Scientific advances supporting the battle against black-grass
by Teresa Rush
Developments in plant transformation and insights gained from the pharmaceutical sector are helping to give a deeper insight into herbicide resistance in black-grass and how it might be combated.
In 2014 a consortium of scientists began work on a four-year BBSRC and AHDB-funded project that set out to use state of the art approaches from molecular biology, weed science, computer modelling and agronomy to identify new resistance control measures in black-grass.
Among the work undertaken as part of the Black-grass Resistance Initiative (BGRI) project were selection and breeding experiments to examine the dynamics of selection for resistance, with the intention of determining the genetic makeup of non-target site resistance for the first time.
The scientists identified the herbicide-resistant populations on which they would work via a national audit of resistance across the UK, during which 71 farms were visited, 138 fields surveyed and over 190 seed populations collected.
Each seed population was grown on and treated with one of three herbicides – Atlantis (sulponylurea), Cheetah (ACCase inhibitor) and Laser (ACCase inhibitor). Over 50,000 plants were screened through these herbicide assays and levels of resistance to the different herbicides were determined.
Speaking at the British Crop Production Council (BCPC) annual weed review meeting in Cambridge, Prof Rob Edwards of Newcastle University, said: “We have known for some time that they [non-target site resistant black-grass plants] are able to detoxify herbicides more rapidly. We also know these plants accumulate more antioxidants. But when we did studies to look at the difference in metabolites between herbicide resistant and herbicide susceptible plants, we found that, curiously, there were very few changes in metabolism.”
So what the researchers did next was to start a search, looking for changes in genes, proteins and metabolites that might serve as genetic markers of resistance.
“We carried out loads and loads of [gene] sequencing and from all of studies we now have a total of 17,000 genes seen to be expressed in black-grass and we know that at least 4000 of these will be randomly altered in their expression if we look at resistant versus susceptible plants.
“Not all those 4000 genes are changed due to resistance but we can see there are some very interesting genetic patterns associated with resistance.
“In particular we zoomed in on the enzymes associated with herbicide detoxification because we know the function of these are important in non-target site resistance.
“We looked at the genes involved in the different phases of detoxification and we chose several of these genes as markers that we could then probe to see whether or not every time we see non-target site resistance in black-grass, is it giving us the same kind of signature in its expression. The answer to that question is clearly ‘no’. We can see that there are different patterns of resistance gene expression in different populations.”
“What this tells us, at big picture level, is that there is more than one type of non-target site resistance, there are sub-types, and that means some types of non-target site resistance may be more effective against sulphonylureas, some may be more effective against ‘fop’ herbicides.
“That immediately tells us there is something there that we may be able to exploit in the future – non-target site resistance isn’t a single one size fits all.”
The next step was to investigate what was happening at the coalface of resistance – protein expression.
“If you want to understand what is happening at a mechanistic level, thousands of gene changes don’t tell you very much, it is like looking for a needle in a haystack.
“So instead we looked at protein expression, proteins are the things that carry out all of the functions within the living system. Suddenly all the complexity started to fall away and we went from hundreds of genes down to a couple of dozen proteins that were typically involved In herbicide resistance.
“And when we looked at what those changes in protein expression looked like – we compared non-target site herbicide susceptible plants, but we also put herbicide susceptible plants through different plant stresses – we found that non-target site resistance didn’t look like any of those other types of stresses; it much more closely resembled changes in gene expression associated with drug resistance in humans.”
The proteins of primary interest to the BGRI researchers were those that are uniquely associated with all populations of black-grass with non-target site resistance. Prof Edward’s group at Newcastle were able to identify the presence of a specific protein called AmGSTF1, the expression of which has found to be elevated in all non-target site resistance populations of black-grass.
“We have got very strong evidence that AmGSTF1, even in this very small number of populations, is intrinsically linked to non-target site resistance,” said Prof Edwards.
To explore the influence of AmGSTF1, the researchers transformed the model plant Arabidopsis with it and were able to show that by inserting the gene coding for AmGSTF1 into Arabidopsis it became tolerant of certain herbicides.
Furthermore, investigation of AmGSTF1 at a molecular level revealed an unusual feature – a large unstructured loop sat over its surface. However, while this was unusual, it had been seen before in nature, in human beings, where it was implicated in resistance to drugs.
“We’ve got to the point where we know AmGSTF1 is critical in a large part of non-target site resistance and when we disrupt its function, we can potentially disrupt resistance. So now we can use AmGSTF1 as a functional biomarker for non-target site resistance. We know that it is only present in plants that are becoming non-target site resistant; can we use that information as a diagnostic to study this resistance out in the field,” said Prof Edwards.
Following on from this finding a simple to use diagnostic for detection of AmGSTF1 in black-grass, using lateral flow technology, was developed with Mologic, a human healthcare diagnostic company.
But, while the development of this tool was a useful practical outcome, the scientists were keen to look at what else they could do with AmGSTF1.
Once again they looked back through the literature generated around human drug resistance and came across the idea of a ‘resistance busting’ approach based on disrupting the function of the cues responsible for controlling multiple drug resistance in cancer cells using a chemical inhibitor.
The researchers found they could use a chemical compound originally developed to treat drug resistance in tumours to disable AmGSTF1’s ability to prevent cell death. They discovered that they could modify the surface of AmGSTF1 and in doing so could show that resistant black-grass plants lost their resistance to herbicides, most notably towards chlorotoluron and phonylureas.
“So now we have this concept – instead of coming up with a new [herbicide] mode of action, why don’t we just bust the resistance mechanism?”
Sadly the cancer therapy compound poses considerable environmental risks and so cannot be used in the field but its activity demonstrates the potential for disrupting resistance mechanisms, opening up the possibility of using herbicide synergists alongside new and existing chemistries to restore chemical control in herbicide-resistant grass-weeds.
“Some way or other we have got to address the resistance mechanism in order to be able to use selective herbicides going forward,” said Prof Edwards.
Edinburgh University, Newcastle University, Rothamsted Research, Sheffield University, Zoological Society of London
This article was taken from our sister publication Farmers Guardian
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