Plant chloraplast viruses as vectors

[Yuriy Fazylov]

Can a plant virus or a phage virus with recombinant chloraplast ligands be used to shuttle in genes?

[Nathan McCorkle]

Seems so:
https://www.google.com/search?q=tmv+expression+virus+transformation

On Thu, Jul 17, 2014 at 10:47 PM, Yuriy Fazylov <yuriy...@gmail.com> wrote:
> Can a plant virus or a phage virus with recombinant chloraplast ligands be used to shuttle in genes?
>

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--
-Nathan

[Nathan McCorkle]

I actually meant:
https://www.google.com/search?q=plant+expression+virus+transformation

--
-Nathan

[Yuriy Fazylov]

Wow. Would you look at that. No violent bursts or air that can take out an eye.

[Mega [Andreas Stuermer]]

I know of a study which tried transposon insertion. It was not really stable because they included the transposase gene though.

It disrupted essential chloroplast genes. The chloroplast genome is very condensed, little junk

[Mega [Andreas Stuermer]]

Additionally, the chlorplasts are protected by a double-membrane...
But, theoretically - sure. Prokaryontic translation machinery will insert foreign DNA quite sure

On Friday, July 18, 2014 7:47:56 AM UTC+2, Yuriy Fazylov wrote:

[Cathal Garvey]

Can we keep the snarky notes off the refrigerator please? :)

On 18/07/14 07:57, Yuriy Fazylov wrote:
> Wow. Would you look at that. No violent bursts or air that can take out an eye.
>

--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com

[Sebastian Cocioba]

IIRC all of these viral transformations are for transient gene
expression. I have yet to find a retroviral vector for stable
transformation in plants. Been pondering about modifying a channel on
the plasma membrane of a plant cell to act as a phage anchor similar to
the motifs that bind coliphages. The real trick is the delivery and
assembly of yet another virus while inside the cell which will bind to
recombinant chloroplast membranes and actually deliver the target DNA.
You would still need to have a transplastomic line to modify in the
first place. Why not just use PEG-mediated naked DNA uptake in
protoplasts? Tougher to get right but way cheaper than a gene gun and
will produce stable transformants. Most successful transplastomic plant
species made using PEG was lettuce.

Also, engineering a virus that infects plants and directly,
permanently, alters their genome might be hard to contain and could
pose a scare to the EPA/USDA here in the states as well as elsewhere.
The thought of an HIV-like particle that can inadvertently infect
any/all crops may not be the best idea regardless of what the transgene
is. There are a few patents for universal chloroplast homologous
integration vectors already filed and there are a number of regions
homologous to many species/genera along the myriad genes of the
chloroplast genome so your single virus (if targeting natural surface
proteins) may infect non-discriminately.

An idea could be to transiently express a channel or membrane protein
ligand via agro which will be expressed in tandem with a visual marker
say anthocyanin and then the same tissue is treated with said
phage-like virus to deliver the DNA goods into the cell. If all goes
well, a mechanism that is not well understood will cause uptake of DNA
directly into chloroplast which will recombine, if properly built, to
form stable transgenic plastids.

After that, one would need to wait and wait for that one transformed
cell to gather enough chloroplasts resistant to spectinomycin so that
one single shoot forms. This shoot will be chimeric in nature and have
a decent amount of wild type chloroplasts still within so the leaf of
the shoot is chopped up and further cultured until more shoots form.
This purification process is done several times to ensure homoplastomy.

Last step would be to Cre-Lox out the resistance marker and viral
ligand sequence via transient agro just to be on the safe side. If all
goes well you'll have a STABLE virus-mediated transformation of
chloroplast genome. About a years worth of lab work but a worthwhile
endeavor.

The current hot topic in plant biotech is plants as biofactories. This
does not require stable transformants so do sift through the articles
and ensure that what you are reading is showing evidence of stable, not
transient, transformations. :)

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Cathal Garvey
Sent: ‎7/‎18/‎2014 5:19 AM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Plant chloraplast viruses as vectors

[Cathal Garvey]

I wonder about resolving heteroplastomy.. there's some interesting
tidbits out there about plastid fusions, right? And I imagine autophagy
pathways are as conserved in plants as animals. I wonder if there's
something you could do to your transformant chloroplasts that would lead
them to induce the cell to undergo some chloroplast spring cleaning,
helping to resolve heteroplastomy early?

