Defense of the dissertation of Iksat Nurgul Nurqanatqyzy for the degree of Doctor of Philosophy (PhD) in the educational program «8D05107 - Biology»
L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Iksat Nurgul Nurqanatqyzy on the topic «Modulation of selective recognition and hydrolysis of viral RNA to stimulate plant resistance to the pathogen» in the educational program of «8D05107 – Biology».
The dissertation was carried out based on the L.N. Gumilyov Eurasian National University in the Rustem Omarov scientific laboratory of plant biotechnology (Astana, Kazakhstan) and Taras Shevchenko Kyiv National University (Kyiv, Ukraine).
The language of defense is kazakh
Official reviewers:
Bisenbaev Amangeldy Kuanbaevich – Academician of the National Academy of Sciences of the Republic of Kazakhstan, Doctor of Biological Sciences, Professor of the Department of Molecular Biology and Genetics of Al-Farabi Kazakh National University (Almaty, Kazakhstan).
Sapakhova Zagipa Beisenovna – Doctor of Philosophy (PhD), Associate Professor, leading researcher at the Laboratory of Breeding and Biotechnology, Institute of Plant Biology and Biotechnology (Almaty, Kazakhstan).
Temporary members of the Dissertation Committee:
Zhambakin Kabyl Zhaparovich – Academician of the National Academy of Sciences of the Republic of Kazakhstan, professor, Doctor of Biological Sciences, General Director of the Institute of Plant Biology and Biotechnology (Almaty, Kazakhstan).
Soltabayeva Aigerim Dauletbekkyzy - Doctor of Philosophy (PhD), Associate Professor, Instructor in Biology, School of Natural Social Sciences and Humanities, Nazarbayev University (Astana, Kazakhstan).
Jatayev Satyvaldy Adineevich – Candidate of Biological Sciences, Associate Professor of the Department of Agriculture and Plant Growing of the S. Seifullin Kazakh Agrotechnical University (Astana, Kazakhstan).
Bekturova Aizat Zhambulovna - Doctor of Philosophy (PhD), a postdoctoral fellow at Bar-Ilan University, Israel.
Academic Advisors:
Masalimov Zhaksylyk Kairbekovich – Doctor of Philosophy (PhD), Candidate of Biological Sciences, Associate Professor, Head of the Department of Biotechnology and Microbiology of the L.N. Gumilyov Eurasian National University (Astana, Kazakhstan).
Alex Shevchenko – Doctor of Philosophy (PhD), associate professor at Taras Shevchenko National University of Kyiv (Kyiv, Ukraine).
The defense will take place on April 29, 2024, at 12:00 PM in the Dissertation Council for the training direction «8D051 – Biological and related sciences» in the specialty «8D05107 – Biology» of L.N. Gumilyov Eurasian National University. The defense meeting is planned to be held online.
Link: https://us06web.zoom.us/j/86278516508?pwd=Jk0PZm4ahGLzEBWI1bPaPy6plvIfbQ.1
Conference ID: 862 7851 6508 Access code: 609988
Address: Аstana, Kazhymukan, 13, building № 3, audience №333.
