Exogenously Applied Cytokinin Altered the Bacterial Release and Subsequent Stages of Nodule Development in Pea Ipd3/Cyclops Mutant

Regulation of plant hormonal status is one of the major targets of symbiotic signaling during nodule formation in legume plants. However, the genetic and hormonal networks that regulate transition to differentiation of nodules are not well-characterized in legume plants. Analysis of plant mutants forming nodules impaired in rhizobial infection allowed us to identify some regulators involved in the control of the later stages of nodule development. In the current work, we extend our earlier studies on the influence of exogenously applied cytokinin on the later stages of nodule morphogenesis using pea sym33 (ipd3/cyclops) mutants impaired in the gene encoding IPD3/CYCLOPS transcription factor. One of the noticeable effects of the influence of exogenously applied cytokinin on nodules in the sym33-3 mutant was an increasing size of these structures. Cytokinin treatment was shown to stimulate bacterial release and increase the percentage of infected cells in nodules. To explore the role of possible regulators of nodule differentiation, we performed searching in pea transcriptome. The transcriptome study in pea P. sativum revealed the importance of the CCS52 regulator, EFD transcription factor, SYMREM regulator, RSD, the MADS-domain/AGL, and SHORT INTERNODE/STYLISH gene families encoding transcription factors in the control of nodule differentiation. Analysis of the expression patterns was verified by real-time PCR in response to exogenously applied cytokinin treatment

Regulation of the Later Stages of Nodulation Stimulated by IPD3/CYCLOPS Transcription Factor and Cytokinin in Pea Pisum sativum L.

The IPD3/CYCLOPS transcription factor was shown to be involved in the regulation of nodule primordia development and subsequent stages of nodule differentiation. In contrast to early stages, the stages related to nodule differentiation remain less studied. Recently, we have shown that the accumulation of cytokinin at later stages may significantly impact nodule development. This conclusion was based on a comparative analysis of cytokinin localization between pea wild type and ipd3/cyclops mutants. However, the role of cytokinin at these later stages of nodulation is still far from understood. To determine a set of genes involved in the regulation of later stages of nodule development connected with infection progress, intracellular accommodation, as well as plant tissue and bacteroid differentiation, the RNA-seq analysis of pea mutant SGEFix--2 (sym33) nodules impaired in these processes compared to wild type SGE nodules was performed. To verify cytokinin’s influence on late nodule development stages, the comparative RNA-seq analysis of SGEFix--2 (sym33) mutant plants treated with cytokinin was also conducted. Findings suggest a significant role of cytokinin in the regulation of later stages of nodule development

Structural Variations in LysM Domains of LysM-RLK <i>Ps</i>K1 May Result in a Different Effect on Pea–Rhizobial Symbiosis Development

Lysin-motif receptor-like kinase PsK1 is involved in symbiosis initiation and the maintenance of infection thread (IT) growth and bacterial release in pea. We verified PsK1 specificity in relation to the Nod factor structure using k1 and rhizobial mutants. Inoculation with nodO and nodE nodO mutants significantly reduced root hair deformations, curling, and the number of ITs in k1-1 and k1-2 mutants. These results indicated that PsK1 function may depend on Nod factor structures. PsK1 with replacement in kinase domain and PsSYM10 co-production in Nicotiana benthamiana leaves did not induce a hypersensitive response (HR) because of the impossibility of signal transduction into the cell. Replacement of P169S in LysM3 domain of PsK1 disturbed the extracellular domain (ECD) interaction with PsSYM10&#8242;s ECD in Y2H system and reduced HR during the co-production of full-length PsK1 and PsSYM0 in N. benthamiana. Lastly, we explored the role of PsK1 in symbiosis with arbuscular mycorrhizal (AM) fungi; no significant differences between wild-type plants and k1 mutants were found, suggesting a specific role of PsK1 in legume&#8211;rhizobial symbiosis. However, increased sensitivity to a highly aggressive Fusarium culmorum strain was found in k1 mutants compared with the wild type, which requires the further study of the role of PsK1 in immune response regulation

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination

Clinical tests based on whole-genome sequencing are generally focused on a single task approach, testing one or several parameters, although whole-genome sequencing (WGS) provides us with large data sets that can be used for many supportive analyses. In spite of low genome coverage, data of WGS-based non-invasive prenatal testing (NIPT) contain fully sequenced mitochondrial DNA (mtDNA). This mtDNA can be used for variant calling, ancestry analysis, population studies and other approaches that extend NIPT functionality. In this study, we analyse mtDNA pool from 645 cell-free DNA (cfDNA) samples of pregnant women from different regions of Russia, explore the effects of transportation and storing conditions on mtDNA content, analyse effects, frequency and location of mitochondrial variants called from samples and perform haplogroup analysis, revealing the most common mitochondrial superclades. We have shown that, despite the relatively low sequencing depth of unamplified mtDNA from cfDNA samples, the mtDNA analysis in these samples is still an informative instrument suitable for research and screening purposes

High-Throughput Sequencing of Circulating MicroRNAs in Plasma and Serum during Pregnancy Progression

Although circulating microRNAs (miRNAs) in maternal blood may play an important role in regulation of pregnancy progression and serve as non-invasive biomarkers for different gestation complications, little is known about their profile in blood during normally developing pregnancy. In this study we evaluated the miRNA profiles in paired plasma and serum samples from pregnant women without health or gestational abnormalities at three time points using high-throughput sequencing technology. Sequencing revealed that the percentage of miRNA reads in plasma and serum decreased by a third compared to first and second trimesters. We found two miRNAs in plasma (hsa-miR-7853-5p and hsa-miR-200c-3p) and 10 miRNAs in serum (hsa-miR-203a-5p, hsa-miR-495-3p, hsa-miR-4435, hsa-miR-340-5p, hsa-miR-4417, hsa-miR-1266-5p, hsa-miR-4494, hsa-miR-134-3p, hsa-miR-5008-5p, and hsa-miR-6756-5p), that exhibit level changes during pregnancy (p-value adjusted &lt; 0.05). In addition, we observed differences for 36 miRNAs between plasma and serum (p-value adjusted &lt; 0.05), which should be taken into consideration when comparing the results between studies performed using different biosample types. The results were verified by analysis of three miRNAs using qRT-PCR (p &lt; 0.05). The present study confirms that the circulating miRNA profile in blood changes during gestation. Our results set the basis for further investigation of molecular mechanisms, involved in regulation of pregnancy, and the search for biomarkers of gestation abnormalities