Pathogen elicitor contamination of bacterial plasmid minipreps

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FlagellinMan
FlagellinMan's picture
Pathogen elicitor contamination of bacterial plasmid minipreps

Hi all,

I am currently using a protoplast system to identify novel inhibitors of plant PTI (Pattern triggered immunity). I bulk-up the plasmid encoding for these inhibitors in E.coli and then isolate plasmid to transfect my protoplasts and express the inhibitor in them and then challange them with flg22. What i noticed is that my no flg22 controls seam to have higher PTI activation than untransformed controls. My suspicion is that some bacterial elicitors are present in the plasmid pre and therefore activate PTI. Has anyone come accross this problem before and if so how did they solve it? Any suggestions?

Many thanks in advance,

Ivan Delgado
Ivan Delgado's picture
Hi FlagellinMan,

Hi FlagellinMan,

Based on your experimental setup description it seems to me that maybe you are adding a tad too much plasmid to your protoplasts. The expression of your inhibitors may be so high that you may be getting the opposite effect (inactivation of PTI instead of activation). One way to make sure this is not the case is to transfect your protoplasts using a range of plasmid concentrations, from too low to too high.