Hello All,

I am having an issue that is conceptually still very confusing for all those who know about the Geminiviral based protein expression vector used to produce recombinant proteins in Plants. We use Nicotiana benthamiana.

Geminiviral vectors enhance the DNA copy numbers delivered into the plant cells mediated by agrobacterium-based infiltration.

When I am expressing cytosolic GFP, the system is working perfectly. We can see up to 10-folds of increase in the protein expression when Rep (a protein needed for the replication of DNA in Geminiviruses) was co-infiltrated which is conceptually correct.

As soon as we target our protein of interest (including GFP) to the Endoplasmic reticulum by adding BiP leader sequence and ER-retention sequence, HDEL, it seems that expression drops significantly in the presence of Rep. Even the basal level of protein expression (without Rep) also drops if I compare it to proteins expressed in the cytosol. It contradicts the concept. DNA copy numbers are increasing in the cytosol so there should be no effect on protein expression in the cytosol or in ER. Its well known that ER is the much-preferred place for recombinant protein production. So its expected to have much higher expression in ER rather than Cytosol.

Please share your experience/suggestions/ tips.

All are welcomed

Thanks in Advance.