Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development.

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identif ied these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might inf luence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium inf lux inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1-3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative inf luence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the f luorophores such as red f luorescent protein (RFP) and yellow f luorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by inf iltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modif ication or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

A high coverage reference transcriptome assembly of pea (Pisum sativum L.) mycorrhizal roots.

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.

Genetic dissection of Rhizobium-induced infection and nodule organogenesis in pea based on ENOD12A and ENOD5 expression analysis.

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.