ИСТИНА

Although high levels of recombinant protein production can be gained via transient expression in plant cells, the mechanism by which tolerance to recombinant proteins is achieved remains unclear. We previously showed that “strong” intron-optimised green fluorescent protein (GFP) encoding Tobacco mosaic virus (TMV)-based vector resulted in protein production accompanied by the formation of large membrane-less GFP protein bodies called Y-bodies, which demonstrated mainly perinuclear localisation. The dimensions of the Y-bodies were heterogeneous, approaching the size of cell nuclei. Experiments with extracted GFP and live cell imaging showed that Y-bodies included actively fluorescent, non-aggregated, tightly packed GFP molecules. The plant cells were likely forming Y-bodies to exclude recombinant proteins from normal physiological turnover. Then, we showed that, in contrast to “weak” non-modified vectors, “strong” intron-optimised vectors induced leaf necrosis at 4 dpi, with the appearance of moribund and dead cells at later time after infection. To understand the mechanisms by which protein overproduction can lead to cell death, we constructed forward and reverse suppression subtractive hybridisation cDNA libraries from N. benthamiana leaves at different time points proceeding and during leaf necrotisation. We selected and isolated a gene encoding protein with 50% homology to Kunitz trypsin inhibitor and later named it plant death factor (PDF). PDF mRNA synthesis was dependent on GFP accumulation. In leaves agroinjected with a 35S-GFP construct, we observed the increase of PDF mRNA level, rising markedly after silencing suppression with Tomato bushy stunt virus p19. We concluded that PDF mRNA accumulation could be induced by GFP mRNA produced from both viral and non-viral vectors. Biologically, PDF mRNA increase is likely to accompany cell death and reduce mRNA and/or protein accumulation. To identify the correct outcome, we performed agroinjection experiments with 35S-PDF and 35S-asPDF constructs, latter contained a part of PDF sequence in antisense polarity (asPDF) to induce host PDF gene knockdown. The results showed that leaf co-injection with a “strong” vector and 35S-asPDF resulted in the acceleration of necrosis formation, whereas a “weak” vector drastically increased GFP production after host PDF gene knockdown. We believe that PDF constrains excessive mRNA and protein synthesis and its knockdown leads to the increased protein accumulation from both the “strong” and the “weak” vectors. In the first case that results in cell death, but in the second case the “weak” vector is able to reach the level of protein synthesis of the “strong” vector. In line with this suggestion, our experiments showed that PDF knockdown may be used for pharmaceutical protein overproduction in plants transfected with “weak” full virus vectors as an alternative to gene-silencing suppressors and “strong” deconstructed vectors. The function of PDF in plant cells is not yet clear, but we suggest that PDF mRNA accumulation is increased when cell resources (translation factors, amino acids, ATP, etc.) are exhausted. PDF may be likened to a “bath plug” or “emergency valve” that prevents resource depletion.