FSBSI ARRIAM
Federal State Budget Scientific Institution
All-Russia Research Institute for Agricultural Microbiology
All-Russia Research Institute for Agricultural Microbiology
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The laboratory maintains an extensive collection of genetic lines of pea (Pisum sativum L.), including more than 100 accessions of cultivated pea with varying degrees of responsivity to inoculation with nodule bacteria and arbuscular mycorrhiza fungi, as well as about 100 pea lines carrying mutations in symbiotic genes. Using transcriptome sequencing technology, studies are conducted on the role of pea symbiotic genes in the development of mutually beneficial symbioses (Zhukov et al., 2021a). The laboratory staff has developed an approach that enables the identification of mutations in symbiotic genes of the garden pea based on RNA sequencing data. (Zhernakov et al., 2019).
Using pea symbiotic mutants, the role of transcription factors (TF) Nsp1 and Nsp2 in the development of arbuscular mycorrhiza in pea was characterized, and the sequence of the Sym34 gene encoding TF Nsp1 was identified for the first time (Shtark et al., 2016). During the analysis of natural pea lines, the sequence of the LykX gene, which controls the specificity of recognition of signaling molecules secreted by nodule bacteria, was identified (Sulima et al., 2017; Sulima et al., 2019). In a joint study with ITMO University staff, computer modeling of the binding of signal molecules to identified plant receptors was conducted (Solovev et al., 2021). Some studies of symbiotic genes of peas have been carried out in collaboration with major European research centres (Ovchinnikova et al., 2011; Couzigou et al., 2012; Magne et al., 2018).
The patterns of formation and features of growth-stimulating activity of bacterial endophytes inhabiting the stems and leaves of pea have been studied. The composition of the endophytic community of several varieties and genotypes of pea has been described (Vasileva et al., 2020), and it has been demonstrated that the effect of treating plants with isolates of endophytic bacteria depends on the genotype of the plant. Using Oxford Nanopore sequencing technology, genomes of associative bacteria were sequenced (Afonin et al., 2021a).
Deep sequencing of the genome of pea cv. Frisson was performed using Oxford Nanopore and Illumina platforms, and a high-quality pea genome assembly with extensive gene annotation, including genes encoding short peptides, was obtained. The gene family encoding nodule-specific cysteine-rich peptides (NCR) was characterized, which play a key role in controlling the differentiation of nitrogen-fixing bacteria in nodules (Zorin et al., 2022). Also, in collaboration with the Department of Genetics and Biotechnology of the Biological Faculty of St. Petersburg State University, gene families encoding the CLE and CEP peptides, which are, respectively, negative and positive regulators of the development of nitrogen-fixing nodules, were characterized (Lebedeva et al., 2022a; Lebedeva et al., 2022b).
Transcriptome studies using RNA sequencing make it possible to advance in understanding the features of the implementation of hereditary information during the development of supraorganismal systems formed by garden pea. Transcriptome sequencing data from various pea varieties and lines are actively used to create molecular markers and analyze candidate genes for the role of symbiotic genes identified during mutation analysis (Zhernakov et al., 2017, Zhernakov et al., 2019). In 2017, a database was created that combines information on 15,000 gene-specific molecular markers of garden pea and facilitates the analysis of candidate genes (Kulaeva et al., 2017). In 2015, the world's first transcriptome assembly of nitrogen-fixing pea nodules was obtained (Zhukov et al., 2015), and in 2020, the transcriptome assembly of mycorrhizal roots of garden pea was obtained as well (Afonin et al., 2020b). Recent studies using modern transcriptome sequencing technologies have described for the first time in the world alternative splicing events characteristic of nitrogen-fixing nodules and mycorrhizal roots of pea (Zorin et al., 2019; Zorin et al., 2020). The work uses the RNA sequencing technology in the modification MACE (Massive Analysis of cDNA Ends), developed by GenXPro (Frankfurt am Main, Germany), which allows obtaining more reliable results of differential gene expression analysis with significantly less coverage than in the case of traditional RNA sequencing. Using MACE sequencing, gene expression was analyzed in the roots of pea supernodulating mutants, as a result of which a connection was demonstrated between the autoregulation system of nodulation and plant responsiveness to inoculation (Zhukov et al., 2021a).
