ИСТИНА

In contrast to mammalian cells, relatively little is known about programmed cell death (PCD) in plants. Recently, we selected and isolated from Nicotiana species a plant death factor (PDF) gene. PDF mRNA level is very low in intact leaves. However, PDF mRNA accumulates significantly in plant cells as they initiate PCD induced by the Tobacco mosaic virus and viral-vector-directed protein overproduction. Although the PDF protein sequence shares 50% homology with the Arabidopsis thaliana Kunitz trypsin inhibitor (KTI), the PDF protein has no trypsin inhibitor activity. To identify the function of PDF, we conducted agroinjection experiments on the PDF sequence with antisense PDF (asPDF) and showed that host PDF gene knockdown resulted in an increase in viral-vector-directed protein production. We believe that the PDF protein constrains both excessive mRNA and protein syntheses, and PDF knockdown leads to increased protein accumulation. To gain a clearer understanding of the mechanism of PDF regulation of PDF mRNA accumulation in intact N. benthamiana leaves, we identified a sequence embedded in the PDF mRNA nested open reading frame (nORF) that encodes a 53-aa protein (53nORF) without homology to the PDF protein. The A. thaliana KTI sequence does not have an ORF similar to the 53nORF. We show that 53nORF fused with the GFP gene is detected in agroinjected N. benthamiana leaves. The biological role of 53nORF in the regulation of the PDF protein was revealed via two approaches. First, we agroinjected leaves with a construct encoding the 53nORF, which drastically suppressed viral-vector-directed PDF mRNA accumulation. Second, mutagenesis of the 53nORF AUG start codon in the sequence of the PDF mRNA increased PDF protein expression levels and accumulation of the corresponding mRNA 8-10-fold in plant cells. We conclude that 53nORF is responsible for constraining the expression of the maternal PDF gene in intact leaves. PDF/53nORF represents the first example of an endogenous nested gene functioning as a repressor of a maternal gene in a plant. Thus, we have uncovered a complex mechanism that may be used in plant genomes to evolve new functions from pre-existing genes.