In part 3 of our series on plant synthetic biology, Orlando de Lange (@) of The New Leaf blog introduces how synbio approaches are being used to develop novel disease resistant crops, overcoming some of the challenges faced by monoculture farming.
The King’s man
In 1970 an unassuming American man with greying hair and large spectacles stood before the King of Norway and was awarded the Nobel Peace Prize. This prize of international renown is set aside for individuals who have made the greatest possible contribution to peace among nations. Who was the king’s man in 1970? A president, a diplomat, a great writer, perhaps? No, he was crop scientist and plant breeder Norman Borlaug, founding father of the Green Revolution.
The Green Revolution was the most intense and best-publicized episode in the agricultural shift that has transformed agriculture across most of the world from a craft to a more industrial process with a clear division between farmers growing crops, and breeders, guided by the latest science, providing seed.
This largely ignored agricultural revolution has arguably kept the worlds population fed and fueled economic growth, but it has not been without its costs or its critics. Part of the process has been the moved from farmers growing landraces, mixed seed with a diverse genetic make up, to monocultures, where every plant is identical. A monoculture promises an even crop and is far easier to breed, but it may have left crops at greater risk of pathogen attack, because if one plant is susceptible all are.
One response to the problematic heritage of the Green Revolution is a growing opposition to science-led breeding and a return to pre-industrial methods. Whilst there is definitely a lot to be learnt from traditional methods I share the wish of many not to throw the baby out with the bath water but instead to invest in making our crops smarter. And in particular I’m going to explore the role that synthetic biology (synbio) could play in this process.
R-genes: the arsenal of resistance breeding
Plants have no antibodies nor do they have a circulatory system allowing specialized immune cells to guard the rest. Each plant cell is a fortress, with those at the outer layers of leaf, stem and root particularly vital to the defense of the realm. Cell walls can be thickened at these layers to provide generalized defenses, but most defense responses are only unleashed once a pathogen is detected. The first layer of defense lies in the cell membrane, where receptors lie ready to pick up common molecular signals produced by pathogens. Pieces of bacterial flagellum are the best-studied example. These membrane receptors trigger a low level response that is good at repelling most invaders. To be successful the invading pathogen disables these defenses by injecting proteins called effectors, which will interfere with the plant immune system. However, plants in turn possess intra-cellular receptors that actually detect the very effectors being delivered by the pathogens to disable the first line of defenses. Pretty clever, right? The nice thing is that all the downstream signaling pieces are conserved; all you need is a specific receptor to make it work against a particular pest of interest.
Receptors on the surface of the plant stand ready to detect invaders and trigger defense responses. Detection can occur at the cell surface or inside the cytoplasm, in either case the downstream signaling processes are conserved and can be considered a ‘black box’ by the breeder, who only needs to interest herself in introducing more receptors into the crop of interest.
The intracellular receptors were noticed as dominant resistance traits by breeders, and termed R-genes, long before the molecular mechanisms of how they function were actually known. If you find some variety or natural relative of the crop your interested that has a certain R-gene you just have to move it across through careful hybridization and hey presto you have a resistant crop. That is until the pathogens find a way to evade the R-gene, which always happens; as so vividly explored by Matt Ridley in The Red Queen, in the game of disease resistance sometimes you have to run as fast as you can just to stay in one place.
Breeding for R-genes has been part of plant breeding for decades, and is still going strong. There is a huge natural diversity of R-genes to select from, and a recent publication describes a new method to harness chemical mutagenesis and next generation DNA sequencing to speed up the process of R-gene discovery (Steuernagel et al., 2016)
The wonderfully modular nature of R-genes has meant that design-build-test cycles, mathematical modelling and complex DNA circuit assembly, the hallmarks of synthetic biology, are unnecessary for working with them. And it’s been pretty successful so far, so why bring synbio into the mix?
Of course R-gene breeding is not the full extent of what has been achieved with conventional methods. I would love to go on about the myriad approaches taken with plant breeding and ‘conventional’ GM, but I need to get to synbio. I would love to delve into the exciting world of homology induced gene silencing that harnesses the power of RNA silencing to defend against viruses and many fungi, but there just isn’t room. Still, if you’d like to read more about that, start with the story of how it saved the Hawaiin papaya industry.
Rise of the plant machines
Conventional methods of plant breeding and insertion of transgenes without synthetic biology can be very powerful. What then is the added value of synbio in all of this?
Systems biology approaches have shown in great detail the extent to which the plant gene expression is reprogrammed during a defense response (Lewis et al., 2015). Thus being able to tune transcriptional flux should be a good way to tune defense response, and clever ways of controlling gene expression is a core competency of synbio.