I know that autophagy can lead to recycling of mitochondria in animals,
I'd expect plastids in plants, too. So, induce autophagy (starvation?
Minor oxidative stress?), and induce mitochondrial damage with something
your transformants are resistant towards (spectinomycin?) so the
untransformed ones are more likely to attract recycling complexes.

Or, study how phages fuse, why, under what conditions, and induce
plastid fusion so there's more cross-over between the plastid pool
within the cell.

Or, pull an IndieBB and make your transformed plastids kill the others
somehow. ;)

[Sebastian Cocioba]

That's a TON of good ideas! Not sure about the inducible autophagy. I
have to admit that since I've been so obsessed with transplastomic
technique, I have embarassingly little knowledge about chloroplast
biology. I know enough to manipulate and hijack some prokaryotic
machinery but little about life cycle.

Im searching for a book by my hero Ralph Bock who underscores all the
important facts about chloroplast and mitochondrial biology of the
plant cell. Its very pricey but may be able to borrow it through
inter-library loan.

The idea of making the chloroplasts secrete a toxin and be resistant to
it if transformed is great but my fear is that it will happen too
quickly and may starve plant of other essential proteins aside from
rubisco that the plant may need and kill the tissue entirely. The
spectinomycin targets 16s ribosome units but the starter dosage during
transplastome selection is relatively tame so it won't kill everything
all at once. Then again the media is supplemented with sugar so
photosynthesis is a luxury. In short, spectonomycin plus secreting
toxins may be too much pressure. Sure as hell want to test your idea
though!

So a construct of such design would look something like this:

HR Flank--Prrn--aaDa--Trps--PpsbA--IndieBB toxin--indieBB
resistance--TpsbA--HR Flank

The flanks would most likely be two transfer rna coding regions and the
construct would be thrown in between as a replacement of the
intergenic "junk" spacer region . Would need to find an area that has
none of the cut sites I use. All minor details.

You could make the indieBB tox antitox circuit polycistronic and throw
in some IEE regions in between the toxin circuit to optimize expression
as an operon and lox sites for good measure. Would need to codon
optimize for chloroplast. Seems doable from a wet lab standpoint. Now
the question is if there is native resistance to indeBB-like toxin.

Maybe invent a protein-based ribosomal repressor specific to
chloroplast? Just throwing thoughts out.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Cathal Garvey

Sent: ‎7/‎18/‎2014 8:52 AM

[Cathal Garvey]

Oh, and because Plastids are polygenomic, you'd also need some clever
way to erase unmodified genomes or induce crossover events.. that's
probably something involving homologous recombination systems like
TALENs or CRISPR which only cleave at the intended target sequence, and
not at successfully injected target sequences.

[Koeng]

The lab I work at is beginning to work with mitochondrial engineering (I'd talk about it but I don't think I'm supposed to), and I heard there was a lab beginning on engineering a phage to infect mitochondria. Not sure how far along that is though. So hypothetically, the idea for chloroplasts should be feasible, but very difficult.

One of my ideas was to import a natural competence system into the mitos or chloros but that'd be pretty difficult and take quite a bit of genetic engineering to the point where its probably not worth it. But hey, it'd be pretty cool to be able to transform any DNA you want and have it recombine into the plastids (if it has homology)

Btw, does anyone know any self selection mechanisms that could be used in mitochondria or chloroplasts? A selfish gene element that spreads itself perhaps, like I-SceI meganuclease. (Its an intron in yeast mitochondria that cuts the tRNA gene. Only when I-SceI's gene is inserted will it stop cutting). That way it could spread throughout the mitochondria, because as you guys know there are like 20-50 mito genomes per cell. (I've only looked at yeast). Or, if you are going for a plasmid system, that's also very possible. The problem with mitochondria is they use really really weird machinery, both transcriptionally and translationally. 

