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Agrobacterium tumefaciens and Cacti

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Crystalito

Rising Star
Senior Member
Here is an idea that came to me recently, please bear with me for an introduction and if in mood check the paper i have attached.


Introduction

Agrobacterium tumefaciens is a gram negative bacterium that can infect dicotyledons and is responsible for the "crown gall disease" in plants.The crown gall appears as a mass of undifferentiated cells that grow on the plant resembling what would look like a tumor.

The "mode of action" of the agrobacterium is fascinating. It usually lives in the soil and when a plant is wounded it is attracted to the wound ,due to chemotaxis being able to locate and move towards sugars and compounds like acetosyringone. The bacterium carries a plasmid ,called Ti Plasmid, that its expression enables it to perform something akin to bacterial conjucation but with the recipient to be the plant cell. It forms a "bridge" with the plant cell and utilising the vir genes, cuts a fraction of the plasmid called "TDNA" ,transfers it in the plant cell and incorporates it to its genome. The TDNA portion contains genes coding for AUXIN production, CYTOKININ production and OPINE production. The first two ,known plant growth regulators, de-differentiate the cells leading to the tumor formation. The opine genes are rather peculiar : they force the cells to make opines which are aminoacids reacted with keto-acids or sugars. So what the bacterium gets out of it? Simply, it feeds off the opines that while the now "genetically modified" plant cannot use ,the bacterium is perfectly capable of utilising!

Given the mechanism of action agrobacterium has received much attention from biotechnology , because if one substitutes the TDNA part with a gene of interest ,it will get carried in the plant cell and incorporated on its DNA, effectively modifying the plant. Here though ,the main interest is NOT genetic modification but the effects of the the "wild type" agrobacterium on the plant and secondary metabolism. It seems ,as far as i have looked around, that such an infection might raise secondary metabolism products by much and some scientists are considering a close relative of tumefaciens, Agrobacterium rhizogenes an option of increasing production of secondary metabolites in hairy root cultures in the lab.

A small collection of articles displaying the effect of the agrobacterium on secondary metabolism (alkaloids, terpenes?) is this :

Functions of rol genes in plant secondary metabolism

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only

Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don

It seems promising, given that such remarks are made within the abstracts :

"The last ones accumulated between 4 (1.1 mg g-1 DW) and 27 (8 mg g-1 DW times more alkaloids than the intact roots (0.3 mg g 1 DW).This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures. "

"Dimeric alkaloid vincristine in the transformed cultures was present at a concentration of 0.011 that was tenfold higher compared to untransformed control cultures. "

Some increase eh?

Searching on literature for cacti i have not found much on tumefaciens, but i found an article on rhizogenes with the title "In vitro analysis of susceptibility to Agrobacterium rhizogenes in 65 species of Mexican cacti" which i am attaching at the end. It seems that the Agrobacterium species CAN infect cacti although at it was thought that it was very rare, at least in nature.Apart from infection ,the paper also examines secondary metabolites using Thin Layer Chromatography and UV light ,Dragendorff reagent and Marquis reagent to visualise secondary metabolism products.So here comes the interesting part :

"Transformed roots induced by A. rhizogenes are
widely used for the study and production of secondary
metabolites from many plant species. With the goal of
verifying if the cacti transformed roots conserve the
biosynthetic capacities found in the normal roots,
alkaloid-like compound production in some of the studied
cacti species was investigated using thin layer
chromatography. In all five species analyzed, and with
the three detection systems used, it was observed that the
patterns obtained in transformed and non-transformed
roots were qualitatively equal (Fig. 3). Also, some of the
spots appear more intense in the extracts obtained from
transformed roots. These results should be confirmed
using more sensitive techniques."

It seems that the secondary metabolism remained the same (same alkaloids) but it seems that in some cases (ones detected as more intense spots) they observed MORE alkaloids. So ,the "increase alkaloidal production" in cacti theory with Agrobacterium starts seeming more plausible!Keep in mind though that this may not be a systematic effect ,but limit itself in the crown gall. If it grows to an appreciable size though ,things could get interesting both biochemically and morphologicaly.



Proposal

In the light of the above ,i wonder if one would want to experiment by infecting cacti samples of "more or less known" potency with Agrobacterium tumefaciens and seeing how this would affect the plant, and propably alkaloidal production.The protocol for such a procedure can be simple ,if one locates crown galls in plants : They could be taken, propably blenderised in some distil water and "subcutaneously injected" in the cacti ,underneath the epidermis or applied in cacti wounds.One could perform a series of such treatments ,given that it might have very low infection rate. My personal recomentation would be to a) have a mixture of different crown galls from different plants, maybe this will ensure more strains in the inocculation liquid , b) using lots of them with little water so the inocculation liquid propably would have high load of bacterium, c)multiple sites of injection : the bacterium attaches to wounded tissue, d) if possible retain the liquid at the injecting site hence the "hypodermic" approach: a "bubble" of liquid could be formed causing also tissue damage if the needle was to be put just underneath the waxy/hard layer of cacti epidermis with orientation almost paralel to the to the axis of cactus growth and the plunger pushed so as the injected liquid to forcefully enter.

