Plantae Presents: Dolf Weijers and Dana MacGregor

Additional Q&A with Dana MacGregor

• Could the VIGS and VOX technology be applied to crops in the field?
  • The biggest hurdle to using VIGS / VOX out in the field is that it relies on a transgenic virus to deliver the genetic change, so there are all sorts of questions regarding how do you control specificity and transmission added to questions of safety and regulation… so although there may be no major scientific reasons that it couldn’t happen there are plenty of issues surrounding regulatory, ethical, public approval..

• Is there a possibility of inserting a gene into blackgrass for release into the environment as a method of biological control
  • The idea of Gene Drive for use in weeds has been explored really well in Paul Neve’s paper and I recommend you check this out (if you haven’t already) for an elegant discussion on the topic https://doi.org/10.1002/ps.5137. However, virus-mediated gene modifications do not create stable transgenics – the virus itself is what is inducing the gene expression change and there is no integration into the black-grass genome –  For the virus effects to be passed from plant to plant, there would need to be direct contact transmission – this might be practical between plants in a patch, but from patch to patch, or field to field… might be a big ask. 

• Hello. Than you for the nice talk. Question: What is the ultimate goal of studying weeds at a molecular level? Why not only focus on the economical crops?
  • In order to fix something, we need to know what is broken. I feel that only when we understand something at the molecular level – what protein is interacting with what or what promoter is on when – then we truly understand what is happening. We are trying to ‘fix’ anthropogenically-driven evolved herbicide resistance in weeds and to do this, we need to know what molecular changes have resulted from overuse of herbicides. As for why not just focus on crops, well no matter how good your crop is, as the data show, if there are weeds competing with it, you can and will not get the full yield.

• How long does the process of transformation take? Is it heritable?
  • The process of transformation takes a couple of weeks to manifest because the virus needs to be propagated through from cell to cell, and leaf to leaf. It is different in each species, but in our hands, VIGS is visible in 5-11 days after infection, and VOX was visible 10-14 days after infection. As for heritability, there are some data that indicate there is potential for the silencing effects of VIGS to be transmitted to subsequent black-grass progeny through seed as there is precedent for this in other species (Bruun-Rasmussen et al., 2007, Jackson et al., 2009, Senthil-Kumar and Mysore, 2011, Bennypaul et al., 2012). As far as we are aware, there are no reports of FoMV-conferred phenotypes being able to be passed to subsequent progeny. 

• Are there instances of auxin herbicide resistance in black-grass? If so, any ideas on the mechanism of resistance?
  • Short answer… not yet … but give black-grass enough time and exposure, it could probably achieve it. Joking aside, synthetic auxin herbicides are mostly used to control broadleaf weeds not monocots like black-grass so they aren’t sprayed often on fields that are infected with black-grass. There is a great recent review at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175398/ on weed’s resistance(s) to synthetic auxin herbicides if you want to know more about details and mechanisms and I would direct you there for more information. 

• Did you find any other candidate gene for herbicide resistance in black-grass?
  • There is a “shortlist” of published genes that have been identified from the transcriptomics and/or proteomics screens I mentioned… but we haven’t been able to test them yet. AmGSTF1 was a logical place to start – it has gotten a lot of attention in the academic and farming press so much more is known about it than any other black-grass gene. But… this is a relatively new project and I haven’t had time to test anything else. I am currently working on securing funding to push through and test more! I’ll eventually be recruiting Technicians / Post-docs to do that work! Meanwhile, I continue to use unbiased methods to explore the genomics and transcriptomics we’re working on to generate new candidate gene lists. Watch this space… 

• Nice technology. What about physiological differences between weed and crop? Hormones distribution, nutritional, etc. It will be great to combine molecular and physiological mechanisms. One can even easily protect from weed by physiological mechanisms.
  • This is a grass weed that grows in grass crops, so physical differentiation between crop and weed is difficult… so much so we wait until the weed is flowering (with its very distinct flower heads standing well above the crop) before we map fields for black-grass presence or abundance. As for phenology, black-grass germinates concomitantly or after the crop has been sown and drops its seeds entirely before the crop is harvested, so the lifecycle is pretty well matched too. There are probably metabolic differences, and I have heard of people using different imaging techniques to separate weeds from wheat, but these are not approaches I am taking.

• There are different physiology in weed and wheat. What effect does this have?
  • How similar could be mechanisms of resistance in Arabidopsis and blackgrass? Arabidopsis, as a small dicot, is quite different from black-grass phylogenetically (see the tree at https://www.nature.com/articles/srep34066 for instance) and they have had very different selection histories and pressures – you will not find Arabidopsis growing in a winter wheat field or black-grass on the kerbsides. But I can and others have used Arabidopsis as a tool – for heterologous expression, for learning about homologous gene functions, for information…, looking in Arabidopsis is like studying apples to learn about pineapple. Although, I never underestimate the power of Arabidopsis to teach us new things.

• Hi, very nice talk, thx so much for your time. How do you deliver VIGS/VOX system into the black-grass? are they stable transgenic plants?
  • These are not stable transgenics. It is really the plant’s response to the invading virus (in our case the transgenic virus) that is inducing the gene expression change so no, there is no integration of anything into the black-grass genome. The methods to get the virus into the plant, we use rub inoculation, which is really nicely explained here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510315/). As for transgenerational effects, for the expression change to be propagated from generation to generation, the virus would have to go through to the seed and be viable in the next generation. There is some evidence this can happen in other species (Bruun-Rasmussen et al., 2007, Jackson et al., 2009, Senthil-Kumar and Mysore, 2011, Bennypaul et al., 2012) so it is possible… but hasn’t been tested in black-grass.

• What contributions can genomics and transcriptomics play in herbicide resistance weed science?
  • The beauty of genomics and transcriptomics is that they present to you what is 1) possible and 2) happening in the plant at the gene level. So with the genome we will be able to investigate what genes are present, in what variations, and how many copies, and then through transcriptome, we can see how much they are expressed and what splice variants are present. Enough of each type of data also allows us to explore differences between the phenotype-genotype relationship – what is similar or different and how it relates to the exhibited phenotypes.

• Would the transient transformation system work in reproductive organs?
  • Maybe… There are some data to indicate that the VIGS effects can be passed through to the next generation in other species (Bruun-Rasmussen et al., 2007, Jackson et al., 2009, Senthil-Kumar and Mysore, 2011, Bennypaul et al., 2012), but this is probably not the case for VOX as there are no reports of VOX being passed into reproductive organs. We also see that GFP declines over time and it isn’t visible throughout the plant’s lifetime – although these data didn’t make it into the paper. There are limitations on how long the virus, with its insert intact, can replicate within the plants… as soon as the insert changes or gets removed from the viral genome, then the effect will not happen. 

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