8 February 2015 marked 150 years since the first of Mendel’s lectures where he presented his results on pea breeding for the first time. These lectures, based on his paper Versuche über Pflanzen-Hybriden (Experiments on Plant Hybridisation), presented the world with a vision of genetics never seen before – and led to him gaining the title ‘The Father of Modern Genetics’.
More than 22 years have passed since the Convention on Biological Diversity was signed. It called for international efforts to conserve the world’s biodiversity, which had long been suffering the effects of human activities. Since then, there has been a lot of debate over what the best way of securing this biodiversity is.
But really, there isn’t a best way. There isn’t a clear consensus, one method to fit all. We can’t choose one way to preserve all the different elements that form what we call biodiversity.
From mid-September until mid-December last year I was on an internship in science policy. Once you count the conference at the start of December and the Christmas holidays, I was left to return to the lab this January after a four-month break.
The aim of my internship was to experience the world outside academic research, and so my project was sent to the back of my mind while I thought about more political topics. Most of my friends did their internships around Norwich, so they had the chance to get into the lab at weekends or in the evenings. But I moved to Cambridge for mine, so I didn’t have the opportunity to nip into the department and get some experiments going. Now, I’m back doing my research, and it’s amazing what’s happened while I was away.
Traditionally, a UK PhD student spends 3-4 years in the lab, researching their project. However, a recent drive to produce ‘well-rounded’ students has led to the development of internship schemes. This means that I’ve left my PhD mid-research for 3 months, and I’m currently six weeks into a placement working in the Centre for Science and Policy, at the University of Cambridge. This centre works in science and policy issues across all areas, from medicine, to physics, to social sciences. I always knew that plant sciences was an important field, and one that’s been booming recently. However, in the past six weeks I’ve been looking from a policy viewpoint and I’ve realised exactly how important it is, and how valuable plant science research will be for our future.
At the John Innes Centre, many of us work on the interactions between plants and bacteria. Most people automatically think of these interactions as harmful and disease-causing (The Sainsbury Laboratory, amongst others, study these). However, plenty of us are more interested in interactions that benefit the plant, not hinder it. These interactions include symbioses. Symbiosis, from the Greek meaning ‘working together’ describes an interaction between two different species. An example of this that is investigated here in Norwich is the rhizobia-legume symbiosis. In this symbiosis the bacteria ‘fix’ nitrogen gas into a biologically useful form that the plant can use. In return the plant provides sugars for the bacteria to use for energy. Since two of us on the blog team work in this area (Izzy and Jo), we thought it would be fun to write something about some of the more unusual symbioses that are out there.
Fig and Wasp
Figs and fig wasps are an example of obligate mutualism – both symbiotic partners need the other partner in order to reproduce. They have co-evolved a unique way in which they rely on each other: figs need the wasps to become pollinated and the fig wasps both need the figs to reproduce and to feed on. Figs (also known as a syconium) are in fact made up of lots of tiny female flowers that cover the inner surface and these are pollinated by female fig wasps. In order to do this, the female wasp crawls inside the fig (through a tube known as an ostiole) and at the same time she will also lay her eggs. Male and female wasps then hatch out into larvae inside the fig and then they reproduce. Male wasps in fact do not survive well outside of the fig, so spend their lives trapped inside the fig! Female wasps will mature and become covered in male pollen from the inside of the fig, and when they tunnel out and escape to find a new fig will pollinate that fig again and the cycle continues. This in itself is enough to put me off eating figs for life, however you will be pleased to know that figs do produce an enzyme which breaks down the wasps that die inside the fig so you aren’t actually eating lots of wasp bodies.
Inside hydrothermal vents and cold seeps deep on the ocean floor live sibloginid worms. Siboglinid, or beard worms are a type of ringed worm which occur (and dominate) a wide range of habitats across the globe. Unlike most animals, however, they completely lack a digestive system. Siboglinid worms are nutritionally dependent on endosymbiotic (meaning one symbiotic partner lives within the other) bacteria, which they acquire from their environment and house in a unique storage organ called a trophosome.
The siboglinid worm has a ‘plume’, an organ which is able to exchange compounds with its environment. Through this plume the worm is able to acquire gases including carbon dioxide, oxygen, hydrogen sulphide and methane. The bacteria within the trophosome are able to convert these gases into organic molecules, which can then be used by the worm as food. The ‘chemosynthetic’ bacteria are also able to convert nitrate into ammonium ions, which can then be used to make amino acids, also provided to the worm.
