowed for the development of transformation protocols to help probe deeper into the genetics, transriptomics and protein products that form the basis of this system. Further, this information is crucial to our engineering attempts aimed at improving crop productivity.

Similarly, next-generation sequencing (NGS) has revolutionized research probing the evolution of C4 photosynthesis and how they may have evolved from their C3 ancestors. Probing the differences between the genetics and gene transcripts between the differentiated cells is steadily helping us understand and pinpoint which genes are absolutely necessary for a functioning C4 pathway. Analysis of C4 meristem tissue as it grows and the adjustment of gene transcription through this growth is also assisting in deciphering what gene regulation is required if we are to successfully install C4 photosynthesis into important C3 crops.

Where to now?

There is still important research to be done and discoveries to be made in C4 photosynthesis research.

Quantifying the amount of fixed CO2 that is leaked from mesophyll cells, under what conditions greater or lesser amounts of CO2 is leaked and what physical properties of the bundle sheath cells may change the rate of flux are all questions that remain to be answered if we are to successfully engineer the system into C3 crops. As with many areas of research, new tools to extract the information and avoid the uncertainty that plagues the more indirect methods of investigation we are currently limited to will greatly assist the research.

Further information about the positioning of important organelles within the cells and the ratios of bundle sheath cells to mesophyll cells are yet to be understood and which could prove to hamper engineering efforts.

Finally, despite the significant leap in the genetic and transciptomic knowledge we have seen of recent times, there is still only a developing understanding of gene regulation in the growing plant tissue to ensure the separation of the two parts of the carbon fixation and decarboxylation process within the tissue. Important transcription elements specific to this demarcation of activity between the cells are still being investigated and isolated.

The future

Significant research effort is being funded to enable the discovery of these important missing pieces of information and into attempting to transfer C4 photosynthesis into important C3 crops. The C4 Rice Project, funded by the Bill and Melinda Gates Foundation, is one such effort that looks very promising.

So, can we supercharge a VW to perform like a Porsche? The progress being made and the dedication to the problem must lead us to a sense of optimism that it will happen within our lifetimes.

C4 Photosynthesis Evolution: why some intermediates may not assist our understanding

C4 photosynthesis, a form of photosynthesis which converts CO2 with greater efficiency than the more common C3 variant, is widely seen as a promising trait which could be used to improve crop growth. This is especially so with the looming concerns over the effects of climate change on food production.

We have previously written about C4 photosynthesis, how plant anatomy and chemistry differs between the two forms of photosynthesis and the possibility of being able to engineer C4 photosynthesis into crop plants.

Further, we have written about an article that suggested that certain parts of wheat plants may already perform C4 photosynthesis and, in a subsequent piece, we wrote about two rebuttals to that assertion.

In the aforementioned article regarding engineering C4 photosynthesis, the use of intermediate crops as a means to assist us to discover how some C3 crops have been able to evolve into C4 crops has been suggested as a means of furthering our knowledge of C4 evolution and to assist us to convert important C3 crops into more efficient light converters.

The article we are writing about this week warns researchers that the use of C3-C4 intermediate crops for such a purpose may not hold the answers to the questions we are asking.

C3-C4 Intermediates

This report recently published in the New Phytologist starts with a short description of C4 photosynthesis and its discovery. Two of these early papers on C4 photosynthesis evolution suggested that plants containing a photosynthetic system which appears to be an intermediate between C3 and C4 photosynthesis may be a proxy for the evolution of C4 photosynthesis.

However, the authors of this paper also point out that it is possible that these intermediates may, instead of depicting an plant in the throws of an evolutionary conversion, be a hybrid plant between two closely related species where one parent uses C3 photosynthesis and the other uses C4 photosynthesis.

The problem this raises is that research of C4 evolution may be misdirected if the intermediate under study is not truly undergoing this process.

To examine this possibility, the authors of the paper looked at the evidence of both natural and experimentally generated C3-C4 hybrids and described the categories of intermediates that have been discovered.

