The key event that initiates nodule organogenesis is the perception of bacterial signal molecules, the Nod factors, triggering a complex of responses in epidermal and cortical cells of the root. The Nod factor signaling pathway interacts with plant hormones, including cytokinins and gibberellins. Activation of cytokinin signaling through the homeodomain-containing transcription factors KNOX is essential for nodule formation. The main regulators of gibberellin signaling, the DELLA proteins are also involved in regulation of nodule formation. However, the interaction between the cytokinin and gibberellin signaling pathways is not fully understood. Here, we show in Pisum sativum L. that the DELLA proteins can activate the expression of KNOX and BELL transcription factors involved in regulation of cytokinin metabolic and response genes. Consistently, pea la cry-s (della1 della2) mutant showed reduced ability to upregulate expression of some cytokinin metabolic genes during nodulation. Our results suggest that DELLA proteins may regulate cytokinin metabolism upon nodulation.

Single three-amino acid loop extension (TALE) homeodomain proteins, including the KNOTTED-like (KNOX) and BEL-like (BELL) families in plants, usually work as heterodimeric transcription factor complexes to regulate different developmental processes, often via effects on phytohormonal pathways. Nitrogen-fixing nodule formation in legumes is regulated by different families of homeodomain transcription factors. Whereas the role of KNOX transcription factors in the control of symbiosis was studied early, BELL transcription factors have received less attention. Here, we report the identification and expression analysis of BELL genes in the legume plants Medicago truncatula and Pisum sativum, which are involved in regulating symbiosis initiation and development. A more precise analysis was performed for the most significantly upregulated PsBELL1-2 gene in pea. We found that the PsBELL1-2 transcription factor could be a potential partner of PsKNOX9. In addition, we showed that PsBELL1-2 can interact with the PsDELLA1 (LA) protein-regulator of the gibberellin pathway, which has a previously demonstrated important role in symbiosis development.

This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA–CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.

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.

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or “camelization” of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

The development of legume–rhizobial symbiosis results in the formation of nitrogen-fixing root nodules. In response to rhizobial molecules, Nod factors, signal transduction is mediated by the interaction of activated receptors with downstream signaling proteins. Previously, some new regulators of the signal pathway, such as phospholipases D, which regulate the level of phosphatidic acid (PA), as well as mitogen-activated protein kinases (MAPKs), have been identified in legumes. Since PA is an important signal messenger, we tested the hypothesis that increasing the level of proteins involved in the reversible binding of PA in plant tissues may have a positive effect on symbiosis. Our findings showed that overexpression of MtSPHK1-PA, encoding the PA-binding domain of sphingosine kinase 1 (SPHK1), stimulated plant growth and nodule development in legume plants. Furthermore, the influence of MAPK6 on the development of symbiosis was studied. Using genetic engineering methods, we increased MAPK6 transcriptional activity in transgenic roots, leading to an increase in the number of nodules and the biomass of pea plants. Therefore, new approaches to obtain plants with an increased efficiency of symbiosis were tested. We report here that both genes that encode signaling proteins may be used as potential targets for future modification using biotechnological approaches.