From the Lab

Tanned Tomato Plants More Resistant to Disease?

Ultraviolet light, specifically shortwave or UV-C, has a demonstrated beneficial effects, both pre-and post-harvest, on a variety of crop plants. What we do not yet know, is how exactly.

For my fourth-year research project, I have joined “Team UV”. I am working with a variety of tomato plant (Solanum lycopersicum) cultivars, Botrytis and a UV machine (sorry, no tanning beds for the plants!). Our main aim is to quantify the expression of a handful of pre-selected genes of interest after a plant has been treated with UV-C. My project will be focussed on incorporating different filters to alter the specific UV wavelength that the plants are treated with.

So why should YOU care?

Ensuring global food security with an increasing population to feed, facing uncertain future environmental conditions, is one of the largest challenges currently facing humanity. Global population estimates for 2050 expect us to easily reach 9-10 billion. Plant pathogens cause approximately 8-15% of global crop yield to be lost, depending on which plant species you look at. It follows that this could pose a problem for future global food security, even without taking into consideration the likely damaging effects of global warming on agriculture. There is, therefore, a lot riding on discoveries of efficient and cost effective ways to manage or prevent disease [1]. The rise in resistance to pesticides, one of the conventional methods of controlling plant diseases, has driven the need for researchers to think a little more outside the box; the greener the better.

Integrated disease management is widely considered more “environmentally friendly” compared to chemical treatment, and is currently believed to be capable of preventing the development of resistance by the pathogen population [2]. Our research will help in this endeavour by improving our understanding of hormetic responses which are induced by UV-C. The long term aim of Team UV is to develop UV-C as a commercial treatment.

Hormesis is the process of inducing beneficial responses through low doses of an agent that would be damaging at higher levels [3]. These responses include changes in gene expression through which they are provided with a means of defending themselves against infection. 

Hormetic UV treatment could contribute to reducing both plant diseases pre-harvest and wastage due to post-harvest rots. This technology will be of most use in areas, such as the tropics, where humid conditions are ideal for pathogen growth all year round and/or industries where phytopathogens cause significant losses [4].

The effects of UV light on plants has been known for >20 years, yet little is known about the exact pathways/mechanisms through which it works [4]. Researchers have noted that it appears to induce plant defence responses, readying the plant for invasion by pathogens. Due to the UV-C wavelength (between 100-280nm, referred to as the “germicidal wavelength”) being completely absorbed by Earth’s ozone layer plants are not usually exposed to it.

Previous work in this area published by Liu et al. in 2015 demonstrated that genes mainly involved in signal transduction, defence response and metabolism were up-regulated. Supporting the observation that a defence response appears to be activated. While, genes that are related to disassembling the cell wall, photosynthesis and lipid metabolism were down-regulated [5]. The key difference between their research and ours is that they carried it out for postharvest UV-C irradiation. Our research will focus on pre-harvest, whole plant treatment, which is far less studied in comparison to post-harvest UV-C fruit treatment.

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Our incredible experimental setup. Left – UV machine. Right- Tomato plant on a box. The picture was taken by Claire.

The filters that I will use are made of different types of glass. One filters out all/most UV-C, but it transmits the majority of UV-A and B, while the other filters out UV-C, most/all of UV-B and transmits most of UV-A and visible light (personal communication Matvez Rupar). By then following up using qPCR on plant resistance related genes we can see if expression levels are affected by using different UV filters. As this is a potentially commercial form of treatment, working out the best doses/wavelengths of UV that are most suitable will stand the technology in good stead.

So what are we doing exactly?

The whole process is quite lengthy. First, and foremost, we need plants to experiment on. In total Claire and I are responsible for >100 plants of 5 varieties.

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Photos of our tomato plants at different stages during their growth. Photos were taken by me.

UV treatment is only effective when carried out at night; otherwise, a process called photo-reversal could result in completely reversing the treatment. Our tomato plants will be treated for ~two seconds using a pulsed UV light source, three times, 48hrs apart and then sampled 24hrs after the final treatment. Our first batch of plants underwent treatment during January, and our second batch is due to finish its last round with the machine very soon. Late nights out in the glasshouses on Sutton Bonington are ahead of us! 

The response of plants genetic expression to damage is rapid. Therefore the process of sampling the leaves of the plant can alter the genetic expression. To limit these changes in expression between irradiation and the RNA extraction steps our samples are taken and then immediately frozen in liquid nitrogenIt is imperative we are careful, and attempt to ensure a pure product, as contaminants have the potential to inhibit qPCR reactants from carrying out their respective functions.

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Showing the holes left behind in the leaves post-sampling. Photo was taken by Claire.

Rather than throwing away our plants at this stage, we decided to take the opportunity to gather more data from them. Creating open wounds the petioles (specific points along the stem) of all plants and infect them with Botrytis cinerea spores. We then measure the length of the lesion 96hrs later three times, with each measurement 24hrs apart. This allows us to say whether the UV treatments had an effect on the spores ability to colonise/form a lesion. Figure 1 shows in a graphical form how this may look.

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Figure 1 – A crude bar chart showing how the different treatment groups may affect Botrytis lesion length on various tomato plant varieties. Average lesion length is measured by hand for the different treatment groups on specific plant varieties. The treatment groups are represented by the key at the top. Half refers to a filtered UV treatment. We expect that UV treatment will reduce the size of the lesions observed on the treated plants, more so than control (untreated) plants.

Before analysing the samples, we checked the quantity of RNA extracted using the nanodrop to test absorbance, then adjusted to ensure each sample had the same concentration of RNA. Reverse transcribed the extracted RNA into cDNA to quantify the expression profile of the sample. After this all that is left is to perform qPCR for all the genes of interest, for all varieties, using SYBR green as our fluorescent nucleic acid dye. Figure 2 is a rough guess at how our results may look.

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Figure 2 – Rough graph idea showing comparison between the mean Cq value three treatment groups (circles) for two different tomato varieties.  Mean Cq refers to the mean number of cycles at which fluorescence exceeds the background levels, corrected by the reference gene actin which has a stable expression in all cells. This allows us to tell whether our gene has been up/down-regulated in comparison and even quantify how much expression occured (will explain in a future blog post!). Yellow = Treated; Red = Filtered treatment; Blue = Control (untreated).

 

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Photo of Me (Hannah). Taken by my wonderful lab buddy Claire.

Links Out:

[1] Feeding the World in 2050 – FAO’s suggestions on how we should go about feeding the 9-10 billion humans that will be around.

[2] Razdan & Sabitha (2009) Integrated Pest Management: Innovation-Development Process, pp 369-389

[3] Shama & Alderson (2005). Trends in Food Science & Technology. 16: 128-136.

[4] Shama, G. (2007). Postharvest Biology and Technology. 44: 1-8.

[5] Liu et al. (2011). Gene. 486: 56-64.

 

 

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