Physiology and Developmental Biology Group - Detailed - Institute of Genetics and Biotechnology
Physiology and Developmental Biology Group
Physiology and Developmental Biology Group - Detailed
Last modified: 21. April 2023
Description of the group
Regulatory activity of RNA interference (RNAi) is based on the action of small (21-24 nucleotide long) RNA molecules. Large population of small RNAs can be produced from long double stranded RNA molecules, small interfering (si) RNAs, or evolutionally conserved sequence specific small RNAs are generated from hair-pin like RNA structures (precursor), micro (mi)RNAs. The importance of RNAi is reflected by its involvement in developmental processes determining important traits of crop plants, in biotic and abiotic stress responses, including heat stresses and trough the activity of RNA dependent DNA methylation in the maintenance and protection of genome stability. Our recent experiments revealed that the biogenesis of miRNAs is not always resulted in the incorporation of miRNAs in the executor complexes. Our data indicate that this phenomenon is linked to the secondary structures of miRNA precursors. We also investigate the role of RNAi machinery connected proteins in determining the loading ability of small RNAs in the executor complexes.
The utilization of genome editing technologies for efficient generation of site directed mutagenesis is a major advance in recent history of molecular biology. The economically important barley and wheat plants other are used to generate CRISPR/Cas9 edited lines. The genome editing technology makes it possible to investigate RNA interference (RNAi), a regulatory mechanism determining many aspects of plant development and stress responses (such as heat stress), in barley possessing a large genome. We assess the efficiency of the technology in trait improvement, such as virus and fungi resistance an enhanced biomass production. Our experiments are representing an important filled by connecting the basic knowledge obtained by the investigation of model organism to economically important crops.
Short presentation of the research projects
- Establishment of genome editing in barley and other crop species for research and crop improvement.
- Development of precision mutagenesis system based on genome elimination in wheat
- Biology of RNA interference in model and crop plants: molecular link between biogenesis and action of small regulatory RNAs.
- Utilization and development of new genome editing tool for producing fungi resistance wheat lines.
Main publications of the group:
Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis.
Dalmadi, Ágnes; Miloro, Fabio; Balint, Jeannette; Varallyay, Eva; Havelda, Zoltan*, Nucleic Acids Res. 2021 Accepted for publication
Genome-Wide Identification of RNA Silencing-Related Genes and Their Expressional Analysis in Response to Heat Stress in Barley (Hordeum vulgare L.).
Hamar É, Szaker HM, Kis A, Dalmadi Á, Miloro F, Szittya G, Taller J, Gyula P, Csorba T, Havelda Z*. Biomolecules. 2020 Jun 18;10(6):929. doi: 10.3390/biom10060929.
AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading.
Dalmadi Á, Gyula P, Bálint J, Szittya G, Havelda Z*. Nucleic Acids Res. 2019 Oct 10;47(18):9803-9817. doi: 10.1093/nar/gkz690.
Creating highly efficient resistance against wheat dwarf virus in barley by employing CRISPR/Cas9 system.
Kis A, Hamar É, Tholt G, Bán R, Havelda Z*. Plant Biotechnol J. 2019 Jun;17(6):1004-1006. doi: 10.1111/pbi.13077.
Expansion of Capsicum annuum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.
Taller D, Bálint J, Gyula P, Nagy T, Barta E, Baksa I, Szittya G, Taller J, Havelda Z*. PLoS One. 2018 Jul 25;13(7):e0200207. doi: 10.1371/journal.pone.0200207
Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.
Kis A, Tholt G, Ivanics M, Várallyay É, Jenes B, Havelda Z. * Mol Plant Pathol. 2016 Apr;17(3):427-37. doi: 10.1111/mpp.12291.
Independent parallel functions of p19 plant viral suppressor of RNA silencing required for effective suppressor activity.
Várallyay É, Oláh E, Havelda Z. * Nucleic Acids Res. 2014 Jan;42(1):599-608. doi: 10.1093/nar/gkt846.
Unrelated viral suppressors of RNA silencing mediate the control of ARGONAUTE1 level.
Várallyay E, Havelda Z. * Mol Plant Pathol. 2013 Aug;14(6):567-75. doi: 10.1111/mpp.12029.
Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation.
Várallyay E, Válóczi A, Agyi A, Burgyán J, Havelda Z*. EMBO J. 2010 Oct 20;29(20):3507-19. doi: 10.1038/emboj.2010.215.
MicroRNA detection by northern blotting using locked nucleic acid probes.
Várallyay E, Burgyán J, Havelda Z. * Nat Protoc. 2008;3(2):190-6. doi: 10.1038/nprot.2007.528.