http://www.pnas.org/content/77/6/3167.full.pdf

Yeast mitos use a ton of wobble codons, which makes the tRNA compact, but also changes some of the codon usage rules, so every gene has to be synthesized and recoded.

http://nar.oxfordjournals.org/content/26/3/689.full

Apparently, there are some retrotransposons in yeast mitochondrias, which could be used for a selfish gene

http://www.sciencedirect.com/science/article/pii/0300908496847560

Probably a more amazing thing would be RNA import into the mitochondria. Don't quote me on this, but I heard of a lab that tried doing this with Cas9. They used a Cas9 with a mitochondrial localization tag to try and engineer the genome and I think they said the sgRNA fell off. I don't know though. http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/targetron.html these things encode an mRNA, but on that mRNA is a protein and this intron element. The protein binds to the intron element and looks for DNA to target for insertion of the element. They've found it to be "retargetable" so you can target new genes. (Mitochondrial localization tag + retarget for the chloroplasts/mitochondria. Recombines in, then make it act as a selfish gene element to transform itself into the rest of the genome copies). Anyway, going back to the RNA transport, it may be possible to import the sgRNA for CRISPR with that mechanism, and then put Cas9 on a mitochondrial localization tag, and then you get mitochondria engineering!

(another article on it) http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035321

(and another) http://schneider.dcb.unibe.ch/PDF/publications/Schneid-TCB02.pdf

I might try the "targettron" sometime if I had more money. They literally charge a thousand bucks for the plasmids I want http://www.sigmaaldrich.com/catalog/product/sigma/tv0010?lang=en&region=US , after tax and shipping. http://www.sigmaaldrich.com/catalog/product/sigma/t2826?lang=en&region=US This one is a bit cheaper. I have designs for it in yeast, but haven't actually gone to construct the plasmid. But hey, if there's anyone out there that wants to try my idea on chloroplasts I'd be more than happy to help. 

Going back to the natural competence idea, here's a good review on natural competence with single membrane bacteria (Bacillus subtilis) http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2009.00164.x/full . I gotta go look for another reference, but there's a similar mechanism in Acinetobacter baylyi, a gram negative. (double membrane!) Acinetobacter baylyi is an easy-to-use bacteria that is biolevel safety 1. If you could combine the genes together somehow (protein fusion or polycistronic expression, with the export machinery to the next membrane) and gene gun that big plasmid into chloroplasts or mitos, just maybe you could get the natural competence to be expressed. If it's expressed, genetic modification becomes easier than modifying the genome. The recombination is really cool because you could actually have plasmids in the cytoplasma or nucleus that uses it's awesome repair system, get imported into the mitochondria, a constant mechanism of repairing mitochondria. (Possibly help aging?)

Anyway, those are just some of my ideas. I think the targetrons could work better than phage. With phage, you'd have to go through genetically engineering both the phage and the mitochondria or chloroplasts to get it in there. With the intron/targetron method, not only do you get constant pressure for recombination, you have an enzyme that will site specifically recombine things for you. And even better, if expressed in the genome there will be constant pressure for the plastids to uptake your DNA (the system goes through an RNA intermediate though, mRNA -> Protein, mRNA  -> Protein finds site -> protein does reverse transcription-> DNA inserted), therefore no selection at all is required. Even better, you can recombine anything you want into it, as the intron has an actual location you can clone things into for site specific recombination! But there is a large "scar" region in that method, but you can recombine lox sites or CRISPR sgRNAs to get whatever you need for downstream engineering in.

Things I think could definitely go wrong:

RNA no bound to protein strong enough, gets knocked off when going into mitochondria (heck like a 100bp of RNA got knocked off Cas9)

RNA intron can't recombine that big of DNA, therefore gets stopped

Just my crazy idea. I might try cloning my construct when I get time. If anyone is willing to try my constructs in plants, contact me. Sorry for rambling :)

-Koeng
(Hopefully someone doesn't take my idea xD)