Abstract (English): General description of the study. The dissertation describes research using the CRISPR/Cas13 system to confer plant resistance to Tomato Bushy Stunt Virus (TBSV). In addition, this study describes the potential to improve the efficiency of the CRISPR/Cas13 system by co-transforming plants with the RNA interference suppressor protein P19 and heat treatment to create effective defense strategies against viral pathogens. The work used methods of molecular biology and genetic engineering. Relevance of the research topic. Plant viruses inflict significant damage to agricultural crop production by seriously reducing crop yield and quality worldwide. In Kazakhstan, viruses cause considerable economic damage to various economically important crops. Thus, the development of new methodological procedures to boost plant resistance to viral pathogens plays a pivotal role in the modern area of plant biology. The CRISPR technology opens up new opportunities for increasing plant productivity and resistance to biotic and abiotic environmental stress factors. In particular, the use of CRISPR in combination with viral sequences as targeting molecules makes it possible to obtain virus-resistant plant lines. According to recent studies, the CRISPR/Cas9 system can be effectively used to induce immunity in plants by selectively targeting viral nucleic acids. In addition, this approach allows specific targeting of genes associated with host susceptibility, especially in the case of RNA viruses. Various delivery systems for CRISPR method, are capable of activating protective mechanisms against transgenes in plant cells. Works have been published on the possibility of using CRISPR/Cas13a in the development of RNA-based antiviral technologies. The key issues regarding the optimization of any CRISPR/Cas system include selecting delivery methods into eukaryotic systems and utilizing optimal promoters to enhance the expression of Cas nuclease mRNA and guide RNAs. One of the factors contributing to low expression of the CRISPR/Cas system in plants is the activity of RNA interference, aimed at the degradation of exogenous mRNA. In this regard, a widely employed method involves co-expressing the target gene with an RNAi suppressor, such as P19. Furthermore, recent studies have demonstrated that the efficiency of the CRISPR/Cas9 editing system can be enhanced in Arabidopsis plants through co-expression with the P19 protein. Therefore, investigating the functional interactions between the RNA silencing machinery and the gene editing system is of great importance. In addition to differences in genome structure, another variable across different biological systems that can influence CRISPR/Cas activity is temperature. Temperature influences many biological parameters, and some of them (such as enzymatic reaction kinetics, chromatin structure, DNA repair pathways) can directly affect the ability of CRISPR/Cas to induce mutations in eukaryotic genomes or to work at the transcript level by degrading RNA viruses. Therefore, temperature may contribute to the observed variation in the efficiency of all CRISPR/Cas systems in organisms. CRISPR/Cas technology has been successfully applied in model plants such as Arabidopsis and Nicotiana and important crops such as wheat, corn, and rice. Information on targeting non-coding genes is sparse. Until now, CRISPR/Cas technology has been used exclusively in human, mouse, or zebrafish cell lines to knock out microRNA or lncRNA genes. Currently, there is no information on CRISPR/Cas13 technology targeting pre-miRNAs and miRNAs in plants. Analysis of the functional characteristics and mechanism of action of microRNAs that cause important stress responses will facilitate the selection of resistant varieties in the future. As one of the model plants, the role and molecular mechanism of miRNAs in response to stress in tobacco, especially miRNAs and lncRNAs, will help unravel the function of stress-related ncRNAs in other species. Single gene resistance has gradually become unsatisfactory for crop production in some areas, and strategies to develop resistant crops in new directions are urgently needed. Thus, the evolution of microRNAs and their directions are an important component of plant evolution. Adaptation to changing environmental conditions, such as exposure to new or modified types of viruses, can be achieved by selecting plant species that express a modified arsenal of microRNAs. Aim of the dissertation: to study the regulation and evaluate the effectiveness of the CRISPR/Cas13 system in the mechanisms of interaction between RNA viruses and plants. Research objectives: 1. To create the pk2GW7-pCas13a construct, providing expression in plant cells; 2. To investigate in planta of Tomato Bushy Stunt Virus (TBSV) silencing using the CRISPR/Cas13 system; 3. To compare CRISPR/Cas13 virus RNA targeting efficiency between GFP-crRNAs vs P19-crRNAs sequences; 4. To study the effect of viral infection