Re: [DIYbio] Plant chloraplast viruses as vectors

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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. ;)

On 18/07/14 11:02, Sebastian Cocioba wrote:
> 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: diybio@googlegroups.com
> Subject: Re: [DIYbio] Plant chloraplast viruses as vectors
> 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.
>>
>

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