Summary auto-generated
Researchers developed a novel gene expression system using tobacco etch potyvirus (TEV) nuclear inclusion (NIa) proteinase to produce multiple viral coat proteins from a single self-processing polypeptide in transgenic tobacco plants. The NIa-based cassette allowed proper cleavage of fusion proteins, generating separate functional coat proteins from TMV, SMV, and incorporating the TEV NIa proteinase itself. Gene constructs were introduced into tobacco, and protein processing was confirmed via Western blot analysis. While transgenic proteins accumulated at unexpectedly low levels—over 100-fold lower than control lines—the proteins that did accumulate remained biologically active and conferred pathogen-derived resistance to TMV, TEV, and potato virus Y (PVY). Plants with mutated cleavage sites accumulated uncleaved fusion proteins, which notably increased in abundance following TEV infection due to protein stabilization rather than increased mRNA levels. This increase was TEV-specific and did not occur with TMV or PVY infection, suggesting interaction with TEV replication products.
Key findings
- The NIa-based expression cassette successfully produced properly processed, biologically active viral coat proteins from single polyprotein precursors in transgenic plants
- Transgenic proteins accumulated at unexpectedly low levels (>100-fold lower than control lines), with C-terminal proteins accumulating less efficiently than N-terminal proteins
- Despite low accumulation levels, transgenic proteins conferred significant pathogen-derived protection against TMV, TEV, and PVY infections
- Mutant non-processed fusion proteins accumulated at greatly increased levels following TEV infection due to protein stabilization, demonstrating TEV-specific stabilization rather than increased gene expression
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Abstract
An expression cassette based on the highly specific tobacco etch potyvirus (TEV) nuclear inclusion (NIa) proteinase has been developed to produce multiple proteins through the translation of a single self- processing polypeptide. Gene constructs encoding TEV NIa, the tobacco mosaic tobamovirus (TMV) coat protein (CP) and the soybean mosaic potyvirus (SMV) CP were used to develop transgenic tobacco plants. Proper processing of the multifunctional polypeptide was demonstrated, leading to accumulation of separate proteins in planta. Moreover, the viral genes expressed in this way were biologically active and conferred pathogen-derived protection to TMV, TEV and potato potyvirus Y (PVY). Transgenic plants were also derived from gene constructs in which the NIa cleavage site was mutated, resulting in the accumulation of the non-processed polyprotein, as predicted. Although transgenic proteins accumulated in low amounts in all the plant lines analysed, accumulation of the mutant non-processed protein form was greatly increased in plants following infection with TEV, but not TMV, apparently as a consequence of protein stabilization.