Nearly every cell in the human body (red blood cells being an exception) contains endosymbionts – or at least, what were once endosymbionts. Mitochondria, the organelle where respiration occurs, were once free-living bacteria, engulfed by the single-celled organisms that are our ancestors. This event is predicted to have happened around 1.5 billion years ago, and led to the first eukaryotic cells. The same theory can also be applied to chloroplasts – which were seen to resemble free-living cyanobacteria. The engulfed bacteria survived, and their ability to respire/photosynthesize was utilized by the host cell. The theory that a eukaryotic cell is a symbiotic union of prokaryotic cells was proposed by Lynn Margulis in 1966 although she initially met criticism for this. Since the theory was put forward, evidence has mounted that strengthens the case for the endosymbiotic theory
We won’t list all of the evidence for it here, since we would probably need an entire blog post to do it, but we thought we’d name a few. One of the most interesting features of these organelles is that they have their own genome, different from the DNA found in the cell nucleus. Mitochondrial genomes show similarity to bacterial genomes, as do the mitochondrial ribosomes. Mitochondria also have double phospholipid bilayer as their organelle membrane, similar to the membrane found on the outside of a cell. The mitochondria also reproduce via binary fission, which is similar to bacteria. If mitochondria are destroyed, the cell is unable to produce new ones using the genes from the nucleus.
Interestingly, many of the genes found in the nucleus are thought to have once been found in the mitochondria, but at some point in the past 1.5 million years have moved location. This ‘horizontal gene transfer’ can be seen in other symbioses too – for example, homocitrate, which is essential to the rhizobia-legume symbiosis, is produced by the plant, despite being required for the bacterial nitrogenase enzyme.
Here we have described just three of the interesting symbioses that are out in the world. However, these three just scratch the surface. There are loads of other examples of different species interacting with each other in a way that benefits both partners. We plan to blog again in the future about some more of the weird and wonderful symbioses out there.
By Jo Harrison and Izzy Webb
Thornhill et al. 2008, Commun Integr Biol 1 (2) 163-166
Sagan 1967 J Theor Biol 14(3): 255–274.
Author’s own photo
Never Eat Anything Bigger Than Your Head & Other Drawings, B. Kliban
During the summer months it’s quite common to find the labs in the Crop Genetics department at the John Innes Centre fairly empty. Although many of us might wish we were on sunny beaches enjoying the sun, the reality is that we’ll generally be found in the less tropical climate of East Anglia attending to our field trials. Trials are very important for plant geneticists and pathologists as they provide a chance to test crop varieties in a natural environment, and also allow data on yield performance, disease resistance and stress tolerance to be collected on a larger scale than in a glasshouse or polytunnel.
But what does running a field trial involve?
Whilst the activity in the plots usually begins to pick up during spring, trials can actually be a year round activity. Generally seeds must be bulked at least 4 months before they’re due to be planted to make sure there is enough to fill a plot. Winter cereal varieties such as winter wheat or barley are generally sown in the field during October to December, so that they’re exposed to the cold temperatures they require to germinate, a process which is called vernalization. Spring cereals can be sown from January to April however, as they don’t need such cold temperatures in order to grow.
Once the seeds have begun to emerge from the soil, irrigation can be set up to ensure that the trials have enough water and fertiliser can be applied to make sure the plants are as healthy as possible. Then depending on the type of trial, plants may be sprayed with herbicides to prevent unwanted weeds, or fungicides which prevent fungal diseases. The time it takes to collect the required data also depends on what type of trait is being investigated. For example, height measurements may be taken after a few weeks of growth, whereas yield data might be gathered at harvest maturity which can be 3 or 4 months after initial planting.
After all the required traits have been recorded (and the weather is co-operating) it’s then time to harvest, either by hand or mechanically using a harvester. This means that yield data can be recorded and the seed can be used for further experiments or analysed for nutrient, fungus or toxin content. It’s also time to start doing some statistics with the data that has been gathered. Whilst two varieties might look different in terms of a specific trait, it’s important to know whether this difference is statistically significant- is the difference real or is it purely down to chance? The results can sometimes be unexpected, but give an indication of which lines would be useful to investigate further. Once this is decided it’s usually time to start planning for next year’s trial!
As you generally only get one chance a year to gather trial data it’s important to plan properly. However, sometimes even the best planned trial can be changed due to problems with drilling, poor seed or even animal damage. For example having control lines within a field plan, such as including dwarf varieties, means it is easy to check the location of specific lines within a field. I’ve found this particularly useful when plots aren’t where I expected them to be due to drilling issues! Using replicates or split plots within a trial can also be helpful, especially if seeds don’t establish well in a certain part of the field, meaning that you don’t completely lose data for that line.
Multi sites and environment
In lab experiments you would usually want replicates to be as similar as possible in conditions to the original. This is not true of field trials. You instead aim to have trials in a wide variety of environments to prove that the effects you see are universal. However running field trials is costly so there is a limit between the ideal number of locations you’d test and what you can actually perform. This year I have 9 different trials, with 5 in Norfolk to allow me to monitor them easily. The others are in Suffolk, Dorset and Ireland. This geographical spread, as well as a showing reproducibility, can also act as a failsafe if the weather conditions cause problems in one area. For example the disease that I work on requires rainy weather to thrive. Thus if Norfolk has a dry summer I may have poor data, however additional trials in different weather conditions can compensate for this.
