Biochemistry of N Gene Function

(Vannesa Handley, University of California, Berkeley)
Employing Agrobacterium inoculation as a delivery system, we are introducing expression constructs containing various portions of the viral replicase into tobacco. These constructs are then scored for their ability to elicit N-dependent HR at the infiltration site. Through use of this technique it was demonstrated that a construct containing a C-terminal 50 kDa portion of the replicase was sufficient to elicit HR while constructs containing more minimal 43 and 46 kDa portions failed to trigger this response

Immunodetection of protein fragments: As a counterpart to the assay above, protein extracts from inoculated plants are prepared and subjected to immunoblot analysis. Replicase fragments were constructed with C-terminal FLAG epitopes to facilitate immunodetection. Interestingly, a 50 kDa fragment is easily visualized on a Western blot while the expected 43 and 46 kDa fragments are not. This result suggests that the failure to elicit could be due to a lack or instability of expressed protein. To address the possibility of protein instability, GFP fusion constructs were created. In a number of plant systems it has been demonstrated that expression of proteins as GFP fusions can produce enhanced stability without functional disruption of the fused protein of interest.

Agrobacterium harboring GFP (a negative control), GFP:p43-FLAG, and GFP:p50-FLAG constructs were generated. Inoculation with GFP strain failed to elicit an HR. Conversely, inoculation with the GFP:p50-FLAG strain led to formation of necrotic HR lesions in the infiltrated tissue. These findings strongly suggest that the failure of p43 to elicit is a function of low (or non-existent) protein levels If the recognition of the elicitor is structure dependent, than perhaps the GFP stabilized p43, though still containing sequences/domains essential for elicitation, is presenting a disrupted or overly labile structure. Conceivably this could reduce the number or affinity of interactions leading to host recognition.