ИСТИНА

Higher plants are constantly exposed to various biotic and abiotic stresses. Wherein, the same defense response genes have different transcriptional activity in leaves and roots. A typical example is the members of Kunitz peptidase inhibitors (KPI) gene family some of which are expressed only in roots but not in leaves others in both parts of plants. We have identified and isolated from the genome of Nicotiana benthamiana a gene NbKPILP encoding Kunitz peptidase inhibitor-like protein. We showed that NbKPILP lost the ability to inhibit serine peptidases but acquired the capacity to influence the intercellular transport of macromolecules in plant leaves providing protection from pathogens. To prove the new function of NbKPILP we have examined the state of intercellular transport in leaves using a 2xGFP (54 kDa) molecule. Normally, plasmodesmata are not able to pass molecules larger than 40 kDa. We used agroinfiltration technique to deliver 2xGFP-encoding plasmid together with NbKPILP-expressing construct into plant cells. Then we counted clusters of GFP-containing cells 24-30 hours post agroinjection using a fluorescent light microscope. NbKPILP overexpression leads to the increase of the intercellular transport activity: more than 50% of the fluorescent signals are distributed between 2-3-4-cell clusters in comparison with the control where the majority of clusters contain 1-2 cells. Another technique for estimation of NbKPILP role in the intercellular transport is the complementation test, when the function of the defective gene is compensated by another gene in trans. We used tobacco mosaic virus (TMV)–based vector encoding GFP to assess the ability of NbKPILP to complement the function of the TMV movement protein (MP) which is known to effectively increase the plasmodesmata size exclusion limit, performing "gating", and to participate in the transport of viral RNA. The mutation in MP resulted in failure of MP synthesis from TMV-GFP(mut) vector led in GFP accumulation only in single cells. The co-injection of the TMV-GFP(mut) and NbKPILP into plant leaves resulted in the complementation of the defective MP transport function: we observed the multicellular GFP-containing clusters. Thus, we have identified the ability of NbKPILP to complement the transport function of TMV MP. We concluded that the NbKPILP interacts with plasmodesmata and stimulates intercellular transport of macromolecules in the plant. This work was supported by the grant of the President of the Russian Federation MD-5697.2016.4 (T.V.K.) and the RFBR grant 16-34-00062. (E.V.S.)