Mind the GAP: understanding how rice plants defend against disease

Image by Mathieu Schoutteten via Flickr (CC BY-NC-ND 2.0)

Image by Mathieu Schoutteten via Flickr (CC BY-NC-ND 2.0)

Rice is the staple food for over 50% of the world’s population, but yields are threatened by rice blast, blight and other diseases caused by microbes. To protect our rice crops against these diseases, it is important that we understand how they can defend themselves against infection.

Plants have two defence systems that to allow them to identify and defend against microbes. The first system is a general response to the detection of molecules that are produced by many different microbes. For example, plants can detect chitin, which is the major component of the cell walls of fungi. So if a plant detects chitin it knows that there is a fungus lurking around and it can activate general defences.

The second system is much more specific to individual species of microbe. For example, the fungus that causes rice blast (Magnaporthe oryzae) produces particular molecules that help it to infect the plant, known as effectors. If the plant is able to detect any of these molecules it is able to identify the fungus as a threat and can activate more specific defence responses. For example, the plant may trigger the death of cells in the area around the fungus to halt the spread of the fungus.

Many proteins involved in plant defence responses have been identified, but it is not clear how they are all controlled to make sure that they are only active at the right times. One way to control proteins is to target them for degradation when they are not needed. A protein called SPL11 suppresses programmed cell death and defence responses in rice plants. It belongs to a family of proteins—called the U-box E3 ligases—that can tag other proteins with ubiquitin proteins to target them for destruction. However, it was not known which proteins SPL11 can target.

Plants that were lacking SPIN6 (16-2, 22-2; RNAi knockdowns) developed lesions on their leaves due to programmed cell death. Image from Liu et al. licensed under CC BY 4.0.

Plants that were lacking SPIN6 (16-2, 22-2; RNAi knockdowns) developed lesions on their leaves due to programmed cell death. Image from Liu et al. licensed under CC BY 4.0.

In a paper recently published in PLOS Pathogens, Liu, Park, He et al. found that SPL11 can add ubiquitin to a protein called SPIN6 (1). Rice plants lacking SPIN6 have higher levels of programmed cell death (see image) and increased resistance to M. oryzae and the bacteria that causes blight (Xanthomonas oryzae pv. Oryzae). The plants also had higher levels of expression of genes involved in defence responses and elevated levels of reactive oxygen species (another defence response).

SPIN6 is a member of a family of proteins—called the RhoGAPs—that are able to control the activity of members of another family of proteins called the ROP GTPases. The researchers found that SPIN6 can inactivate a ROP GTPase called OsRAC1, which is a key protein in rice defense responses that is activated when rice cells detect chitin.

OsRAC1 is involved in both general and more specific rice defines responses so SPIN6 may be involved in linking these systems to enable the plants to mount an effective defence. The equivalent of the SPIN6 protein in the model plant Arabidopsis thaliana also suppresses programmed cell death and defence responses in plants, so it is likely that other plants have proteins with similar roles. The next challenge is to find out how the activity of SPIN6 is controlled when a microbe invades to better understand what role it plays in defense.

Reference: 1) Liu, J., Park, C.H. He, F., Nagano, M., Wang, M., Bellizzi, M., Zhang, K., Zeng, X., Liu, W., Ning, Y., Kawano, Y. and Wang, G. (2015) The RhoGAP SPIN6 associates with SPOL11 and OsRac11 and negatively regulates programmed cell death and innate immunity in rice. PLOS Pathogens. http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004629

The potential of fungi to control invasive plants

By Aimee Fowkes (@AimeeFowkes)

A plant species is considered to be “invasive” in an area if it is not native and has a tendency to spread. Invasive plants can have a damaging effect on the new environment they find themselves in because they can outcompete the native flora and so interfere with the food web. Also, they may not have any natural enemies in their new location so they often show improved fitness and can grow uncontrollably.

I. glandulifera is also known as Himalayan balsam, Indian balsa and policeman’s helmet (owing to the shape of its flowers).  Image by ArtMechanic via Wikimedia Commons (CC BY-SA 3.0).

I. glandulifera is also known as Himalayan balsam, Indian balsa and policeman’s helmet (owing to the shape of its flowers). Image by ArtMechanic via Wikimedia Commons (CC BY-SA 3.0).

Impatiens glandulifera is a large flowering plant that originates from the western foothills of the Himalayas. It was introduced into Europe in the early nineteenth century as an ornamental plant, and it has since spread across Europe and North America. It grows on riverbanks, waste grounds and in woodlands.

