Laboratory for the Molecular Basis of Plant Stress Resistance

Scientific results:

To develop approaches to the creation of new lines and varieties of potato that can adapt to the climate change and its consequences, we have studied the combined effect of viral and bacterial infection and on potato plants. At the initial stage we conducted the phenotypic and physiological analysis of 19 varieties/ variety lines of potatoes from the collection of the selection genetics center of «Doka-Gene Technologies» Ltd («DGT» LLC) that vary in resistance to increased temperature, potato virus Y potyvirus (PVY) and Clavibacter michiganensis bacteria that are individually or collectively susceptible to these stresses. For the phenotyping of potato plants reacting to individual and combined stresses, new approaches have been proposed that combine classical (physiological) and molecular biological, microbiological and biophysical methods. Our researchers have found new types of interactions between stresses: (a) virus-bacterium (interference or synergism), virus-thermal stress (synergism) and bacterium-thermal stress. The collected data is important for  practical plant production and can be recommended for the zoning of potato varieties in the context of the climate change. It has been demonstrated that Clavibacter michiganensis is widespread in Russia on potato plants and sugar beet, which confirms the practical importance of the proposed model for the study and improvement of the resistance of potato plants to combined stresses.

We have created transgenic lines of potatoes with hyperexpressed genes of nuclear proteins - coilin, poly (ADP-ribose) polymerase (PARP) and fibrillarin. The Laboratory has produced model potato plants with coilin, PARP and fibrillarin expression inhibited by RNA interference. It has been demonstrated that a  hyperexpression of the corresponding genes leads to increased sensitivity of potato plants to combined stresses, while a deficit of these proteins leads to increased resistance.

We have conducted a proteomic profiling of potato varieties contrasting in their resistance to thermal stress and infection by the YBK virus that had been determined at the initial stage —  of the varieties Gala (resistant) and Chicago (sensitive) at early and late stages of infection. To study the quantitative changes in the proteomes of both varieties, we performed a mass spectrometry analysis of proteins related to markers for relative and absolute quantification (iTRAQ). The Chicago variety demonstrated rather weak deviations in the quantitative and qualitative composition of the proteomes in response to individual and combined stresses (viral infection and temperature increase) in comparison with the Gala variety. We have researched the molecular mechanisms of the resistance of the Gala variety to stresses. This variety was compared to the sensitive Chicago variety, and we confirmed the results of the proteomic profiling  and demonstrated that the resistivity of potato plants to stresses is determined by the mechanisms induced by salicylic acid and the thermal shock related to thermal shock proteins. Using a yeast two-hybrid system, the methods of co-immunoprecipitation and FLIP-FRET, we have determined the proteins interacting with the nuclear proteins  fibrillarin and coilin that participate in the responses of a plant to biotic and abiotic stresses. As a result of the conducted research, we have determined candidate proteins determining the resistance of the Gala variety to various types of  stress factors: we have identified more than 10 potential target genes controlling the resistance to combined biotic/abiotic stresses. One of the most significant conclusions is a demonstration of the interaction of the classic mechanisms of resistance mediated by salicylic acid with new noncanonical functions of the cell nucleus performed by fibrillin, coilin and PARP.

One of the key results of our research is the detection of new functional relations of proteins determining the level of resistance of plants to stresses with RNA metabolism. Using proteomics methods, we have determined that the synthesis of proteins participating in the RNA methylation cycle (the main ferments of the methionine cycle), takes place at the high level  in the stress-resistant potato  variety is suppressed in the sensitive potato variety in conditions of thermal stress and viral infection. This data has been confirmed in additional research of the expression of the corresponding genes and the levels of the main metabolites of the methionine cycle and methionine in the sensitive and the resistant varieties in conditions of combined stress. It has been shown that applying exogenous methionine to sensitive plants increases the resistance of potato plants to infection with PVY at an increased temperature. Therefore, the resistance or sensitivity of plants with respect to  the PVY  infection in conditions of thermal stress in different varieties of potato can be regulated at the level of the methionine cycle that determines the efficiency of transmethylation reactions.

Using methods of transgenic hyperexpression, dsRNA-induced RNA interference and CRISPR/Cas gene editing, we have determined that the protection system of a plant is regulated  by proteins of nucleolus – fibrillarin and Cajal bodies - coilin. The inhibition of the expression of fibrillin and coilin in potato plants leads to molecular changes in salicylic acid-dependent protection pathways and strengthens the protective reaction of the plant, leading to the resistance of plants to stresses. The results observed in laboratory experiments were confirmed by data of tests of potato plant lines edited in the coilin gene in field conditions against the natural infection background. Field experiments have shown that the coilin gene is accompanied by a more intensive growth and an increased productivity  with an increased resistance to infection with PVY. At the same time, these nuclear proteins can differentially influence the viral infection, increasing the  infectivity of PVY and simultaneously reducing the pathogenicity of the tobacco rattle virus (which is dangerous to potato), which is necessary to consider when developing protection measures against viruses and stresses. This data confirms the suggestion that a close interaction between protection pathways and processes in the cell nucleus exists.

