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. 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''. The plant sentinels will produce a signal, visible to the naked eye output, within 48 hours of detecting the pathogen. The sentinels would allow growers to apply appropriate agrochemical application before the diseases progress to symptomatic pathogenesis in neighbouring crops. Sentinels will also allow scientists to gather epidemiological data about plant diseases. Our Green Canaries will reduce crop losses whilst decreasing the necessity for continual use of agrochemicals, contributing to more sustainable and less environmentally damaging farming practices. 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 Golden Gate technology.
The Experiments
Initially we selected several promoters known to respond to plant pathogens. We chose PDF1.2, a plant promoter that induced by the hormone methyl jasmonate, produced naturally by plants in response to various biotic and abiotic stresses. We also chose PR1, which responds to salicylic acid (another plant hormone produced in reposes to infection). Lastly, we identified two promoters of plant genes that are induced by TALES (transcriptional activator-like effectors), small molecules secreted by specific plant pathogens, ''Xanthomonas oryzae'' and ''Xanthomonas campestris''.
Next, we selected several coding sequences that, when expressed in plants, might produce a visible signal. First we chose BaxI to rapidly induce cell death. Next we 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.
Initially we tested our promoters by using them to drive expression of GFP. We also used a well-characterised constitutive promoter to drive the expression of our visible signals. When our circuits were cloned, we 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. Because we wanted to avoid working directly with the plant pathogens, to induce the promoters we painted the leaves with the appropriate plant hormone, or expressed the TALE protein at the same time.
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 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 is the shipping backbone, we also made some "flipper" parts. These "flip" Golden Gate parts into standard biobricks.
The methods that we used for GoldenGate cloning, transfecting the ''A. tumefaciens'' and plant chassis and other useful protocols are given on our lab protocols page.
Results
The promoters that respond to plant hormones were both 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.
!![insertBS3/AvrBS3 images and legends]!!
We were unable to see expression of chromoproteins in our plant chassis, even when expressed from a known strong, constitutive promoter. We were able to induce cell death using the Bax1 coding sequence from mice, however.
!![insert BAX image and legends]!!
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