If one has access to commercial Agrobacterium strains or somehow can get his/her hands on them ,this would also be a nice idea .The first method though is low tech, requires one to purchace almost nothing just looking around for crown galls which can be easily spotted. A google image search on them will show you how they look.

Tell me what you think of the above please.
 

Attachments

  • agrobacterium.pdf
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Many kinds of infections could induce mescaline production in cacti. I guess the only good part about agrobacterium is its not necessarily a true pathogen so it won't kill the plant while most other pathogens might kill it.

The same kinds of effects may be inducible depending on soil microbes as well and perhaps endophytes.
 
Plus i think you will get a rather productive callus ,both in biomass and alkaloids which can be harvested and processed.I wonder also how it will look and if it will cause differences in cacti morphology : people seem to like "monstrose" and other weird growths so its worth a shot.I think the increases described in the papers seem quite big so its also a quantitative approach.
 
This is one of the coolest ideas I've read on the nexus.

Assuming you were able to inoculate cacti with one of these strains, one thing I'd worry about is the galls containing something else you were not bargaining for, like potentially hazardous agrobacterium products.* You might be able to sidestep these concerns by thoroughly purifying your extract, but then you'd need good analytical tools (like GC/MS) to make sure your final product truly pure.

I'm really interested to know 1) how big the galls can grow & how much of a cactus can be converted into galls, and 2) exactly how much mescaline production increases in galls. Think about it, if you could convert half a cactus' mass into galls, and each gall produced 10x the amount of mescaline, then a single transformed 1-foot tall plant could produce as much alkaloid as a normal 5-foot plant...

*I have some distant recollection of learning somewhere the galls were inedible because they were toxic, but perhaps I am making this up?
 
As far as toxins go i have not heard anything of toxin production but still if you find anything please add it here.The novel compound in the cacti would be opines, but many opines exist in nature for example in octopus.By all means though i urge others to search more aqbout this issue : after all this is just an idea for someone with quite some time in their hands or feeling experimental.

Now on the questions:

1)I think the gals are like tumors : the cactus is not turned into a gall, rather a gall forms and grows. How big? Hmmm difficult to answer, in trees they can get humongous.The only way to answer it is to actually try it.I would suggest multiple sites of application and making sure that a) the innoculant stays in good contact with the wound (subcuttaneous can be a nice idea) b)making the innoculant concentrated and if possible from different crown galls of plants. Come to think of it, the inocculant itself might contain "wound signals" such as acetosyringone that could furtherly promote infection.

2)Exactly how much? This question cannot be answered since there is no study on mescaline specifically, but IF it follows the trends of other examples then we might have a nice multiplication of potency.Thinking of it ,even doubling it could be worthwhile.
 
Just read this thread now and wow.

iv noticed aloe spp's being infected with tumor like growths - totally wild massive undifferentiated growth - and i would be willing to try this experiment.
Would the first step be to - go take some photos of the infected aloes - then collect some infection samples ?
 
Yup, first of all ID correctly as a crown gall, and then collect part of the tumor.I would go for younger growths if possible,its more propable to have live,reproducing bacteria in there.
 
This line of research is very interesting to me, and as a bored biologist looking for something to use his training on, this is a very intriguing. I currently have access to a stocked lab, numerous large SP cacti and miles of forest to search for crown galls; in the interest of science, I'm going to attempt your proposal, quantitatively study the results and post them here when ready (probably a few months). Tomorrow the hunt for A. tumefaciens begins...
 
Nzym said:
This line of research is very interesting to me, and as a bored biologist looking for something to use his training on, this is a very intriguing. I currently have access to a stocked lab, numerous large SP cacti and miles of forest to search for crown galls; in the interest of science, I'm going to attempt your proposal, quantitatively study the results and post them here when ready (probably a few months). Tomorrow the hunt for A. tumefaciens begins...

You will be interested in this project using Agrobacterium-mediated gene transfer

 
Here are a couple links I had from looking into the previous posts project that would also be of interest

agro.org

more info

A source for the organism

Invitrogen + A. tumefaciens LBA 4404


And this piece

YEP medium for Agrobacterium culture


This medium can be used to culture Agrobacterium tumefaciens either on agar plates (add the agar) or in liquid broth culture (don’t add agar). For minipreps, you should grow about 5-8 ml of broth culture.

fill a 200 ml beaker with approximately 150 ml of ddH2O (eyeballing is ok), put a stirrbar in there and place on a stirrplate
weigh out and add, stir until dissolved:
2.5 g bacto-peptone
2.5 g bacto yeast extract
1.25 g NaCl
adjust volume to 250 ml
pour into 500 ml autoclavable bottle

weigh out 3.75 g of bacto agar and add to bottle; do not shake
place stirrbar in bottle;
place lid loosely on bottle, tape in place with autoclave tape; label and autoclave for 15 – 20 min
remove from autoclave and let stir until comfortable to touch; pour into regular petri plates
let dry in laminar flow hood with lids partly open until no more condensation is detected on plate lids; place plates back into plastic sleeve and store at 4 OC until use


culture Agrobacteria at 28 OC in the dark, note that they will take 3-4 days before they are ready to use
parafilm plates before storing at 4 C, make glycerol stocks for long-term storage at -80 OC

