Plants are able to discriminate and respond to structurally related chitooligosaccharide (CO) signals from pathogenic and symbiotic fungi. In model plants Arabidopsis thaliana and Oryza sativa LysM-receptor like kinases (LysM-RLK) AtCERK1 and OsCERK1 (chitin elicitor receptor kinase 1) were shown to be involved in response to CO signals. Based on phylogenetic analysis, the pea Pisum sativum L. LysM-RLK PsLYK9 was chosen as a possible candidate given its role on the CERK1-like receptor. The knockdown regulation of the PsLyk9 gene by RNA interference led to increased susceptibility to fungal pathogen Fusarium culmorum. Transcript levels of PsPAL2, PsPR10 defense-response genes were significantly reduced in PsLyk9 RNAi roots. PsLYK9’s involvement in recognizing short-chain COs as most numerous signals of arbuscular mycorrhizal (AM) fungi, was also evaluated. In transgenic roots with PsLyk9 knockdown treated with short-chain CO5, downregulation of AM symbiosis marker genes (PsDELLA3, PsNSP2, PsDWARF27) was observed. These results clearly indicate that PsLYK9 appears to be involved in the perception of COs and subsequent signal transduction in pea roots. It allows us to conclude that PsLYK9 is the most likely CERK1-like receptor in pea to be involved in the control of plant immunity and AM symbiosis formation.

Background and Aims Recent findings indicate that Nod factor signalling is tightly interconnected with phytohormonal regulation that affects the development of nodules. Since the mechanisms of this interaction are still far from understood, here the distribution of cytokinin and auxin in pea (Pisum sativum) nodules was investigated. In addition, the effect of certain mutations blocking rhizobial infection and subsequent plant cell and bacteroid differentiation on cytokinin distribution in nodules was analysed. Methods Patterns of cytokinin and auxin in pea nodules were profiled using both responsive genetic constructs and antibodies. Key Results In wild-type nodules, cytokinins were found in the meristem, infection zone and apical part of the nitrogen fixation zone, whereas auxin localization was restricted to the meristem and peripheral tissues. We found significantly altered cytokinin distribution in sym33 and sym40 pea mutants defective in IPD3/CYCLOPS and EFD transcription factors, respectively. In the sym33 mutants impaired in bacterial accommodation and subsequent nodule differentiation, cytokinin localization was mostly limited to the meristem. In addition, we found significantly decreased expression of LOG1 and A-type RR11 as well as KNOX3 and NIN genes in the sym33 mutants, which correlated with low cellular cytokinin levels. In the sym40 mutant, cytokinins were detected in the nodule infection zone but, in contrast to the wild type, they were absent in infection droplets. Conclusions In conclusion, our findings suggest that enhanced cytokinin accumulation during the late stages of symbiosis development may be associated with bacterial penetration into the plant cells and subsequent plant cell and bacteroid differentiation.

In this study, we demonstrated the successful transformation of two pea (Pisum sativum L.) cultivars using Agrobacterium rhizogenes, whereby transgenic roots in the resulting composite plants showed expression of the gene encoding the green fluorescent protein. Subsequent to infection with A. rhizogenes, approximately 70%–80% of pea seedlings developed transgenic hairy roots. We found out that the transgenic roots can be efficiently nodulated by Rhizobium leguminosarum bv. viciae and infected by the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. The morphology of nodules in the transgenic roots was found to be identical to that of nodules observed in wild-type roots, and we also observed the effective induction of markers typical of the symbiotic association with AM fungi. The convenient protocol for highly efficient A. rhizogenes-mediated transformation developed in this study would be a rapid and effective tool for investigating those genes involved in the development of the two types of symbioses found in pea plants.

In legumes, perception of rhizobial lipochitooligosacharide-based molecules (Nod factors) and subsequent signal transduction triggers transcription of plant symbiosis-specific genes (early nodulins). We present genetic dissection of Nod factor-controlled processes in Pisum sativum using two early nodulin genes PsENOD12a and PsENOD5, that are differentially up-regulated during symbiosis. A novel set of non-nodulating pea mutants in fourteen loci was examined, among which seven loci are not described in Lotus japonicus and Medicago truncatula. Mutants defective in Pssym10, Pssym8, Pssym19, Pssym9 and Pssym7 exhibited no PsENOD12a and PsENOD5 activation in response to Nod factor-producing rhizobia. Thus, a conserved signalling module from the LysM receptor kinase encoded by Pssym10 down to the GRAS transcription factor encoded by Pssym7 is essential for Nod factor-induced gene expression. Of the two investigated genes, PsENOD5 was more strictly regulated; not only requiring the SYM10-SYM7 module, but also SYM35 (NIN transcription factor), SYM14, SYM16 and SYM34. Since Pssym35, Pssym14, Pssym34 and Pssym16 mutants show arrested infection and nodule formation at various stages, PsENOD5 expression seems to be essential for later symbiotic events, when rhizobia enter into plant tissues. Activation of PsENOD12a only requires components involved in early steps of signalling and can be considered as a marker of early symbiotic events preceding infection.

