difference between plant viruses vs animal viruses - (Feb/27/2014 )

I have been working on avian viruses for 8 years, but recently I joined a team that wants to express avian viral proteins in plants. The work involves working with plant plasmids, probably called binary plasmids.

1- I don't know how to transform this kind of plasmids after insertion of a gene. I used to tranform animal plasmids into top10 and later extract plasmid. Can I still use top10?

2- Is the plant viral RNA extraction similar to animal viral RNA extraction? With trizol?

they use (most often) agrobacterium tumefaciens to transform the plants. ANd yes you can propagate the plasmids in top10 cells first.

And yes for the second question too. Trizol is also used.

The binary plasmids just refer to the fact you use 2 plasmids. You dont use 1 because it would be too big. You put certain genes on 1 plasmids (vir genes, its disarmed) and the "real" gene (the one you are interested in) on the second plasmid.

Thanks Pito. How big is too big for agrobacterium? Our team has bought 2 plasmids containing the full length genome of tomato yellow leaf curl virus from an Israeli institute. Now, these two plasmids are about 10 kb each I've been told. I haven't checked the sequences yet. Maybe the information that I was given is wrong, but to me 10 kb is not that big to have the full genome split into two. I will check with them on Monday about the exact size.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150518/

Read the above paper , its a good review.

What you do is , simply put, remove all the genes that the A. tumefaciens needs to infect plant cells. You put these on a helper plasmid. The "real" plasmid (with your gene of interest) just keeps your gene of interest and the left and right border (needed for the transfer of the gene).

+ of course all the genes needed for survival (like an ori in e.coli, markers....)

The orginal TI plasmid is about 140-250 kb large (gigantic!)

The size of the t-genes (the genes really transfered to the plant) is about 20-25kb (15 genes +-) , so yeah, your 10kb should work... perhaps even in 1 plasmid, 20kb in total.

(altough, I am not so sure I understand the 2 plamids part ... you mean you have a "gene" that is 20kb lang and its divided in 2 plasmids or the gene is shorter, I can not imagine the plasmid itself is just the gene of interst??)

Anyway: I am prretty sure you can simple use the gene as a whole for the transformation rather than working with 1 gene.

Keep in mind: agrobacterium transformation does not really work well in monocots.

Also: organelles are not transformed.

I am not so sure why you need agrobacterium transformation if you are working with a virus? Why not use the virus itself to transform the plants?

Curtis on Sat Mar 8 10:21:30 2014 said:

Thanks Pito. How big is too big for agrobacterium? Our team has bought 2 plasmids containing the full length genome of tomato yellow leaf curl virus from an Israeli institute. Now, these two plasmids are about 10 kb each I've been told. I haven't checked the sequences yet. Maybe the information that I was given is wrong, but to me 10 kb is not that big to have the full genome split into two. I will check with them on Monday about the exact size.