Categories of Intermediates

Intermediates of photosynthesis were categorised in a previous article by Sage et al (2014) which distinguished four categories of intermediate:

  1. Proto-Kranz: the plant contains bundle sheath cells or Kranz-like cells which have some similar traits to C4 species such as being larger and orientated in a centripetal position.
  2. C2 type 1: contains the same characteristics as proto-Kranz cells, but which also shuttles glycine produced in the mesophyll cells during photorespiration to the Kranz-like cells where its is decarboxylated and the photorespired CO2 is concentrated and partially refixed by Rubisco. This refixation of CO2 that would otherwise be lost in photorespiration in C3 and proto-Kranz photorespiratory cells improves efficiency.
  3. C2 type 2: the same as C3 type 1 save that the refixation of CO2 appears to rely largely on enzymes normally found in C4 photosynthesis, although their expression is lower than that found in C4 and C4-like species. C2 type 1 cells, by comparison, rely largely on a glycine shuttle and C4 enzymes are much less active.
  4. C4-like: a rarely found intermediate that shows increased activity of enzymes used in the C4 photosynthetic cycle and reduced reliance on C3-active enzymes.

Adapted from the Sage article is a table which contains a comprehensive list of plant species that fit within each criteria and under which category they sit. The Sage article also provides the below diagram illustrating the evolution from C3 to C4 photosynthesis.

C3 evolution

Figure from Sage et al (2014) showing the differences between C3, C4 and each intermediate category of photosynthesis. The figure in the article contains a lengthy description of the illustration (and the article is freely available).

The possibility of photosynthetic hybrids

Hybrid intermediates were experimentally attempted prior to the discovery of naturally occurring hybrids and have since been successfully created experimentally in a number of closely related species. Experimentally created hybrids have resulted in a mix of intermediate photosynthetic systems that were scattered throughout the spectrum between C3 and C4 photosynthesis. Intermediates generated would contain some components that related to C4 photosynthetic systems but would retain some C3 properties and similar properties would be passed in varying degrees to the segregating populations generated from the experimental hybrid.

In all, the previous research demonstrated the hybrids (C3 x C4, C3 x C3-C4, and C4 x C3-C4) could be classified in accordance with the criteria contained in the Sage paper to classify naturally occurring hybrids. The problem this causes, the authors suggest, is that the hybrids cannot be differentiated from true evolutionary intermediates on the basis of the Sage criteria.

The Naturally Occurring Hybrids

Naturally occurring photosynthetic hybrids have been considered likely in a number of papers where lineages have contained C3, C4 and some intermediate species. The article provides examples of a number of papers which observed or considered likely that hybridisation is the cause of the intermediate species.

The concern raised by the authors is that studies looking at phylogenetic trees of such lineages rarely look specifically for hybridisation as a cause of the photosynthetic intermediates. Therefore, the cause of the identified intermediates may be mistaken as being due to evolutionary changes rather than being due to hybridisation.

Conclusion

On the basis of the similarity between experimentally created photosynthetic hybrids and natural intermediates and their inability to be distinguished from each other using the Sage criteria, the authors believe that greater care should be taken when considering which intermediate is to be used to study C4 evolution. Taking the point further, actively excluding the possibility of the intermediate under study being of hybrid rather than evolutionary basis would be best practice before embarking on its use for the purpose of studying how C4 may have evolved and linking it to research of how we may create a similar evolution.

Finally, although hybrids may not be good candidates for evolutionary study, such hybrids aren’t without any worth. The hybrids experimentally created and discussed in the papers cited by these authors could be themselves be the basis of further research that could assist us to improve photosynthetic efficiency in food crops.

Abscisic Acid and Plant Stress Tolerance – A Mini Review

Frontiers in Plant Science recently collaborated with two other journals in putting together a number of articles related to the research topic “Mechanisms of abiotic stress responses and tolerance in plants: physiological, biochemical and molecular interventions”. As part of the topic a mini review was published reviewing what we know about abscisic acid signalling and abiotic stress tolerance, what further information we need to acquire and what we may be able to do with the knowledge for future food production.

Introduction

Biological chemistry is mind-blowingly complex yet crucial to developing our understanding how organisms grow, reproduce, protect themselves and die, amongst others. How plants, stuck in one spot and forced to deal with whatever conditions may befall them, have the ability to respond to biotic and abiotic stress is important to know if we are going to be able to adapt crops to changing environmental conditions and improve food production security and efficiency.

Abscisic Acid (“ABA”) is one of a small number of phytohormones that play a significant role in how plants develop and grow. The amount of ABA produced by a plant is known to be affected by extracellular stresses and is considered important in assisting plants to adapt to and withstand abiotic stresses when they arise as well as being involved in such processes as seed development.