on plant microRNAs; 5. To study the influence of cotransformation of the RNA interference suppressor - protein P19 to increase the efficiency of vectors; 6. To determine the effectiveness of temperature effects on the expression of the CRISPR/Cas13 system. The object of study. Nicotiana benthamina, Tomato Bushy Stunt Virus, CRISPR/Cas13. Research methods: design and construction of Cas13 orthologues for expression in plants, construction of crRNA-GFP and crRNA-P19 expression constructs, LR Gateway recombination cloning, transient expression, in vitro transcription, inoculation with viral material, rapid analysis of viral particle purification, SDS-PAGE, Western blotting, rapid viral particle detection assay, immunoprecipitation, microRNA isolation, NATIVE PAGE, 8-oxoguanine ELISA, liquid-liquid phase separation, qRT-PCR, horizontal agarose gel electrophoresis. Scientific research innovation. The proposed research topic is currently not being studied in Kazakhstan. The effect of various thermal treatments on Cas13 expression levels during CRISPR-mediated viral RNA degradation in plants has been poorly studied. In addition, this work is the first to examine the role of the expression of the P19 suppressor protein as a means of increasing the efficiency of the gene-editing system to produce pathogen-resistant transgenic plants. The effect of different levels of P19 expression on CRISPR-mediated microRNA editing in plants has been poorly studied. Taken together, the results provide a platform for focusing attention on the most pressing issues related to rapid viral evolution and viral evasion of the CRISPR/Cas system. Theoretical and practical significance of the research. The scientific and technical impact of the project results is to obtain new fundamental data on the interaction between viruses and plants using gene editing technology. The obtained scientific results can be used in the educational process in the field of biology and biotechnology. These studies can provide the basis for applied research and development to create virus-resistant plants. The economic efficiency of the results of this work lies in the technology enabling the generation of agricultural organisms with specified characteristics, which contributes to increased productivity and reduced production costs. Based on the results of this research work, key plant species important for food security could be engineered to use CRISPR/pCas13 mechanisms against one or more viruses and possibly other pathogens. The results of this dissertation work became the basis for grant funding for 2023-2025. under the project “Zhas Galym” AP19174389 “Modulation of the CRISPR/Cas13 gene editing system to stimulate antiviral resistance of plants” with the support of the Ministry of Education and Science of the Republic of Kazakhstan, as well as for program-targeted funding of scientific, scientific and technical programs BR21882269 “Use of genome editing technology to increase productivity economically important cultivated plants" task 3 "Development of a CRISPR/Cas13 gene editing system to ensure antiviral resistance of plants" of the Committee on Science of the Republic of Kazakhstan for 2023-2025. The main points put forward for the defense of the dissertation: 1. An in silico design was carried out to assemble Cas13 and crRNA directed against the wild-type TBSV virus and its mutants for further expression in N. benthamiana plants. The generated pK2GW7-pCas13a construct was transiently expressed in plants. The perspective of universal application of the CRISPR/Cas13 system in combating all viruses belonging to the Tombusvirus genus was considered by creating crRNAs specifically targeting the P19 protein of the TBSV virus. 2. Molecular interference against TBSV was studied using P19 protein expression as an indicator of viral particle titer in the presence of the CRISPR/Cas13 system. Successful intervention of the CRISPR/Cas13 system against the suppressor protein P19 has been demonstrated. 3. The targeting efficiency of the CRISPR/Cas13 system to viral RNA was compared between different crRNA-GFP and crRNA-P19 sequences. All crRNAs showed a decrease in GFP expression levels in inoculated leaves under UV light compared to controls. Additionally, lower but detectable levels of GFP signal reduction were found in the GFP crRNAs, whereas higher levels of reduction were observed with crRNAs targeting P19. The potential for better targeting of crRNA complementary to the P19 sequence of the TBSV genome compared to another target is associated with the function of P19 in the viral genome as a suppressor of the host RNA interference defense mechanism. Any dose-dependent change in P19 levels may affect virus propagation in the plant cell, as shown in TBSV mutants and the formation of siRNA-P19 complexes. On the other hand, higher GFP signals were observed in the control with nonspecific nsgcrRNA and N. benthamiana plants inoculated with wild type, confirming the effectiveness of the CRISPR/pCas13a system for viral interference. 