People usually react with shock when I reveal that I can go weeks without using a PCR machine! But the equipment favoured by the field scientist is often very different to those that you would associate with a scientist. My most important piece of equipment is my clipboard; it is so much easier for data collection than having to write on the ground or leaning on the back of a passerby. Other important parts of key field gear include wellingtons, sunhats and suncream depending on the weather. Beyond this, the equipment you need depends on what you are working on, from a simple metre sticks for height measurements to combine harvesters for measuring yield. As for me, my favourite piece of field equipment is my field cushion, as it has prevented my knees being completely destroyed when taking measurements at ground level.
What do we do in the field?
Chris: My field trials are about how the fungus Zymoseptoria tritici spreads in the crop canopy and how this relates to differences in yield. So my main task in the field is scoring the disease. This is done by inspecting leaves at different heights in the canopy and scoring the lesions formed by the fungus. This process is complicated by the presence of other disease and abiotic damage so you need to be trained in what you are looking for. In addition to this a wide variety of physiological traits are being measured in the plots to identify differences in the physiology of the lines used. At the end of the experiment, both diseased and undiseased plots of these lines will be harvested allowing for comparisons of yield between the lines. Across my different sites the combination of the disease, physiology and yield data should allow us to understand the septoria-yield trade off better.
Rachel: My trials look at the resistance of a heritage barley variety to Fusarium Head Blight (FHB) which is a major disease of cereals caused by toxin-producing Fusarium fungus. Resistance to FHB has been linked to plant height, with taller varieties being more resistant. However, tall varieties are less favoured in agriculture, so I’m aiming to see if FHB resistance and a shorter height trait can be combined into one variety. This involves spraying the crops with Fusarium to generate disease, and then scoring for disease resistance, height and other important traits. My trial will be hand harvested and then the grain analysed to determine the toxin content of each line to see if the disease resistance score correlates with a reduction in toxin production.
By Chris Judge and Rachel Goddard, both 3rd year PhD students in the department of Crop Genetics
In the last year, a brand new technique for genome editing has appeared with the potential to revolutionise the way in which scientists engineer genomes. It provides the ability to make cuts in the genome at precisely controlled locations, resulting in the silencing of that particular region. This technique is known as CRISPR. To make such a precise cut, however, requires the active protein to find a unique 20 base pair region within the vast sequence space of the entire genome.
The CRISPR system originates from the bacterial immune system in which a segment of the bacteria’s genome contains short elements of viral DNA (protospacers), which act as guides to attack invading viruses by disrupting their genetic code . This segment (known as the CRISPR array) contains so-called ‘Cas’ proteins capable of cleaving segments of DNA, that are guided into place by the protospacers stored in the array. The bacteria are able to add new stretches of foreign DNA to this array to develop immunity in future encounters with the invader.
The genome editing technology [2,3] takes this CRISPR array structure and replaces the viral DNA sections with lengths of the target genome that are intended to be silenced. This can then be inserted into the target genome by standard methods and, when transcribed, the Cas protein finds its way to the point in the genome with precise sequence complementarity to its guide. However, despite this technique’s success, the mechanism enabling the Cas-RNA complex to find its target was unknown, until now.
In this week’s nature cover article , Sternberg et al. show that a particular Cas protein, Cas9, can only bind to regions containing a three-nucleotide motif region, known as a PAM, that is found adjacent to protospacers. When searching for a needle in a haystack, you would not pick up every piece of hay, compare it to a picture of a needle, and then replace it when it didn’t match. Similarly, Cas9 does not try every point on the genome and compare the local sequence to its guide. It first limits the number of places to search by only binding to PAMs. This could be likened to the first step in searching for a matching phone number; you only compare the numbers with the same area code.
Of course, there are still a large number of PAM sequences in a given genome (or else the technique would be somewhat limited), but it vastly simplifies the problem. Furthermore, the PAM acts as the start point for the guide/genome sequence comparison, which proceeds one nucleotide at a time in sequence. The process is such that if the first 2-3 nucleotides do not match, then the complex rapidly disengages from that region and can move on to try a different location. These factors combined ensure that the complex spends as little time as possible at incorrect locations.
This work is an impressive demonstration of how biological systems are able to solve difficult problems. An invading virus needs to be dealt with quickly if a bacterium is to survive. The CRISPR system therefore needed to find a rapid way of finding a target within a large search space. It achieved this by utilising a regular motif present within the target genome, choosing to only incorporate stretches of DNA adjacent to these motifs. It seems the trick to finding a needle in a haystack is to choose a smaller haystack to lose it in.
- Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331-338 (2012).
- Mali, P. et al. RNA-guided human genome editing via Cas9. Science 339, 823-826 (2013).
- Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013).
- Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62-67 (2014).
By Matthew Evans- a second year PhD student in the lab of Dr Richard Morris