355px-Abscisic_acid.svg

Abscisic Acid phytohormone. Credit: Wikipedia

ABA is produced in all parts of plants but accumulate in roots and terminal buds of growing plants, forming the basis of the hypotheses that they play a role in communication between the two ends of the plant during periods of stress. In the context of stress response, it plays a crucial role in assisting the plant in times of dehydration, thermal stress, strong UV absorption and uptake of heavy metals. The paper cites previous studies which have shown that mutant plants lacking ABA biosynthesis show decreased tolerance to fluctuations in their environments compared to the wild-type, while overexpressing plants have increased resilience.

ABA table 1

Table 1 from article. ABA regulation of stress effects on plants.

Drought Stress

Water deficiency in plants results in an osmotic stress in plant cells which cause cell desiccation and a resistance to water uptake. ABA levels rise in these conditions with one study showing that the levels of ABA under drought stress can be 40 times higher than when the plant isn’t stressed. Studies involving transgenic plants overexpressing ABA genes suggest that the hormone assists plants by maintaining membrane stability and causing changes in metabolite accumulation when water availability is low. Further, ABA is believed to be involved in stomatal closure during drought periods when water retention is of paramount importance to the plant. Studies which have applied ABA exogenously have also been shown to result in increased resistance to drought stress.

Drought stress can also result in the production of ethylene, a chemical which is commonly used in agricultural industries to bring on fruit ripening due to its effect of bringing on early senescence. However, early senescence in crops not yet at the stage of being useful for food production is problematic. Studies have shown that increased ABA production reduced ethylene production associated with this type of stress and others such as UV stress (see below).

Whilst ABA acts to protect the plant from water stress, it is generally agreed that the protective mechanisms initiated also result in decreased plant growth above that caused by the water stress itself. Plants subjected to drought stress with purposefully lowered ABA levels were demonstrated to have reduced growth. Further, plant shoot growth may be inhibited by ABA even when appropriately watered.

Heavy Metal Stress

Heavy metals such as cadmium, iron, mercury, copper and chromium, which are generated as a result of human activities and which can pollute agricultural land, can be taken up by plants leading to a toxicity caused by the reactivity of these metals. Reacting with cellular components results in energy loss, lowered photosynthetic capacity, reduced growth and early death.

Heavy metal absorbance results in increases in ABA levels. Testing of cadmium-tolerant and cadmium-susceptible rice cultivars has shown that the ABA level of the tolerant cultivar exceeded that of the susceptible one. Further, the application of exogenous ABA to the susceptible cultivar increased resistance to cadmium.

Chromium and copper can cause intracellular stress and resultant production of oxidants and free radicals. The uptake of these metals in plants has also been shown to result in ABA biosynthesis. Conversely, reduced intake of heavy metals leads to a reduction in the levels of ABA, indicating that ABA plays some role in the stress response.

UV Stress

Ultraviolet radiation absorbed by plants can result in the creation of reactive oxygen species within the plant, resulting in damage to cellular components which leads to retardation in plant development and growth.

Ethylene production increases with UV-B radiation exposure, causing early senescence in plant tissues. The presence of ABA in the plant tissue reduces ethylene production and thereby reduces the detrimental effects of the radiation exposure, a discussed above in relation to drought stress. In fact, research has demonstrated that a plant under one form of stress will also have an increased resistance to other forms of stress such as in the case drought stress and ultraviolet radiation stress.

The importance of Abscisic Acid

As can be taken from the examples provided, the production of ABA has been linked to the response of plants to such stresses as radiation, drought and heavy metal uptake. In some cases, research has linked stress tolerance to the presence of ABA and susceptibility to the lack of ABA present. However, the precise mechanism of action is not well understood.

The article also provides some background into the production of ABA and hints that, while we know many of the genes and proteins involved in its pathway to production we are still missing a complete understand of a number of steps and how the many branches of the ABA reaction link and/or work together.

Conclusion

This mini-review helpfully outlines some of the research surrounding ABA involvement in stress tolerance and the importance of understanding this if we are to enhance our crops.

It must be said that the review was in parts difficult to read and understand, perhaps due to to some issues with a translation to English, and that there was some unnecessary repetitiveness through the piece.

However, it is a good starting point for further research into abiotic stress tolerance and future food production.

A New Method to Accurately Measure Transgene Copy Number in Crops

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.

dPCR

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.

Conclusion

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-

 

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.

Activation and Repression of Transcription Factors via a Synthetic Construct in Arabidopsis thaliana

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.

Gene Transcription

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.

cosri-fig-1a

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.

cosri-fig-1b

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. thalianthat 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.

Conclusion

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.