How to construct a begomovirus infectious clone?

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Guillermo Domínguez Huerta

Hi Harun,

Don't bother to build a partial construct (like 1.4 or 1.7mer). You just need 2 INTERGENIC REGIONS in the same insert. Amplify the begomovirus with RCA, do a partial digestion with a one-cutter enzyme, isolate the dimer, and clone into a pBin19 or any other binary vector. Transform it in E.coli, and if after some anaylses everything makes sense, proceed to extract the plasmid from E.coli and transform it into Agrobacterium.

Is your begomovirus monopartite o bipartite?

How long is the genome? assuming that is aproximately 3 kb, you won't problem to have a 2mer cloned in a plasmid.

Anything you need, you can contact me!

Article One-step cloning approach for construction of agroinfectious...

Harun Kimathi Ringeera

Thank you Guillermo Domínguez Huerta

my begomovirus is monopartite,2.774 nt. I appreciate your advice. I think this is easier than the PCR method. i have an RCA product but my concern was how to cut a partial sequence to combine with the full sequence clone.

Can i use biolistic (gene gun) during inoculation as an alternative to agroinoculation?

monopartite viruses are difficult to inoculate using biolistics - it's not that it never works, simply you'll get a lower frequency of infected plants. If you are going to go ahead with biolistics, you better use high concentrated RCA product of your begomovirus.

Partial digestions can be tricky! I'd try with a good amount of starting material (RCA product), try several short times of digestion (don't discard the Idea of trying a few seconds also!), and inactivate the enzyme very quickly!!!

With a 2mer of the viral genome you will have perfectly functional infectious clone if you transfer it to a binary vector like pBin19.

Let me know how it goes

Best,

Guillermo

Hi Guillermo,

Thank you for the knowledge. I have read the paper you suggested, i think the agroinoculation has a higher efficiency, fast and lower cost. I have used much time trying to get the partial clone. i will go for the agroinoculation. i will share the outcome.

I was able to construct a monopartite infectious clone of TolCV. Digesting a partial dimer with BamH1 and PST1 (450nt) was not successful. Increasing the insert size, i changed PST1 to Xba1 and 1.2kb size digested from full sequence RCA clone was successfully ligated to Pcambia 2300 vector.

Congrats!

Did you try to inoculate Agrobacterium carrying your infective clone into your plant host?

Cheers