From the Lab

Shining a Light on the Disease Response in Tomato

Welcome to my MSci Blog!

The majority of this year, my fourth here at Nottingham, is spent preparing and undertaking a research project. The working title for my project is “How Does UV-C Irradiation affect the Disease Response of Tomato Plants” under the supervision of Professor Matt Dickinson and Dr Matevz (Mat) Rupar from The School of Biosciences Crop and Plant Science Division.

 

Why is this research important?

The world’s population is predicted to increase from 7.3 billion in 2016 to 9 billion by 2050[1]. Naturally, this will pose a problem for food security. Our research will contribute to the understanding of the hormetic responses induced by UV-C light. Hormesis is the process of inducing beneficial responses through low doses of an agent that would be damaging at higher levels[2]. These responses include changes in gene expression through which they are provided with a means of defending themselves against infection.

Below is a video which will help to explain what UV-C is. Only the first two minutes are relevant to this discussion[3].

The long term aim is to aid in the development of UV-C as a commercial treatment as there are an increasing number of diseases that are becoming resistant to chemical treatment, predicted to increase with climate change, which has resulted in even harsher chemicals being used. Legislation has already banned the use of some of these[4] which leaves the crops susceptible to infection, with farmers having no way to protect their crop and their livelihood.

I hope this research will give a clearer idea of the genes involved in the molecular responses after UV-C treatment and it will aid our understanding of how UV-C may be used as an alternative to chemicals.

 

The Plants

We are using tomato plants (Solanum lycopersicum) in our project because they are relatively quick and easy to grow; from seed to treatable plants in approximately one month.

Tomato plants are covered with fine hairs called trichomes which attach to each other when the leaves brush against those of another plant. This has made them difficult to deal with and it takes patience to move the plants around without causing damage to the leaves when they attach themselves to another plant. Figure 1 is a picture of our plants in the greenhouse and as you can see they are rather close to each other so this attachment happens quite often, especially with the cultivars that do not grow particularly straight.

uvc1

Figure 1 The greenhouse where we treated our plants

There are two of us working together in the same greenhouse so there are about 80 plants! These are divided into eleven groups: control and treated for Shirley and Ailsa Craig and ethylene mutants RinNor and Nr. I am working with the Ailsa Craigs and ethylene mutants and Hannah with the Shirleys.

Unfortunately, we will have to kill the plants before any ripe fruits are produced.

 

Ethylene mutants

Ethylene is a major plant hormone involved in current and future plant defences. Ethylene deficient mutants are more susceptible to necrotrophic fungi such as B. cinerea[5]. Previous work with tomatoes has shown that ethylene is upregulated when a plant is treated with UV-C[6] however my work will look further at the gene expression that also changes with this.

 

Treatments

Treated plants will be irradiated with UV-C light (100-280nm)[2] three times, 48hrs apart and then sampled 24hrs after the final treatment (figure 2a and 2b). We will then cut the petioles of all plants (figure 3) and infect with Botrytis cinera spores, measuring the size of the lesion 96hrs later three times, with each measurement 24hrs apart.

 

Figure 2a The equipment used to treat the plants. Figure 2b The leaves after sampling.

img_2423

Figure 3 The cut and inoculated petioles of the plants

Low doses of UV-C light treatments have been shown to induce a hormetic response in plant which beneficially changes the regulation of defence genes[2]. The aim is to then analyse how gene expression has changed in the treated plants and in addition how this treatment has affected lesion size and the plants’ response to infection. It has been shown that preharvest UV-C treatment on tomatoes reduced lesion size on the fruit[7] so this is expected to show in our results on the plants.

 

What stage are we at now?

We recently completed our training in the lab under the guidance of Mat in the different techniques we will have to use which include RNA extraction, reverse transcription and quantitative polymerase chain reaction (qPCR).

We are currently using these techniques to validate the genes that may be expressed in our plants when we get to that stage. This is being done using leaf matter from plants treated and sampled by Mat. We have just finished RNA extraction and reverse transcription of these samples.

With our own plants, we have just treated and sampled our first set of plants (shown in figures 1, 2 and 3) and inoculated them with Botrytis. We have also planted a new set of plants to do a repeat in one months’ time when they have grown.

Figure 4 Top – The lab where we are analysing the gene expression. Left to right bottom – Mat’s samples in ice, preparing tubes for sampling our plants and Hannah pipetting.

What are we going to do next?

Having just finished RNA extraction and reverse transcription of the validation samples we are now going to do the same to the samples we collected from our own plants. Once we have completed this we will run qPCR on both the validation and our own samples using SYBR green which fluoresces and the amount can be recorded by the equipment to analyse how much expression of a certain gene is happening depending on what primer we are using.

After that it will be a case of analysing all of our data while we wait for our second set of plants to grow to a treatable size to decide if there is anything we need to repeat.

References

[1] Augustin MA, Riley M, Stockmann R, Bennett L, Kahl A, Lockett T et al., 2016. Role of food processing in food and nutrition security. Trends in Food Science & Technology. 56: 115–125.

[2] Shama G, 2007. Process challenges in applying low doses of ultraviolet light to fresh produce for eliciting beneficial hormetic responses. Postharvest Biology and Technology. 44: 1-8.

[3] Video courtesy of Fernandez E, 2016. https://www.youtube.com/watch?v=Ts5zHlXRsOU&t=167s

[4] Charles MT, Goulet A and Arul J, 2008. The effects of biological and chemical treatment on gray mold disease in tomatoes grown under greenhouse conditions. Postharvest Biology and Technology. 47(1): 41-53.

[5] Broekgaarden C, Carls L, Vos IA, Pieterse CMJ and Van Wees SCM, 2015. Ethylene: Traffic Controller on Hormonal Crossroads to Defense. Plant Physiology. 169(4): 2371-2379.

[6] Liu C, Cai L, Han X and Ying T, 2011. Temporary effect of postharvest UV-C irradiation on gene expression profile in tomato fruit. Gene. 486: 56-64.

[7] Obande MA, Tucker GA and Shama G, 2011. Effect of preharvest UV-C treatment of tomatoes (Solanum lycopersicon Mill.) on ripening and pathogen resistance. Postharvest Biology and Technology. 62: 188-192.

All images are my own.

 

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