4. The interaction of the suppressor protein P19 with the function of plant miRs was studied in a biological context. Specific primers for microRNAs and their precursors have been developed. The relative expression of plant microRNAs in the presence and absence of the CRISPR system was compared. The differential effect of P19 protein on the expression of miRs 168 and 162 was found to be greatest during the early stage of viral infection. The results showed that the effect of P19 on plant miR functions was not in the development of symptoms but in plant antiviral responses. Expression of two of these miRNAs was also detected in the presence of the CRISPR/Cas13a system directed against wild-type TBSV. 5. To increase the efficiency of vectors, the effect of directed co-transformation of the RNA interference suppressor - the P19 protein - on the accumulation of the target gene was studied. Increased expression of the recombinant protein was obtained in plant tissues in samples co-transformed with P19 protein. Based on the data obtained, a working model of the functional interaction of plant RNA interference and the CRISPR/Cas13 system was created. The role of the P19 protein as a modulator of the regulation of the CRISPR/Cas13 system in plants was studied. 6. A new technique has been developed to increase the efficiency of the CRISPR/Cas system using temperature modulation. Based on the obtained results, it was determined that 370C is the most optimal temperature for achieving increased expression of the pCas13a protein. Personal contribution of the author. Review of literature related to scientific research, determining the goals and objectives of the dissertation, conducting practical work, discussing research results, conducting statistical analysis, writing a doctoral dissertation, preparing work by regulatory requirements, and introducing the research results into the educational process for students of the specialty “Plant Biotechnology” of the L.N. Gumilyov Eurasian National University was carried out with the personal participation of the author. The dissertation work was carried out to the highest standards of intellectual integrity and the prevention of falsification of scientific data, falsification, plagiarism, and false co-authorship. Connection of work with research programs. The dissertation work was carried out within the framework of grant funding 2021-2023 AR09258746 «Regulation of CRISPR/Cas13 gene editing system by viral protein expression to confer antiviral plant resistance» by the Ministry of Education and Science of the Republic of Kazakhstan at the L.N. Gumilyov Eurasian National University in the scientific laboratory of plant biotechnology named after Rustem Omarov (Kazakhstan, Astana) and the Department of Virology of the Taras Shevchenko National University, Research Institute of Biology and Medicine (Ukraine, Kyiv). In addition, the methods used in the dissertation were optimized during training at Ben-Gurion University (Israel, Negev) and the University of Silesia (Poland, Katowice). Approbation of research results. The results of the research and the main provisions of the dissertation were reported and presented at international and national conferences: - «Bioresources and Viruses» X International Conference - 2023, Kyiv, Ukraine; - «Current problems of microbiology, biotechnology and biodiversity» International scientific and practical conference - 2022, Astana, Kazakhstan ; - «ǴYLYM JÁNE BILIM – 2022» XVII International Scientific Conference of Students and Youth - 2022, Astana, Kazakhstan ; - «Biological diversity of Asian steppes» IV international scientific conference - 2022, Astana, Kazakhstan; - «BIOLOGY AND BIOTECHNOLOGY OF THE XXI CENTURY-2021» International Scientific Forum of Young Scientists - 2022, Astana, Kazakhstan; - «TENDENZE ATTUALI DELLA MODERNA RICERCA SCIENTIFICA», international scientific and practical conference, 2020, Stuttgart, Germany; - II International Scientific and Practical Conference, 2020, Ekaterinburg, Russian Federation. Articles. The results of the dissertation are presented in 19 published articles, including 1 article (49%) and 1 thesis (Q2) in journals with an impact factor above zero, recommended in the Web of Science and Scopus databases, 4 articles in journals recommended by CQAFSHE MSHE RK, 12 theses and articles at international scientific conferences, as well as 1 patent for a utility model. Scope and structure of the dissertation. The dissertation is presented on 96 pages and consists of definitions and abbreviations, an introduction, a literature review, materials and methods, research results and their discussion, a conclusion, list of 120 sources used, 9 tables, and 59 figures. Acknowledgments. This work is dedicated to our respected mentor, рrofessor Rustem Omarov, who greatly contributed to our knowledge, and the development of science and was a role model to follow.