RNA Interference, known as RNAi, is a biological process that leads to the silencing of gene expression. A lot of plant viruses are RNA viruses including grapevine leafroll-associated virus and grapevine red blotch virus. Yen-Wen Kuo, Assistant Professor in the Department of Plant Pathology at the University of California, Davis is researching ways to induce RNAi in grapevines to target virus. Growers may have heard of double-stranded RNA sprays which are intended to initiate RNAi. The challenge has been that double-stranded RNA breaks down quickly in the elements. The Kou lab is working to improve this process and look for alternatives that will have little impact on the ecology.

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Transcript

Craig Macmillan 0:00

Our guest today is Yen-Wen Kuo. And she is Assistant Professor in the Department of Plant Pathology at UC Davis. I'm Craig Macmillan, your host, and I'm very excited to have Dr. Koh here with us today. Welcome.

Yen-Wen Kuo 0:11

Thank you for having me.

Craig Macmillan 0:13

So you've been doing some interesting work the lab on interference RNA, and also how it affects plant viruses and possibly insects in the future. Can you explain for those of us that did not take genetics like we were supposed to in college, what interference RNA is and how it works?

Yen-Wen Kuo 0:29

Sure. So RNA interference is a biological process in which certain types of RNA RNAs can trigger RNA interference. And then once it's triggered, it will produce specifics more RNAs, that can regulate gene expression, by degrading or binding to the target RNAs containing a homologous sequence containing a similar sequence of those small RNAs. So this is a general concept of RNA interference, we also call it RNAi is very complicated the whole process. And there are different pathways and mechanisms included in the RNA interference. RNAi is a primary and effective antiviral defense in plants, but also found in some fungi and insects and lower eukaryotes. And because of all these different mechanisms, scientists and researchers, they they work on different aspects of this mechanism for either plants or animals. And they're also looking for different potential and better ways to use RNAi for different applications.

Craig Macmillan 1:45

So if I understand correctly, you have cell and there is DNA in that cell, and there's genes that code for certain things. And so the RNA is was transmitting or was carrying information from that's encoded with the gene out into the world to do something, is that a fair explanation?

Yen-Wen Kuo 2:05

So the genome there in plants or animals and human is their DNA genomes is DNA, and then the DNA will transcribed into RNA. And those RNA, some of the messenger RNAs can translate into proteins. So it's a how the central dogma from DNA makes RNA and then RNA makes protein. In the old days, we thought that oh, the protein is the important things because the protein can have different functional, different functions in different ways to to regulate everything in the body or in different organisms. But then afterwards, we found that actually RNAs they have many different forms and they can function at the RNA level. So it can interfere with gene expressions and many different things.

Craig Macmillan 3:03

And how does this apply to plant viruses because you've done some really exciting work with Gemini viruses, I believe with grapevine virus a Tell me a little bit about that work and how that works.

Yen-Wen Kuo 3:15

A lot of plant viruses, they are RNA viruses, a lot of those devastating viruses in grapevines, for example, grapevine leaf roll associated virus or grapevine red blotch virus they. So grapevine leaf roll associated viruses and RNA virus and grapevine red blotch is DNA virus. So there are different types of viruses. And so my work is trying to use different viruses making them into viral vectors to induce RNAi in Grapevine plants, to target those important viruses causing diseases in the field for the grapevines. And because so for example, when the viruses they are infecting plants, they will trigger RNAi in the plant, so that plants can protect themselves from virus infection. And because of that, we're trying to develop viral vectors can trigger RNA interference to target those viruses that's causing diseases. The work I have on the grapevine Gemini virus A that GGVA is to either develop the virus into viral vectors to target RNA virus first. So that's the initial plan for us to use. GGVA the grapevine Gemini virus A target grapevine leaf roll associated viruses. So before we eventually target that virus, we have to do a lot of different tests. We need to know if the clones the constructs or DNA constructs we have of this, GGVA can actually affect Gravelines plants, so we have to do that. And then we want to see if we can develop it into viral vector to carry the sequence we want them to express in grapevines to do the work we want them to do. So then we use it to target genes in the plants to see if they can silence the genes in the plants. So then we did that, we found that yes, we can use that viral vector to silence genes in plants. And then now we try to see that if we can use this viral vector to target other RNA viruses, or other grapevine RNA viruses, because we are actually at the same time developing different viral vectors, and one of them is GBA, is grapevine virus, a another's name, it can be very confusing. GGVA is a DNA virus. GVA is an RNA virus totally different to viruses. So since we have both viruses in the lab, so first, we try to prove the concept. We use the GGVA, the DNA virus, to target the GBA wild type virus, to see if we can see any effects. The GBA infection viral titers in the infected grapevines. So this is what we're working on right now. And so eventually, we want to use this viral vector, and potentially other viral vectors to to target grapevine leaf roll associated virus. And maybe we can use it to target mealybugs too.

