SUMMARY

The use of plants as biofactories has been proposed as an easy, economical and safe option for the biotechnological production of heterologous proteins and other products for use in medicine or industry. Likewise, plant viruses can be a very efficient tool for the production of these products, thanks to its natural capacity to infect, invade the plants and efficiently express their genetic information. Among them, it is Tobacco etch virus (TEV), a positive strand RNA virus of the family Potyviridae widely used in studies on molecular biology of plant viruses and plant-pathogen interactions. Based on an infectious clone of this virus, this study has developed a disarmed viral vector that allows the simultaneous expression of multiple heterologous proteins in plants.

Since the only available infectious clone of this virus was unstable during amplification in bacteria such as Escherichia coli, in the first step towards the development of the expression vector, three new plasmids were designed and constructed with the same TEV infectious clone that allowed infecting plants with in vitro transcribed RNA (pMTEV), directly with DNA (p35TEV) or by agroinoculation (pGTEV). In the construction, sequences considered redundant were eliminated in the corresponding plasmids and the various functional elements were directed in a logical way to increase the stability of the viral cDNA during propagation in bacteria. The three new plasmids were much more stable than the ancestral and allowed infecting plants efficiently through transcribed RNA, directly with plasmid DNA or by agroinoculation.

The expression vector was constructed based on the plasmid for agroinoculation pGTEV. In the vector, the viral cistron encoding the RNA-dependent RNA polymerase (protein NIb) was replaced by a cassette containing the cDNAs of different heterologous proteins to be expressed. These cDNAs were flanked by sequences corresponding to natural and artificial cleavage sites of viral protease NIaPro to ensure the proper processing of the heterologous proteins from the viral polyprotein. Several versions of the expression vector were constructed, among which are the vector expressing three fluorescent proteins, red (mCherry), yellow (Venus) and blue (mTagBFP), and another expressing two transcription factors (Delila and Rosea1) from Antirrhinum majus  biosynthetic pathway of anthocyanins. When inoculating transgenic tobacco plants (Nicotiana tabacum), which supplement the NIb function through a transgene, the col-localized expression of the three fluorescent proteins was detected both at tissue and subcellular level. The efficient processing of each heterologous protein from the viral polyprotein was also observed. Finally, it was proved that the vector that lacks the RNA polymerase cistron is unable to infect plants in which this function is not supplemented, which is an important component of biocontainment for the technology. In the case of expression of the two transcription factors that activate anthocyanin biosynthesis, the inoculation of transgenic plants expressing NIb caused a strong accumulation of these compounds in tissues infected with the virus that acquired a strong reddish color. These results support the usefulness of the new vector to express heterooligomeric proteins that require a proper assembly of its multiple components and in plant metabolic engineering.

Next, to improve the expression levels of the heterologous proteins from the viral vector based on the TEV, the effect of inserting a second viral suppressor of RNA-induced silencing was analyzed. The results on a vector expressing the red fluorescent protein mCherry showed that co-expression of the P19 protein from Tomato bushy stunt virus (TBSV) increases the expression by approximately 50%, and the 2b protein from Cucumber mosaic virus (CMV) increases it by 20%. In contrast, the 16K protein from Tobacco rattle virus (TRV) has no effect, whereas the HC-Pro protein from another potyvirus, Plum pox virus (PPV), has a negative effect. In a second strategy to improve the disarmed expression vector based on the TEV, the transfer of other cistrons of the viral genome to the host plant was studied. Using a transient expression strategy in Nicotiana benthamiana plants, new cistrons were identified that could be eliminated from the vector and supplemented in trans were, which will improve the vector capacity to accommodate heterologous genetic information.

Finally, the studies about transcription factors from the anthocyanin pathway led to the development of a new system to visually track the dynamics of a viral infection process in plants. The system is based on the expression of the transcription factor Rosea1 (Ros1) of 25.7 kDa that induces colored anthocyanin accumulation in the tissues where the virus replicates and moves. The amount of red pigment was observed to correlate with viral load, so that the marker was found to be quantitative. In addition, this marker was shown to be very stable during serial infective passages in tobacco plants. In addition to TEV infection in tobacco plants, the marker was shown to be helpful in tracking the infection of turnip mosaic potyvirus in Arabidopsis thaliana plants and Tobacco mosaic virus or Potato virus X in N. benthamiana plants.