Grants:
- Establishment of genome editing in barley and other crop species for research and crop improvement. (NKFI K125300) (PI: Zoltán Havelda)
- Development of precision mutagenesis based on genome elimination in wheat (NKFI FK 134264) (PI: András Kis)
- Biology of RNA interference in model and crop plants: molecular link between biogenesis and action of small regulatory RNAs. (NKFI K134924) (PI: Zoltán Havelda)
- Agribiotechnology and precision breeding for food security (RRF-2.3.1-21-2022-0000 7) (PI: András Kis and Zoltan Havelda)
Detailed presentation of the works
The CRISPR/Cas9 system is an efficient, and highly specific genome editing technology which has been introduced into basic and applied sciences with stunning velocity. It is inevitable to establish this ground breaking technology in Hungary and exploits its potential in basic and applied researches. In the planned experiments the economically important barley (Hordeum vulgare) will be used to generate CRISPR/Cas9 edited plants by adapting and optimizing the plant tissue culture and molecular technics already available in our laboratory. The genome editing targets will be selected to investigate basic biological processes of RNA interference components, the main research field of the laboratory, to understand its role in barley development and stress response. CRISPR/Cas9 system can also provide precise genome modifications directly in valuable local cultivars, even the introduction of multiple traits, saving the time-consuming backcrossing procedure of conventional breeding approaches. In addition, CRISPR/Cas9 systems has the potential to act in trans providing alternative approaches circumventing the drawbacks associated with traditional genetically modified organisms. We will also assess this characteristic of CRISPR/Cas9 system by targeting genes involved in trait improvement of barley. Moreover, wheat, as one of the most important crops in Hungary, will also be used as a target for genome editing.
The CRISPR/Cas-based techniques represent a major breakthrough in targeted genome editing of living organisms. In the case of wheat, the technology has already enabled the specific reconstruction of previously characterized mutations, resulting in economic benefits. In terms of implementation, the most efficient method is to integrate the gene encoding the Cas enzyme responsible for DNA cleavage and the guideRNA's gene that designates the target sequence, into the plant genome and then to remove it after mutagenesis. However, as a result of transgenic integration, small and untraceable pieces of DNA can be incorporated into chromosomes, which are difficult to identify. Therefore, various DNA-free transient techniques have been developed, such as the biolistic delivery of the Cas-guideRNA ribonucleoprotein complex into tissue cultures, but its mutation frequency is very low. Our aim is to develop a more efficient genome editing method for wheat that does not require transformation of the target genome.
RNA interference (RNAi) is indispensable regulatory mechanism present in almost all eukaryotes controlling developmental processes, stress responses and genome integrity. The ARGONAUTE1 (AGO1) is the main effector component of the RNA-induced silencing complex (RISC) predominantly responsible for micro RNA (miRNA) mediated repression of target mRNAs. In our previous work we revealed the presence of high level of free miRNA species in the cytoplasm, unbound to AGO1. We also demonstrated that distinct miRNA precursors are responsible for the altered AGO1 loading of various miRNAs. In the proposed work we would like to elucidate the fine molecular mechanism of this newly identified regulatory system which determines the biological activity of miRNAs by sorting only a subset of the produced miRNA pools into the effector complexes. MiR168, targeting AGO1 mRNA, typically accumulates in AGO1 unbound, free, forms in the cytoplasm. In our experiments, we will use modified precursor structures to map structural feature regulating the loading efficiency of miR168 into AGO1. We will also investigate the biological role various miRNA biogenesis proteins in regulating the accumulation/production of protein unbound miRNA species. We also intend to translate our results to economically important plants to extend our understanding to the practical utilization of the gained data. As a result of our research we hope to uncover the action of a pivotal regulatory mechanism in the miRNA pathway which can help to improve the artificial miRNA-based research tools and provide data about the action of miRNAs determining important traits of crop plants.
The MLO genes of cultivated wheat lines will be disabled by a newly developed genome editing tool to establish powdery mildew resistant wheat varieties.
Group members
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Ágnes Dalmadi, post-doctoral fellow M.Sc.: Phone: +36-28/430-494 / 4155 |
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Dr. András Kis, post-doctoral fellow MSc: Agricultural engineer, Szent István University, Faculty of Agriculture and Environmental Sciences, 2010. Phone: +36-28/430-494 / 4155 |
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Fabio Miloro, research assistant, PhD student MSc: biologist, Turin University, Plant Biotechnology, 2019. Phone: +36-28/430-494 / 4155 |
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Mohammad Ali, Stipendium Hungaricum studentMATE, GBI, Department of Plant Biotechnology, MSc: Plant biotechnology, Szent István University, Gödöllő, 2019. |
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Poldán Erzsébet, laboratory assistant |
| MSc student: | Barbara Sorbán-Kiss, Agricultural biotechnologist MSc, MATE, 2020. |