SB
 
Very very interesting contributions for both of you! I wonder how the project will turn out, i think at least its worth a try, given that in its simplest form its not THAT demanding. It might take some time and tries to develop a steady crown gall in cacti, i can imagine some infection "dying" or not developing into a gall (being fought/contained by the cactus). As in every "theory" or "prototype protocol" it might need some tweaking around, rarely ideas carry over from paper to practice flawlessly the first time they are tried.

Also, is the "hair on cacti" for real? I always thought it was a "surrealistic art project",something like a rather laborious prank.If its for real, then wow, those guys did a nice work , guided expression when the spines should be would require knowing very well the cacti genome.
 
Well, after conducting some more preliminary research, it turns out that though A. tumefaciens can infect some cacti, it's very rare and the galls aren't permanent (1, 2). A. tumefaciens seems to be able to infect plant cells in general (of receptive species, including cacti), though cacti rarely form a tumor, and when they do form they grow very slowly (14 months for 2.4cm!?)**; plant anatomy may dictate whether or not a tumor is formed and the bacteria can proliferate, though I couldn't find any direct studies. Maybe something about its mechanism (i.e., forced excess of growth hormones) is only effective on normal woody or herbaceous plant anatomy, and the rare occurrence of tumors on cacti is caused by some rare variable that allows the cluster to form in the modified anatomy?

Even so, it's still an intriguing idea and I will continue. I have several ideas in place and have read up on a few methods for inoculation of cacti.

I meant to write more but now I'm late, so I'll respond after the hunt.

*1 2

** Crown Gall on Sahuaro, Michael Levine. Of course, the sahuaro is hardly a san pedro, so who knows?
 
My thoughts were doing an invitro Agro attack on the cactus genome to ativate the hormonal effects in the cactus culture then propagating the resulting culture which A: survive and B: which are carrying the agrobacter changes. This would produce a GMO cactus with the hormonal variation found in crown gall, in theory that the organism is unable to produce an actual "gall" to me is not an indication it has no hormonal effect at all. An alkaloidal examination of the crown gall found on Carnegia would be of primary utility.
At any rate this project is on the burner for next winter after a move to bigger digs and a better space for such projects. I have several friends working in recombinant DNA as resources for reagents and occasional analysis and I would love to use the A tumefaciens in similar fashion to the "hair" project in exploring the active ligands in the Tricho genus.IMO it is an opportunity to perhaps at least learn quite a bit about trait linkage as it relates to overall alkaloid production if not a super-plant.
SB
 
Ah Nzym, the research you share is nice. I wonder also if results can be different "in wild" vs "the lab", meaning that propably purposefully trying to infect cacti you might have better success : try many innoculation points, different innoculation methods and if possible rather concentrated agrobacterium innoculants. I wonder also if using a mashed crown gall vs pure agrobacterium will make a difference ,given that injuring the crown gall to retrieve it might make the plant cells make substances that agrobacterium uses as signals or helpers for docking on the cells (acetosyringone comes to mind...).
 
Well, the hunt was not a success, though I only had an hour or so at my disposal. Next weekend a few friends and I will try again for longer, and we will be able to cover more ground.

Giving it some more thought, even if the ultra-productive tumor thing doesn't pan out, the ability to create the GMO cacti that you described is within the technological limits of the home microbiologist if they can purchase the custom DNA strands; 1 and 2 both present similar in vitro methods of producing viable transgenic cacti.

soxy bastard, what concerns me about your last post is that plant tissues infected by the wild-type agrobacter will be producing up to 200x the normal auxin and cytokinin levels... which will probably rec havoc on development... not to mention the drain on them caused by their production of useless opines.

But thinking about that led me to the idea that perhaps the methods outlined above can be used to, for example, add an extra copy of the gene for O-methyltransferase S-adenosylmethionine (refer to the figure below that outlines the biosynthetic pathway for mescaline), resulting in an overexpression of the enzyme that's mainly responsible for converting dopamine to mescaline*. This could then cause an increase in the rate of mesc production by pulling the reaction to the right... or it could just as likely muck everything up because O-methyltransferase S-adenosylmethionine might be used in multiple biosynthetic pathways... but I'm through with research for tonight.



* Another candidate protein is the one responsible for the rate-limiting step of the pathway... but since I don't know the biochemical kinetics more research is needed, though I do think I once saw a paper that had the relevant data. I'll try to dig it up later.
 
well i went and collected some galls from where i saw them last time - they were red and green and chunky and alive last time i saw them on the aloes - this time they had dried out - like raisins of their previous selves - can they be brought back to life ?
 
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