Conventional control of I. glandulifera using chemicals has so far failed because it tends to grow in places that are inaccessible, or where use of herbicides is prohibited. This has led to investigations into an alternative solution, biological control.

Biological control is the use of one organism to reduce the population of another pest species, for example, by importing a natural enemy of the pest species into the new environment. Insects have been used in biological control since the nineteenth century, but it is only recently that people have started looking into using fungi to control weeds and invasive plants. There are several cases of fungi being used as biological control agents and their narrow host ranges reduce the risk that they will have an impact non-target species.

A team of scientists went to areas where I. glandulifera is native and searched for natural enemies of the plant. Between 2006 and 2010, Tanner et al. gathered various insects they found feeding on the plants and samples of plants showing symptoms of fungal infection. Here is a photo diary of the team’s search in 2008:

Further testing showed that most of the sampled natural enemies were able to infect other species of plants that are closely related to I. glandulifera so they were all rejected, except for one fungus known as Puccinia komarovii. The researchers had found a variety of the fungus almost exclusively infects I. glandulifera, which is now known as P. komarovii var. glanduliferae. This fungus belongs to a group of disease-causing fungi known as rusts, named after their yellow/orange pustules of spores that can be seen on the leaves of plants. In general, the rusts have very narrow host ranges, and they will only infect a few closely related species.

Why could this fungus make a good biological control agent?

Tanner et al. tested P. komarovii var. glanduliferae against 75 species of plants and it could only infect I. glandulifera and the closely related I. balsamina. In fact, the other species that are closely related to I. glandulifera showed high levels of resistance to the pathogen, suggesting that the risk of cross-infection is low. Importantly, the fungus does not infect economically important species in the Impatiens genus, or a UK native species called I. noli-tangere (touch-me-not or yellow balsam). While infection of I. balsamina is unwanted, it is non-native to the UK and has very little economic value so is of little concern.

It is important that the biocontrol agent can survive in the new environment. P. komarovii var. glanduliferae originates from the Himalayas so it has evolved to cope with a range of temperatures. The spores can infect between 5 oC and 25 oC so they should be able to survive the British climate. As previously mentioned, I. glandulifera is problematic in river bank habitats in the UK. When the leaves drop from the plants in the Autumn, any spores on the leaves will also be incorporated into the soil. Although any spores below the flood plain could be washed away, it is thought that spores on plants in more stable areas (above the flood plain) should be able to survive the winter and infect seedlings the following spring.

Rust spores are carried on the wind and are able to cover huge distances. A species of rust (P. chondrillina) was previously used to control skeleton weed in Europe and within the first year, it was shown to have spread 300 km. Using a rust fungus can overcome the problems associated with chemical control, its specificity makes it more environmentally friendly, and it is less expensive because if the rust is able to establish itself it will not need repeat applications.

I. komarovii var. glandulifera was initially investigated under strict quarantine, but recently, the Department for Environment, Food and Rural Affairs (DEFRA) in the UK gave permission for outdoor trials to be conducted in England. If the trials are successful, this fungus could become the 29th fungus to be used to control a weed and it may encourage more interest in exploiting rusts as biological control agents.

About the author: Aimee Fowkes is a masters student at the University of Nottingham. She is interested in plant-pathogen interactions and is currently doing a project looking at the effect of an elicitor on defence responses in broad bean. She has recently started her own blog: When Plants Fight Back. Follow her on twitter (@AimeeFowkes)

References:

Barton et al. (2011) Predictability of pathogen host range in classical biological control of weeds: an update. Journal of the International Organization of Biological Control.

CABI: Biological Control of Himalayan balsam. http://www.cabi.org/projects/project/32944

Shelton, Biological Control: A Guide to Natural Enemies in North America. http://www.biocontrol.entomology.cornell.edu/cite.php

Pal & McSpadden Gardener (2006) Biological Control of Plant Pathogens. The Plant Health Instructor.

Tanner et al. (2013) Impacts of an Invasive Non-Native Annual Weed, Impatiens glandulifera, on Above- and Below-Ground Invertebrate Communities in the United Kingdom. PLOS one.

Tanner et al. (Accepted article available online) First release of a fungal classical biocontrol agent against an invasive weed in Europe: biology of the rust, Puccinia komarovii var. glanduliferae. Plant Pathology.

The promise of drought-tolerant and disease-resistant barley

Image by Gerste Ähren (CC BY-SA 3.0)

Image by Gerste Ähren (CC BY-SA 3.0)

Barley is the fourth most important cereal crop in the world (behind rice, maize and wheat) and is grown in both temperate and tropical climates. It was one of the first grains to be domesticated and has a variety of uses including in the alcoholic drinks beer and whisky, as a foodstuff (especially in the Middle East) and for animal feed. Like other crops, the barley yields can be affected by many different pests and diseases, but also by adverse environmental conditions such as drought or low soil nutrients.

