Abstract
The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.
Highlights
The tobacco virus resistance gene N contains four introns
Using the full intron-containing gN-Int1234 and intron-less cDNA sequence (cN) genes with the 35S promoter instead of the native N gene promoter (NP2.3), we show that the presence of these introns can enhance the transcript level either with or without elicitor expression
Our finding that the frameshift mutant N gene, gNfs-Int1234, exhibited as high transcript level as gN-Int1234 further supports the conclusion that the interaction between N and p50 is unnecessary for the intron-mediated up-regulation of the N transgene expression under the 35S promoter
Summary
The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. RT-qPCR was conducted using tobacco leaves expressing gN-Int1234 or gNfsInt1234 without the p50 co-expression and it was determined that gN-Int1234 and gNfs-Int1234 produced similar levels of transcript at both 24 and 36 hpi (Fig. 2d), indicating that N protein production is dispensable for high accumulation of these intron-containing transcripts.
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