Craig Macmillan 6:35

How are these vectors introduced to the plant?

Yen-Wen Kuo 6:38

We modify from the previous reports how people try to deliver those constructs the plasmids into grapevines. Most of the experiments or the assays, from before, they needed to have grapevine plants grown from in vitro, on media or from embryos. But that's really a lot of work. And it will be harder to have applications in the field. So then we develop vacuuming filtration method that we can directly vacuum infiltrate those plasmids that those DNA construct plasmids directly into the greenhouse grown grapevine plants. So those plants are propagated from the cuttings and then those plants, they are usually maybe 12 to 19 inches high above the soil when we infiltrated those plasmids into those grow vine plants. So this is an we got pretty good results, we successfully introduced those DNA constructs into the grapevine plans and those constructs can be infectious and initiate the whole the virus replicate in the grapevine.

Craig Macmillan 7:50

So is this something that can be done in a nursery then with new plants? And basically, they then would come with the vector or is it something you could do in the field?

Yen-Wen Kuo 7:57

Yes, I think the plan is that we can introduce those plasmas in the nursery in greenhouse plants before we plant them into the field. So then the plants that's planted into the field, they can have this viral vector to protect the plants from specific viruses.

Craig Macmillan 8:18

Got it. That's really neat. That's a great idea. And it's pretty cool. So that's fantastic. And in the work that you're doing so far, it sounds really exciting. And it sounds like the direction that you're kind of going in the future is with leaf roll virus that you mentioned. And then also, interaction with mealybugs you mentioned. Can you tell me more about that? What's that work all about?

Yen-Wen Kuo 8:39

Because this virus does GGVA and other viral vectors we're working on to a lot of viruses infecting grape vines, their phloem limited virus, so this GGVA is also phloem limited, meaning that the virus is can only infect the tissues around or in the phloem is restricted. It doesn't go to like mesophyll cells or epidermal cells in infected plants, because mealybugs they feed on phloems. So we think if they can pick up those RNA interference signals, may be those RNA interference signals those small RNAs can target mealybugs too. So we can choose different target sequences in mealybugs. Hopefully you can see some effects for many bucks to to prevent that from transmitting viruses or have lethal effects for mealybugs. That's the plan. Hopefully we can do that. But we have to do tests to see how the efficacy and everything though it can have mealybugs, because there are previously they are different studies they use RNAi on insects, and many people prove that they can see some effects. We hope that the viral vector approach can also use for really apply this into the field for grapevine plants.

Craig Macmillan 10:00

What kind of index on insects are we talking about?

Yen-Wen Kuo 10:03

Depends on what target genes or sequences we choose. For my first choice, I would like to have a target that can prevent the transmission of the virus by mealybug, that will be my choice. I'm not sure if it's good to kill the insects, if it's going to affect the ecology too much. So if we can make the mealybug not transmitting the virus or other diseases, I think there will be a very good first step if we can see a lower transmission rate. And and then we can see if we need to adjust from there.

Craig Macmillan 10:40

That is amazing. And we haven't, yeah, the little bit of research that I did we have we do have proof of concept basically on this in other cropping systems. Is that right?

Yen-Wen Kuo 10:55

Yes,

Craig Macmillan 10:55

Can you tell me a little bit more about that, because that might give us some some vision of where we might go in the vineyard industry.

Yen-Wen Kuo 11:01

So, the RNAi applications, people are already trying to do some of those works. So, one example is that before people can spray double stranded RNA into the field. So, let me talk a little bit about the introduction of why using double stranded RNA. So, there are different types of RNAs that can induce RNA interference, certain types, one of them is double stranded RNA, either double stranded RNA or the single stranded RNA, they can form into a secondary structure in folding into a structure like a hairpin RNA, those are found to be able to induce RNA interference. And there's also other things like artificial micro RNAs, there are different types of RNAs that can induce RNAi and most convenient ways to make double stranded RNA. And people have been synthesizing the double stranded RNA or using bacteria to produce those double stranded RNA and then spraying to the field to get some protection for the plants. It worked at some level, but it's just not stable enough. Although double stranded RNA is more stable compared to single stranded RNA, steroids and RNA can be degraded in the field with the sun and everything the whole environment it can be degraded, people started to look for ways like bio clay to protect the RNA, and then so, they can spray in the field. So, the RNA can last longer and cause the effects. So, those double stranded RNAs can be absorbed by the insects, they can pick up from the surface of the plant or the plant can absorb those double stranded RNA into the plants. So, those are different ways and people started to see some effects on that, but still, we have to improve those different methods delivering double stranded RNA or other types of RNA to induce RNA interference in the plant. So, they are different different approaches. So, one of that is now we are trying using virus to introduce the RNAi to induce the RNAi in the plants. So, people are trying different ways to deliver those specific RNAs to induce RNAi to target specific diseases, sometimes not just viral diseases, that they will try to target fungal disease or something else and insects. This is what many different groups they are trying to do also previously, another way is to try to make transgenic plants. So if we can make plants to express those RNAs that can induce RNAi targeting to specific diseases, then you don't need to really use any tool to the deliver because the transgenic plants itself can produce those RNAs doing to induce RNAi plants. So that's also another way that people are trying to do we call that host induced gene silencing HIGS, and the virus induced gene silencing is the way my group is working on and we call it VIGs vigs. So there are different ways that which we would use to introduce those RNAs to induce RNAi in the plants.