In July last year, I wrote about how a variety of barley called Golden Promise was genetically-modified to be more drought-tolerant (see previous post). This variety carries an extra copy of an existing barley gene—called HvSNAC1—that promotes the closure of pores (stomata) on the surface of barley leaves to reduce water loss when water is scarce (1). However, it was not known if making the plants more drought tolerant in this way could alter the ability of the plants to resist diseases caused by invading microbes.

Researchers at the John Innes Centre and SRUC studied that the ability of this variety to resist infection by several fungi that can cause diseases in barley (2). McGrann et al. found that the plants are more resistant to infection by a fungus called Ramularia collo-cygni. This fungus causes a disease called Ramularia leaf spot, a newly emerging disease that is currently affecting barley crops in Europe.

How can a single gene make plants more drought-tolerant and disease-resistant? One possibility is that the HvSNAC1 gene could promote the closure of stomata, which is the main way that the fungus can enter barley leaves. However, the experiments show that when the barley plants were treated with the fungus, the stomata of the plants carrying an extra copy of HvSNAC1 remained just as widely open as the control plants.

The symptoms of Ramularia leaf spot tend to appear late in the season when the leaves are starting to die back (senescence). Leaf senescence was delayed in the barley variety carrying an extra copy of HvSNAC1, suggesting that this may be linked to the increased resistance, but how is not clear.

It is important to note that although HvSNAC1 does increase the resistance of the barley to infection with R. collo-cygni, it does not have any effect on resistance to diseases caused by powdery mildew (Blumeria graminis f. sp. hordei), eyespot (Oculimacula yallundae) and several other fungi. However, producing a crop variety with increased resistance to even one fungal disease is still a step forward.

This research shows that it is possible to improve a crop’s tolerance for extreme environmental conditions and improve its resistance to a fungal disease at the same time.

References:

1) Al Abdallat et al. (2013) Overexpression of the transcription factor HvSNAC1 improves drought tolerance in barley (Hordeum vulgare L.). Journal of Molecular Breeding. (link above is to the freely available PDF, for closed access version on publisher’s website click here)

2) McGrann, G.R.D. et al. (2015) Contribution of the drought tolerance-related Stress-responsive NAC1 transcription factor to resistance of barley to
Ramularia leaf spot. Molecular Plant Pathology.

Studying plant genes with a paintbrush and a baking tin

Image by J. Murray. Used with permission.

Image by J. Murray. Used with permission.

A paintbrush and a baking tin might seem unlikely equipment for scientists to use to study plant genes but both feature in a study recently published in the journal Plant Cell. Intrigued? Let me explain…

Imagine for a moment that you are a research scientist studying how legume plants (e.g. peas, beans) set up friendly relationships with soil bacteria called rhizobia. The rhizobia provide the plants with much needed nitrogen. In return, the plant provides the rhizobia with carbohydrates and a home within the plant roots in special organs called nodules.

To enter the nodules, the rhizobia first have to infect into the root, which is a complex process with multiple stages. We currently only know about a few of the plant genes involved, so to identify more genes, you would like to start by getting an overall picture or “profile” of which genes are switched on (expressed) as the rhizobia infect into the root.

Bacteria (stained in blue) infecting into a root hair cell as seen under a light microscope. Image by the author.

Bacteria (stained in blue) infecting into a root hair cell as seen under a light microscope. Image by the author.

Most of the studies that have profiled how large numbers of genes are expressed in plants have used samples from large pieces of plant tissue, for example from whole roots or leaves. However, rhizobia do not infect plant cells evenly across the whole root and most of the cells in the deeper layers remain uninfected, so if whole root samples are used it may be difficult to spot the genes involved in infection. The rhizobia start by infecting individual cells on the surface of the root called root hair cells (see image). How could you separate out the root hair cells from the rest of the root?

Some of my friends at the John Innes Centre, UK have addressed this question. Breakspear, Liu and colleagues (2014) froze the roots of the legume Medicago truncatula in liquid nitrogen to preserve the genetic material, and then removed the hair-like part of the root hair cells by brushing them with a small paintbrush (1). However, in the first attempts, the fragments of root hairs stuck to the sides of the container holding the liquid nitrogen so they switched to using a Teflon-coated (non-stick) baking tin, which allowed the root hair fragments to be transferred into tubes so the genetic material contained could be extracted for gene expression profiling (using fancier equipment).