Arbuscular mycorrhiza (AM) is an ancient mutualistic symbiosis formed by 80–90 % of land plant species with the obligatorily biotrophic fungi that belong to the phylum Glomeromycota. This symbiosis is mutually beneficial, as AM fungi feed on plant photosynthesis products, in turn improving the efficiency of nutrient uptake from the environment. The garden pea (Pisum sativum L.), a widely cultivated crop and an important model for genetics, is capable of forming triple symbiotic systems consisting of the plant, AM fungi and nodule bacteria. As transcriptomic and proteomic approaches are being implemented for studying the mutualistic symbioses of pea, a need for a reference transcriptome of genes expressed under these specific conditions for increasing the resolution and the accuracy of other methods arose. Numerous transcriptome assemblies constructed for pea did not include mycorrhizal roots, hence the aim of the study to construct a reference transcriptome assembly of pea mycorrhizal roots. The combined transcriptome of mycorrhizal roots of Pisum sativum cv. Frisson inoculated with Rhizophagus irregularis BEG144 was investigated, and for both the organisms independent transcriptomes were assembled (coverage 177x for pea and 45x for fungus). Genes specific to mycorrhizal roots were found in the assembly, their expression patterns were examined with qPCR on two pea cultivars, Frisson and Finale. The gene expression depended on the inoculation stage and on the pea cultivar. The investigated genes may serve as markers for early stages of inoculation in genetically diverse pea cultivars.

In this paper, we have analyzed changes in the proteomic spectrum of pea Pisum sativum L. roots during inoculation with rhizobial bacteria with the aim of revealing new regulators of symbiosis development. To study the changes in the proteome spectrum of pea roots, a differential twodimensional (2-D) electrophoresis was performed using fluorescent labels Cy2 and Cy5. The images obtained made it possible to identify differences between the control variant (uninoculated roots) and the root variant after inoculation with Rhizobium leguminosarum bv. viciae RCAM 1026 (24 hours after treatment). 20 proteins were revealed and identified, the synthesis of which was enhanced during the inoculation of pea roots by nodule bacteria. To identify the proteins, a mass spectrometric analysis of tryptic peptides was performed on a quadrupole-time-of-flight mass spectrometer combined with a high-performance liquid chromatograph. Among such proteins, the beta-subunit of the G protein and the disulfide isomerase/phospholipase C were first found, whose function can be related to the signal regulation of symbiosis. This indicates that G-proteins and phospholipases can play a key role in the development of early stages of symbiosis in peas. Further experiments are expected to show whether the beta-subunit of the G protein interacts with the receptors to Nod factors, and how this affects the further signaling. Other proteins that might be interesting were annexin D8 and D1, protein kinase interacting with calcinerin B, actin-binding protein profilin, GTP-binding protein Ran1. They may be involved in the regulation of reactions with calcium, the reorganization of the actin cytoskeleton and other important processes in plants. The study of the role of such regulatory proteins will later become the basis for understanding the complex system of signal regulation, which is activated in pea plants by interaction with nodule bacteria.

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.

Developing seeds of some higher plants are photosynthetically active and contain chlorophylls (Chl), which are typically destroyed at the late stages of seed maturation. However, in some crop plant cultivars, degradation of embryonic Chl remains incomplete, and mature seeds preserve green colour, as it is known for green-seeded cultivars of pea (Pisum sativum L.). The residual Chl compromise seed quality and represent a severe challenge for farmers. Hence, comprehensive understanding of the molecular mechanisms, underlying incomplete Chl degradation is required for maintaining sustainable agriculture. Therefore, here we address dynamics of plastid conversion and photochemical activity alterations, accompanying degradation of Chl in embryos of yellow- and green-seeded cultivars Frisson and Rondo respectively. The yellow-seeded cultivar demonstrated higher rate of Chl degradation at later maturation stage, accompanied with termination of photochemical activity, seed dehydration and conversion of green plastids into amyloplasts. In agreement with this, expression of genes encoding enzymes of Chl degradation was lower in the green seeded cultivar, with the major differences in the levels of Chl b reductase (NYC1) and pheophytinase (PPH) transcripts. Thus, the difference between yellow and green seeds can be attributed to incomplete Chl degradation in the latter at the end of maturation period.