The Laboratory has produced transgenic plants with hyperexpression of the Rab28 nuclear protein, which is one of the key proteins participating in the formation of the response to the impact of abscisic acid (ABA). The expression of the Rab28 gene is activated in response to the processing of ABA and in plants caught up in a drought. These transgenic plants manifested an exceptional resistance to drought and the resistivity to viral infection at normal temperature and in conditions of thermal stress. The results of field experiments of these plants against a natural background confirmed the laboratory data showing a significantly increased stability  against viral infection in Rab28 transgenic lines of potato. However, such a significant characteristic of an agricultural plant as productivity turned out to be equal or even 2-2.5 times lower than the productivity of the source variety. Thus, the gene that is useful for one feature can impair other features of the plant. Irreversible changes in gene expression (hyperexpression or, to the contrary, exclusion of expression) using editing methods relying on CRISPR-Cas technology or during the creation of transgenic plants complicates the use of such lines in  plant farming. Presently, our research efforts are focused on the development of methods of temporal regulation of the expression of genes based on RNA interference.

In the process of the implementation of the project it has been demonstrated that short peptides, including those with nuclear localization, play an important role in the processes of growth and development of plants. Due to the fact that these processes overlap with signaling pathways that ensure the response of a plant to stress, these peptides are of considerable interest from the viewpoint of induction and stability against both individual and combined biotic/abiotic stresses. In this regard, for the testing of biologically active peptides we have developed two methods (Russian Federation patents have been obtained): 1) a system for testing  growth-inducing and biologically active compounds based on gametophore cultures of green mosses; 2) cellular test systems based on spores of green mosses for the analysis of the biological activity of low-molecular compounds.

At the final stage of the implementation of the mega-grant, we conducted a multiplex sequencing  of coding and noncoding RNA (RNA-seq) from a potato plant of the Chicago variety, which is sensitive to stresses, in conditions of abiotic (increased temperature)/biotic (infection with PVY) and combined (PVY and increased temperature) stresses. It has been shown that  individual and combined (PVY and increased temperature) stresses cause significant changes in gene expression, influencing the transcription of both coding and noncoding RNA. Among the newly identified genes responsible for infection with PVY we have found genes encoding ferments participating in the catalysis of the formation of polyamines and PolyADP-ribosylation. We have also identified a series of new noncoding RNA that are differentially synthesized in response to individual or combined stress and include antisense RNA and RNA with regions linked via siRNA. The data obtained with the use of nanopore RNA sequencing broadened the knowledge on the potential role of alternative splicing and epitranscriptomic RNA methylation. These results provide significant information for the future research of the functional relations between viral infections and transcriptome reprogramming, RNA methylation and alternative splicing.

It has been demonstrated that upon activation of the mechanism of specific RNA interference caused by the endogenic use of  dsRNA against PVY, the nonspecific protective response according to the type of immunity induced by pathogen-associated molecular patterns, does not manifest itself and is not able to protect the plant from infection with an unrelated virus. Therefore, exogenous dsRNA is a highly specific and active trigger of RNA interference, ensuring a high level  of  «exclusion» of the homologous gene and, consequently, such an approach is promising not only for research in the field of  functional genomics but also for applications in practical plant farming.

We are currently developing approaches to the creation of RNA-oriented technologies based on RNA interference, which are aimed at protecting plants from pathogens and regulating the expression of genes of plants to give them valuable features. The research is conducted on both  model N.benthamiana plants and on potato plants.
To create RNA preparations aimed against relevant pathogens (viruses) infecting potato plants in various regions of Russia. We have conducted an analysis by next-generation sequencing (NGS) of RNA (RNA-seq) of leaf and tuberous samples of potatoes from various regions of Russia with a a subsequent bioinformatics-based survey of consensus sequences of potato viruses, forecasting and synthesis of universal double-stranded RNA against the determined virus strains. The antiviral activity of the predicted and accumulated dsRNA against the Potato Virus Y is assessed in laboratory tests involving infected potato plants.

We are conducting experiments in the exogenuous use of dsRNA preparations as well as artificial artificial small RNA against the genes of phytoene desaturase and magnesium chelatase for the inhibition of their expression in N.benthamiana plants for the creation of a functional RNA model of gene interference of the plant as a fast visual test of the efficiency of RNA preparations in the context of their delivery to cells of the plant. 

Implementation of research results:

The technologies and preparations developed during the implementation oof the project, as well as potato lines,  were transferred to «DGT» Ltd for the testing and implementation of the results of the project into agricultural practice. Among the transferred results are:

1. Genetic-engineering structures and preparations of double-stranded RNA for the inhibition of the reproduction of a number of plant viruses on the basis of RNA interference;
2. A technology for the production of double-stranded RNA against plant viruses;
3. Lines of transgenic plants with hyperexpressed genes of coilin and Rab28;
4. Lines of potato modified in the coilin gene using the CRISPR/Cas technology. 

Education and retraining of personnel:

• Lecture courses have been developed or are currently in the process of development for the program «Plant biotechnology».

• We have conducted internships of postgraduates and young researchers at the James Hutton Institute (United Kingdom).

• Members of the academic staff of the Laboratory paritcipated in the staging of the Russian Potato Forum as well as presented keynotes at the event («Doka-Gene Technologies» LLC, Rogachyovo village, Moscow region, August 2018). The forum was conducted in collaboration with representative of the Russian Academy of Sciences, the Ministry of Science and Education of Russia and the Presidential Administration of Russia. At the forum, a program of the implementation of the results of our work on the mega-grant have been presented.

• On the basis of the results of the implementation of the mega-grant, we organized and staged the symposium (conference) «Molecular mechanisms of plant resistance to environmental stresses and diseases» (November 2020) that featured keynotes by members of the academic staff and invited speakers.

• Three Candidate of Science dissertations have been prepared and defended.