Breakspear, Liu and colleagues used this method to study gene expression in the root hairs of Medicago truncatula at 1,3 and 5 days after they added rhizobia to the plants (1). They found that many genes were more highly expressed in these plants than in the control plants that had no infection.

The ARF6a gene and several other genes that were switched on during infection can also be switched on when plants are treated with the plant hormone auxin, which is known to have a role in making root nodules, but it has not yet been shown to be involved in infection. Further experiments on ARF6a showed that it is strongly switched on in infected root hairs only and M. truncatula plants missing the ARF6a gene had lower levels of infection than normal plants. These findings suggest that auxin is involved in promoting infection.

Many of the other genes that are highlighted in this study are involved in responses to other plant hormones, or communication with the rhizobia. These genes could be interesting subjects for future studies. The gene expression data is itself a useful resource for other scientists and has been deposited in an online database, The Medicago Gene Expression Atlas, which contains lots of other data from the various tissues and organs of M. truncatula in different situations.

This is just one example of how everyday items can find a new use in science experiments. Clingfilm (food wrap) and foil are essentials in most biology labs. Toothpicks, cotton wool, fabric netting and sewing thread are just a few of the everyday items I’ve used in the lab. Since problem solving by individual scientists is behind many methods used in science, it is perhaps not surprising that everyday items are involved.

Reference:

1. Breakspear, A., Liu, C. Roy, S., Stacey, N., Rogers, C., Trick, M., Morieri, G., Mysore, K.S., Wen, J., Oldroyd, G.E.D., Downie, J.A. and Murray, J. (2014) The root hair “infectome” of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for auxin signaling in rhizobial infection. Plant Cell.

Nitrogen fertiliser: the fallen hero of agriculture

Spraying fertiliser on oilseed rape near Barton Grange, North Lincolnshire,  UK. Image by David Wright - geograph.org.uk (CC BY-SA 2.0)

Spraying fertiliser on oilseed rape near Barton Grange, North Lincolnshire, UK. Image by David Wright – geograph.org.uk (CC BY-SA 2.0)

In many ways, the development of nitrogen fertilisers was a big success story for agriculture. All plants need nitrogen to grow, but most can only absorb it from the soil in the form of ammonia or nitrates. Unfortunately, there are limited amounts of ammonia and nitrates in soil, which limits plant growth and poses a challenge for agriculture. Animal dung and plant waste are naturally rich in ammonia and nitrates and have been used for centuries to promote plant growth, but it was the development of an industrial process to manufacture ammonia in the early 20th century that revolutionised agriculture. Synthetic nitrogen fertilisers are now widely used in all but the World’s poorest countries, and are estimated to have increased crop yields by 35-50% since the 1940s (1).

However, nitrogen fertilisers have some major drawbacks. When applied to fields, only about 50% of the nitrogen in fertiliser is actually absorbed by plants (2). The excess can be washed out of the soil into rivers and lakes where it promotes the growth of algae, leading to algal blooms that can kill other life forms by blocking out light and lowering the oxygen content of the water. Also, excess nitrogen fertiliser in soil can be converted into nitrous oxide by some bacteria. Nitrous oxide is a very potent and long-lived greenhouse gas, and worryingly, the levels of it in the atmosphere are rising. Agriculture is a major contributor to the increase and accounts for over 80% of total nitrous oxide emissions (3). Continue reading

Olives: A gift from the gods

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Olives in Athens. Photo taken by Brian John Abbs (c) 2014, used with permission.

By Kirsty Jackson (@kjjscience)

Olives, a symbol of peace and victory, are a tasty treat and make an excellent oil that is used all over the world. I knew that olives originated along the Mediterranean, but whilst on honeymoon in Athens, Greece, I was shocked by how many olive trees I saw. They were everywhere, lining the streets in the same way that Plane trees do in the UK. The soft squishy fruits were all over the pavements and the local pigeons had the shiniest feathers I had ever seen – I assume from their olive rich diets. The only other trees I saw along the pavements were tangerines, but I never discovered why this was.

Row of olive trees in Athens. Photo taken by the author.

Row of olive trees in Athens. Photo taken by the author.

There is an explanation for why olives are so prevalent in Athens that goes back to an ancient Greek myth. Athena, the Greek Goddess of war and wisdom, and Poseidon, the Greek God of the Sea, were fighting over who should be patron of the city. Both being Gods, they were very evenly matched and the people couldn’t decide who would be best. Athena suggested that each God should give the city a gift and the one that was deemed to be the most useful by the people of the city would be the winner. Continue reading