What is Green Canary?

A sentinel plant which warns of the presence of plant pathogens by producing a visible signal.

Abstract

Food security is a prominent challenge faced by the increasing global population. Currently about 40% of crop losses are due to pests and diseases. Our aim is to reduce crop losses whilst decreasing the use of agrochemicals, contributing to more sustainable and less environmentally damaging farming practices. Applying synthetic biology approaches, we aim to produce proof-of-concept, sentinel plants that will diagnose the presence of two pathogens, Xanthomonas oryzae and Xanthomonas campestris. These Green Canaries will produce a signal, visible to the naked eye output, within 48 hours of detecting the pathogen. This will allow growers to make appropriate agrochemical application before the diseases progress to symptomatic pathogenesis in neighbouring crops. Green Canaries will also allow scientists to gather epidemiological data about plant diseases. Furthermore, we are constructing a series of BioBricks that will allow Golden Gate assembly to assist cloning of transcriptional units within the iGEM standard. These important developments will aid future iGEM teams to work with plant chassis as well as utilise the Golden Gate parallel assembly method.

The Experiments

Initially we selected several promoters known to respond to plant pathogens. We chose PDF1.2, a promoter from ''Arabidopsis thaliana'' that is induced by the hormone methyl jasmonate, produced naturally by plants in response to various biotic stress. We also chose PR1, which responds to salicylic acid (another plant hormone produced in reposes to infection). Lastly, we identified two promoters from capsicum and rice plants that are induced by TALES (Ttranscriptional Activator Like Effectors). Effectors are small molecules secreted by specific plant pathogens, Xanthomonas oryzae and 'Xanthomonas campestris that enable the pathogen to invade. They bind to a very small region of their cognate promoter, inducing expression. We then selected several coding sequences that, when expressed in plants, might produce a visible signal. First we chose BaxI from mice, which is known to induce cell death. We also chose genes that would make the plants go white (de-green) by breaking down chlorophyll and chromoproteins to colour the leaves canary-yellow or deep-blue. Ultimately, we planned to make a plant that would de-green in the presence of any pathogen and then re-colour to a new colour that identified the specific pathogen it had sensed.

[insert Alistair's diagram of the leaf from the banner here!!!]

We planned to test our disease-responsive promoters by using them to drive expression of GFP, which we know is easy to detect in the leaves of our chassis. To test our visible signals, we used a well-characterised constitutive promoter. To express our circuits in plants we assembled them in 'E.coli then transferred them to a second chassis, Agrobacterium tumefaciens, which has the ability to transfer DNA into plant cells. We then injected the leaves of our plant chassis with cultures of 'A. tumefaciens' and monitored the plants for the expected signal. To avoid working directly with plant pathogens that require special permits, we painted the leaves with the appropriate plant hormone, (or simultaneously expressed the TALE protein) to test our inducible promoters.

Materials and Methods

We used GoldenGate cloning to assembly our constructs. This was very efficient as it allowed us to assemble a whole transcriptional unit (promoter, coding sequence and terminator) in a single step. We could then combine several transcriptional units into a multi-gene contract in a second step. We used Nicotiana benthamina as the plant chassis for testing our plant circuits. ''N. benthamiana'' is a widely used experimental plant from the solanaceous group of flowering plants that includes tomatoes, potatoes and capsicums. We chose it because it is possibly to obtain high-levels of transient expression in just a few days. Although this transient expression only lasts about a week, it is much quicker that making a stably integrating genes into a plant genome, which takes months!

Because we had to submit our parts to the registry in the iGEM shipping backbone, we also made some "flipper" plasmids. These plasmids "flip" Golden Gate MoClo parts into standard biobricks. The methods that we used for cloning and transfecting as well as other other useful protocols are given on our lab protocols page.

Results

The promoters that respond to plant hormones were successfully able to drive expression of GFP. Expression was strongly up-regulated in response to the hormone but a background expression level was also observed. This may have been cause by our transient delivery method, A. tumefaciens, which the plant recognises as a pathogen, even though the strain that we used is not capable of causing disease.

!![insert PDF1 and PR1 images and legends]!!

The promoters from Xanthomonas oryzae and Xanthomonas campestris, however, only induced expression of GFP in the presence of the corresponding TALE ,indicating that expression from this promoter was very tightly regulated.

!![insertBS3/AvrBS3 images and legend]!!

We were able to induce cell death using the coding sequence from the Bax1 gene from mice. This produced a lesion on the plant leaf where the A. tumefaciens carrying our circuit had been delivered. We were unable to see expression of chromoproteins in our plant chassis, even when expressed from a known strong, constitutive promoter. It may be that the colour was masked by the green chlorophyll. The next step would be to attempt to express the chromoproteins simultaneously with a de-greening circuit that would remove the chlorophyll, to to express them in the roots, which are devoid of chlorophyll.

!![insert BAX image and legend]!!

Conclusions and Future Work

We were able to express circuits in plants that were switched on or up-regulated in response to signals of pathogen invasion. We were able to demonstrate promoters that respond to general stress signals that could be used even when the sentinel was unable to diagnose the specific pathogen. We were also able to demonstrate very tightly regulated expression in response to a particular pathogens.

We conclude that our project is viable and that we would continue with our original plan to produce plants that induce a mild readout (e.g. de-green) in the presence of any pathogen. The second step would be to use the pathogen-specific promoters to induce a second signal to diagnose the specific pathogen. Once these circuits had been tested in the transient system, we would make stably transformed plants that could be tested with the actual pathogens in laboratory and, if successful and permits were granted, in field conditions.

Finally, we would investigate the switches that would shut off the circuit as presently the Green Canaries are single use, unable to return to green once they have encountered a pathogen.