Craig Macmillan 14:31

And right now you are at the greenhouse stage, if I understand correctly.

Yen-Wen Kuo 14:35

Yes.

Craig Macmillan 14:36

Have you introduced mealybug into your experiments into your work yet?

Yen-Wen Kuo 14:40

Not yet. We are just working on targeting grapevine virus first to see the effects. So where we have to continue monitoring those tested plants to see if the effects can last long, and the efficacy and how good they can be. So now we're at four for five months, so it's still we can see the targeted virus is being suppressed in a very, very low titer. So GVA can cause some symptoms in the grapevine plants when they see the plans are infected. But we have to peel off the bark to see the symptoms, we want to see that after targeting to the GBA virus, we saw that the viral titer is very low, if we can see that, also, the symptoms is not there anymore, is now like wild type, when when the virus was infecting in the plants alone, if we can see the difference, we don't even see the symptoms there will be really great. And this part, hopefully I can collaborate with the collaborators, Maher, he's run the foundation plan services, he can help my group on this, to see that how good the effects can be using this GGVA viral vector. So after that, if we can successfully target two different viruses, then we will start to work to change the target sequence in this viral vector to target mealybugs. So that's after the virus work.

Craig Macmillan 16:12

Yeah, well, that's very exciting. This is a really fascinating idea, and obviously is still relatively new. And I think it's really great that you and everybody else is working on this sounds like there's tremendous potential, and I hope that you folks continue on are able to continue on, is there one thing really related to this topic, you would tell growers one thing that you would advise them or you would educate them with?

Yen-Wen Kuo 16:34

I understand that there could be some concerns and maybe doubts, questioning RNAi applications in the field, because before, they already probably heard about the spray of double stranded RNA or other methods, and they saw some effects but not stable enough. So they may have some concerns or doubts, I think many scientists are trying different delivery methods that can be applied efficiently in the field. And we will do different types of tests and trials to make sure we work on any potential issues of this technology before applying them in the field and try not to affect the whole ecology or anything in the field too. And obviously, the current approaches we have are not enough to keep certain grapevine diseases, at low enough incidence. So we have to explore more potential control approaches before those diseases get worse, and adjust the ways to manage those different grapevine diseases with this changing environment. And I think hopefully, we can all work together to achieve this same goal. And I understand this is something new, I hope everyone can keep an open mind and willing to work with us to do different trials and see if we can improve different approaches to control different diseases.

Craig Macmillan 17:58

Well, I hope so too. grape growers are very creative. And they're always looking for solutions to their problems that very much fit what you're describing. And it sounds to me, this could be another tool in the IPM toolbox that may not be the single solution may not be a silver bullet. But it sounds very exciting that it may play a very important role to improve the efficacy of other techniques we have, which is great. Where can people find out more about you?

Yen-Wen Kuo 18:22

So because I will, setting up my lab, so hopefully I can have a lab website soon. I don't have accounts at Twitter or Instagram.

Craig Macmillan 18:34

Neither do I.

Yen-Wen Kuo 18:36

I don't use social media a lot. So my email that people can reach me through the email. And hopefully, when this is up or in your podcast, I will have my lab website set up so people can find us our work, my lab website.

Craig Macmillan 18:53

And we will have links and everything else that we can find posted on the episode page at the Vineyard Team podcast website. I want to thank you for being on the program. This was really, really interesting and is a kind of a view into the future of what's possible. Yeah. Our guest today was Dr. Yen-Wen Kuo. She is with the Department of Plant Pathology at the University of California Davis. And I want to thank you for being on the podcast.

Yen-Wen Kuo 19:20

Thank you for having me on the show. I really appreciate this opportunity to talk about research to explain some details about our work to the course and hopefully, I answer some questions that growers might have. I look forward to in the future maybe collaborating with different people to make this thing to work.

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189: RNA-Based Vaccination for Grapevine Viruses

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