May 23, 2023
Written by: Peter Newman
We’ve talked in the past about the concept of the dimmer switch. There are times when the mood tells us it’s time to turn the lights down low, put on a bit of Barry White, and talk in sweet, dulcet tones. Mmmmmm!
And then there are times when you turn the lights up to full noise, like at last drinks at the pub, and the tones aren’t so dulcet!
One more, please!
This dimmer switch analogy has been used to describe the turning up of P450 genes to overproduce an enzyme to detoxify a herbicide.
Now we have a new concept for you.
The person who turns up the dimmer switch.
The dimmer switch can’t turn itself up, right? It takes someone to realise that it’s closing time and we need the lights up to send the message to the unruly crowd that it’s time to go home.
This person is ‘upstream’ of the dimmer switch, and they are called the TF. The ‘Transcription Factor’.
What on earth are we on about here? Well, it’s the research of visiting Chinese researcher to AHRI, Dr Lang Pan and his group, and Associate Professor Qin Yu plus others from AHRI as part of a global effort to understand metabolic herbicide resistance.
The researchers identified 91 P450 enzymes in a weed called Beckmannia syzigachne (Slough grass), the most problematic weed in wheat and canola in China, as well as in other countries. They then identified a single P450 enzyme linked to metabolic resistance to three ALS herbicides.
And then they went one step further. They identified the TF. The Transcription Factor is the enzyme that binds to the P450 gene and tells it to turn up the dimmer switch. This results in overproduction of that particular P450, which in turn breaks down the herbicide before it can do its job.
The global push to understand metabolic herbicide resistance is now gaining speed, and the research has come a long way. This is high-level science that mortals like myself find very difficult to understand. Thankfully, we have great researchers like Dr. Todd Gaines who can ‘dumb it down’ for us with analogies like the dimmer switch to give us a chance of understanding it. Thanks, Todd.
How did the researchers find 91 dimmer switches, then find the single person in that pub who had the ability to turn it up?
Step 1. Look for known target-site mutations
The researchers sequenced the ALS gene (think group B/group 2 herbicides) and found that none of the common target-site mutations existed. There are nine known target-site mutations, and none were present in the 50 seedlings tested that had already survived Mesosulfuron methyl (e.g. Atlantis®). This suggested that the resistance was not due to a target-site mutation. Rather, herbicide metabolism studies revealed the resistance is due to enhanced mesosulfuron detoxification.
Step 2. Identify all of the P450 genes
The researchers identified 91 P450 genes in the resistant Slough grass, and five of these were identified to be up-regulated (dimmer switch turned up a bit). This was then narrowed down to two candidates that had high-level expression (dimmer switch turned up to full noise). Further testing identified the main culprit. The gene for this P450 was cloned and its further expression tested to confirm that they had found the P450 likely responsible for the resistance.
Step 3. To be sure to be sure – enter Rice calli
This gene was then over-expressed in Rice calli and the calli and the regenerated rice seedlings then exposed to mesosulfuron methyl. They found the transgenic rice seedlings became resistant! Conversely, when this similar gene was knocked out from rice, the rice became more sensitive to mesosulfuron methyl. The researchers were now confident that they had found their guy. BsCYP84Q32. Even sounds like a suspicious character!
Further testing was done, including looking for differences in metabolites, and the researchers now knew they had definitely found the P450 responsible for the ALS resistance. It was due to demethylation. Big word, but essentially it means that the methyl group was knocked off the molecule by the P450 – so mesosulfuron methyl became the artist “formally known as”, and changed its name to mesosulfuron (with poor old methyl left harmlessly floating around the cytoplasm).
Step 4. Who turned up the dimmer switch?
Not content with simply knowing that the dimmer switch was turned up resulting in the over-expression of a P450, the researchers continued their crime scene investigation to find out who turned up the dimmer switch. You guessed it, it was BsTGAL6, a Transcription Factor that binds to the promotor sequence of the P450 gene, turning up the dimmer switch to over-express the P450 gene, and therefore overproduce the BsCYP84Q32 P450.
Researchers around the world felt as though they had picked the easy fruit in understanding herbicide resistance by describing many target-site mutations. Metabolic resistance is a much more complex beast, and our researchers are now not only describing the specific enzymes involved, they are also working out what is causing the up-regulation of these enzymes.
Will the next step be to stop the person turning up the dimmer switch (reverser resistance), to find how many switches the person can turn up (regulating multiple events) and to find who tells the person to turn up the dimmer switch (haha)? Stay tuned!
Metabolic resistance to acetolactate synthase inhibitors in Beckmannia syzigachne: identification of CYP81Q32 and its transcription regulation
Posted in: AHRI Insight, Gene discovery for herbicide resistance