I’ve mentioned already that the plant needs to detect invading pathogens with a set of receptors, and that this information needs to be passed downstream, ending in gene expression changes that trigger a defense response. But while the plants are busily trying to integrate information from their various receptors the pathogens are not sitting idly by. It is vital to a successful invasion that the pathogen block or subvert this process. This is generally achieved in the form of effector proteins that are injected directly into the plant cell where they bind, break and bamboozle host-signaling proteins. Synthetic biology could be put to good use producing orthologous signaling pathways for the plant immune system. That is to say pathways that receive the same inputs and produce the same outputs but with all the steps in between carried out by synthetic proteins, keeping them safe from interference from invaders.
Successful pathogens secrete numerous effector proteins that bind and inactivate defense signaling components (left). An orthologous receptor and signaling pathway could overcome this (right).
Little has been achieved in this direction so far, though the wealth of tools for creating programmable transcription factors, such as TAL effectors and CRISPR, is sure to be a boon. And there is perhaps a precedent in the first truly plant synbio project, the creation of modified cress plants able to detect TnT in the soil via a synthetic signaling pathway (Antunes et al., 2011).
Sentinel systems are another way that synthetic biology could boost the power of crops to defend themselves. Allowing crops to share information with one another in the field about an oncoming pathogen attack.
In a generalized sentinel system the natural pathogen detection system is hooked up to production of a synthetic system for production of a volatile chemical. That chemical is picked up by a synthetic receptor in other plants, triggering natural defenses. The sentinel and receptor parts could all be in all plants or these functions could be separated.
This has been proposed in a recent article, from Rothamsted research in the UK, where they have already invested significantly into the production of transgenic plants that secrete a pheromone making them less attractive to insect pests (Bruce et al., 2015). On the detection side, the plant synbio group of Charles Stewart Neal at Georgia have developed tobacco plants with leaves that change colour during pathogen attack (Liu et al., 2013). These two processes of detection and chemical release have yet to be hooked up. If they were hooked up and additionally that chemical triggered could be picked up in other plants in the field, you would have a really powerful sentinel system. This general concept has been explored with the plant hormone auxin produced and detected by a synthetic sentinel system in populations of yeast cells (Khakhar et al., 2015).
Another distinctly synbio contribution would be the use of multi-component systems to precisely control expression of defense genes. A notable example that sticks in my mind was the recent product between a plant synbio lab at UC Berkeley and a plant pathology lab at UC Davis (Gonzalez et al., 2015). They used a bi-partite inducible system to achieve a tightly-inducible artificial immune response circuit in plants. They placed expression of an R-gene under chemically inducible control, combining two independent control pathways to keep R‑gene expression off until the chemical is added.
Generally, transgenes introduced into plants are always on or are driven from a chemically inducible promoter. Such inducible promoters tend to be leaky. If the gene triggers a severe defense response leaky expression can be very costly for crop yield. By introducing a mechanism for the same chemical to control both the promoter and stability of transcripts expression of the gene is brought under control. This concept was explored by Gonzalez et al.
The long and winding road
Clearly most of the ideas presented here are still in the early R & D stage, and it is far from clear what practical impact they might have for crop development. It must be admitted that the jury is still out on what the added value of plant synthetic biology will be in improved disease resistance. I’ve placed my bets along with my time and energy into this enterprise. And no I don’t expect it to solve all problems or make other approaches redundant, but I do hope that the engineering spirit, expanding the profile of resistant crop varieties, will keep the promise of the Green Revolution delivering for future generations.
References for your reading pleasure
Antunes MS, Morey KJ, Smith JJ, Albrecht KD, Bowen T a, Zdunek JK, Troupe JF, Cuneo MJ, Webb CT, Hellinga HW, et al. 2011. Programmable ligand detection system in plants through a synthetic signal transduction pathway. PloS one 6: e16292.
Bruce TJA, Aradottir GI, Smart LE, Martin JL, Caulfield JC, Doherty A, Sparks CA, Woodcock CM, Birkett MA, Napier JA, et al. 2015. The first crop plant genetically engineered to release an insect pheromone for defence. Scientific Reports 5: 11183.
Gonzalez TL, Liang Y, Nguyen BN, Staskawicz BJ, Loqué D, Hammond MC. 2015. Tight regulation of plant immune responses by combining promoter and suicide exon elements. Nucleic Acids Research: gkv655.
Khakhar A, Bolten NJ, Nemhauser J, Klavins E. 2015. Cell-cell communication in yeast using auxin biosynthesis and auxin responsive CRISPR transcription factors. ACS Synthetic Biology: 150623113028004.
Liu W, Mazarei M, Rudis MR, Fethe MH, Peng Y, Millwood RJ, Schoene G, Burris JN, Stewart CN. 2013. Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants. Plant biotechnology journal 11: 43–52.
Lewis, et al. 2015. Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000. 27: 3038–3064.
Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, Hatta A, Ayliffe M, Bariana H, Jones JDG, et al. 2016. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nature Biotechnology.
The header image was downloaded from flickr under the creative commons license, photograph ‘walk to Caxton 4 / Monoculture 3’ by Andrew Fogg