Biology subjects

Nekrasov, V.

Publications and source records attributed to Nekrasov, V..

6 recordsLinked to original sources

bioRxiv2026

Integrating Semi-Dwarf Traits into Diverse Wheat Landraces through CRISPR/Cas9, Base Editing and Prime Editing

The semi dwarf stature of modern wheat varieties is conferred by RHT1 alleles derived from a single Japanese cultivar. These alleles are absent in landrace collections such as the Watkins collection. This constrains the direct use of rich genetic diversity preserved in Watkins landraces. These ancestral accessions, adapted to diverse local environments, harbour valuable traits absent from elite cultivars. Here, we demonstrate a precision breeding approach that integrates CRISPR/Cas9, cytosine base editing, and prime editing to introduce semi-dwarfing alleles into selected Watkins landraces. Our strategy overcomes problems caused by the tall stature of most Watkins accessions, providing rapid and precise modification of the Rht1 locus to confer semi-dwarf phenotypes. High editing efficiencies achieved across multiple Watkins landrace wheat lines confirm the robustness of our approach. By unlocking previously untapped genetic variation and enabling targeted trait integration, this study lays the foundation for modern landrace-based breeding programs, supporting sustainable wheat improvement and global food security.

molecular biology↗

bioRxiv2021

Efficient CRISPR/Cas-mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

The CRISPR/Cas technology has recently become a molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. The primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.

plant biology↗

bioRxiv2021

The Arabidopsis pattern recognition receptor EFR enhances fire blight resistance in apple

Fire blight disease, caused by the bacterium Erwinia amylovora (E. amylovora), is responsible for substantial losses in cultivated apple worldwide. An important mechanism of plant immunity is based on the recognition of conserved microbial molecules, named pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), through pattern recognition receptors (PRRs), leading to pattern-triggered immunity (PTI). The interspecies transfer of PRRs represents a promising strategy to engineer broad spectrum and durable disease resistance in crops. EFR, the Arabidopsis thaliana PRR for the PAMP elf18 derived from the elongation factor thermal unstable (EF-Tu) proved to be effective in improving bacterial resistance when expressed into Solanaceae and other plant species. In this study, we tested whether EFR can affect the interaction of apple with E. amylovora by its ectopic expression in the susceptible apple rootstock M.26. Stable EFR expression led to the activation of PAMP-triggered immune response in apple leaves upon treatment with supernatant of E. amylovora, as measured by production of reactive oxygen species and the induction of known defense genes. The amount of tissue necrosis associated with E. amylovora infection was significantly reduced in the EFR transgenic rootstock compared to the wild-type. Our results show that the expression of EFR in apple rootstock may be a valuable biotechnology strategy to improve the resistance of apple to fire blight.

plant biology↗

bioRxiv2020

Remarkable recent changes in genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 alleles present in historic Z. tritici isolate collections, with up to ~ 18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of alleles allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted re-sequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world between 2013-2017. However, in contrast to the data from studies of historic isolates, our study revealed a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of alleles, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence alleles tend to be eliminated in subsequent populations.

microbiology↗

bioRxiv2019

A modular cloning toolkit for genome editing in plants

BackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.\n\nResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.\n\nConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

plant biology↗

bioRxiv2019

A CRISPR/Cas9 mediated 53 kb deletion of the NRC4 gene cluster of tomato does not affect bacterial flagellin-triggered immunity

Plants utilise cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) to fend off invading pathogens. Although PRR- and NLR-triggered immunity are generally thought to activate distinct pathways, they can induce similar outputs. However, whether these two pathways converge at some point to potentiate and strengthen the immune response remains unclear. For instance, the extent to which the tomato NLR helper NRC4 is implicated in response to the bacterial flagellin peptide flg22 needs to be elucidated. One challenge is that the tomato NRC4 gene cluster consists of three paralogues and the related NRC5 gene. Here, we took advantage of the CRISPR/Cas9 system to generate a tomato mutant with a 53 kb deletion that encompasses the four NRC genes. Although this mutant failed to respond to the NRC4-dependent NLR Rpi-blb2, it remained unaltered in flg22-induced responses. We conclude that the NRC4 genes are not essential for flg22-induced responses in tomato.

plant biology↗