Mapping-by-sequencing using NGS-based 3′-MACE-Seq reveals a new mutant allele of the essential nodulation gene Sym33 (IPD3) in pea (Pisum sativum L.)

Large collections of pea symbiotic mutants were accumulated in the 1990s, but the causal genes for a large portion of the mutations are still not identified due to the complexity of the task. We applied a Mapping-by-Sequencing approach including Bulk Segregant Analysis and Massive Analysis of cDNA Ends (MACE-Seq) sequencing technology for genetic mapping the Sym11 gene of pea which controls the formation of symbioses with both nodule bacteria and arbuscular-mycorrhizal fungi. For mapping we developed an F2-population from the cross between pea line N24 carrying the mutant allele of sym11 and the wild type NGB1238 (=JI0073) line. Sequencing libraries were prepared from bulks of 20 plants with mutant and 12 with wild-type phenotype. MACE-Seq differential gene expression analysis between mutant-phenotype and wild-type-phenotype bulks revealed 2,235 genes, of which 514 (23%) were up-regulated and 1,721 (77%) were down-regulated in plant roots inoculated with rhizobia as a consequence of sym11 mutation. MACE-Seq also detected single nucleotide variants between bulks in 217 pea genes. Using a novel mathematical model we calculated the recombination frequency (RF) between the Sym11 gene and these 217 polymorphic genes. Six genes with the lowest RF were converted into CAPS or dCAPS markers and genetically mapped on the complete mapping population of 108 F2-plants which confirmed their tight linkage to Sym11 and to each other. The Medicago truncatula Gaertn. (Mt) homologs of these genes are located in a distinct region of Mt chromosome 5, which corresponds to linkage group I of pea. Among 94 candidate genes from this region only one was down-regulated—the pea Sym33 homolog of the Mt IPD3 gene which is essential for nodulation. Sequencing of the Sym33 allele of the N24 (sym11) mutant revealed a single nucleotide deletion (c.C319del) in its third exon resulting in a codon shift in the open reading frame and premature translation termination. Thus, we identified a novel mutant allele sym33-4 most probably responsible for the mutant phenotype of the N24 (sym11) line, thereby demonstrating that mapping by MACE-Seq can be successfully used for genetic mapping of mutations and identification of candidate genes in pea

Transcriptomic Insights into Mechanisms of Early Seed Maturation in the Garden Pea (Pisum sativum L.)

The garden pea (Pisum sativum L.) is a legume crop of immense economic value. Extensive breeding has led to the emergence of numerous pea varieties, of which some are distinguished by accelerated development in various stages of ontogenesis. One such trait is rapid seed maturation, which, despite novel insights into the genetic control of seed development in legumes, remains poorly studied. This article presents an attempt to dissect mechanisms of early maturation in the pea line Sprint-2 by means of whole transcriptome RNA sequencing in two developmental stages. By using a de novo assembly approach, we have obtained a reference transcriptome of 25,756 non-redundant entries expressed in pea seeds at either 10 or 20 days after pollination. Differential expression in Sprint-2 seeds has affected 13,056 transcripts. A comparison of the two pea lines with a common maturation rate demonstrates that while at 10 days after pollination, Sprint-2 seeds show development retardation linked to intensive photosynthesis, morphogenesis, and cell division, and those at 20 days show a rapid onset of desiccation marked by the cessation of translation and cell anabolism and accumulation of dehydration-protective and -storage moieties. Further inspection of certain transcript functional categories, including the chromatin constituent, transcription regulation, protein turnover, and hormonal regulation, has revealed transcriptomic trends unique to specific stages and cultivars. Among other remarkable features, Sprint-2 demonstrated an enhanced expression of transposable element-associated open reading frames and an altered expression of major maturation regulators and DNA methyltransferase genes. To the best of our knowledge, this is the first comparative transcriptomic study in which the issue of the seed maturation rate is addressed

Receptor-Like Kinase LYK9 in Pisum sativum L. Is the CERK1-Like Receptor that Controls Both Plant Immunity and AM Symbiosis Development

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

Transcriptome Analysis of Alternative Splicing Events Induced by Arbuscular Mycorrhizal Fungi (Rhizophagus irregularis) in Pea (Pisum sativum L.) Roots

Alternative splicing (AS), a process that enables formation of different mRNA isoforms due to alternative ways of pre-mRNA processing, is one of the mechanisms for fine-tuning gene expression. Currently, the role of AS in symbioses formed by plants with soil microorganisms is not fully understood. In this work, a comprehensive analysis of the transcriptome of garden pea (Pisum sativum L.) roots in symbiosis with arbuscular mycorrhiza was performed using RNAseq and following bioinformatic analysis. AS profiles of mycorrhizal and control roots were highly similar, intron retention accounting for a large proportion of the observed AS types (67%). Using three different tools (SUPPA2, DRIMSeq and IsoformSwitchAnalyzeR), eight genes with AS events specific for mycorrhizal roots of pea were identified, among which four were annotated as encoding an apoptosis inhibitor protein, a serine/threonine-protein kinase, a dehydrodolichyl diphosphate synthase, and a pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH1. In pea mycorrhizal roots, the isoforms of these four genes with preliminary stop codons leading to a truncated ORFs were up-regulated. Interestingly, two of these four genes demonstrating mycorrhiza-specific AS are related to the process of splicing, thus forming parts of the feedback loops involved in fine-tuning of gene expression during mycorrhization

Association Study of Symbiotic Genes in Pea (Pisum sativum L.) Cultivars Grown in Symbiotic Conditions

In garden pea (Pisum sativum L.), several symbiotic genes are known to control the development of mutualistic symbioses with nodule bacteria (NB) and arbuscular mycorrhizal fungi (AMF). Here, we studied whether the allelic state of the symbiotic genes was associated with the growth parameters of pea plants under single inoculation with NB and under double inoculation with NB + AMF. Using different statistical methods, we analyzed the dataset obtained from a pot experiment that involved 99 pea cultivars, 10 of which were characterized as having shortened internodes due to the presence of the natural mutation p.A229T in the developmental gene Le. The plant’s habitus strongly influenced most of the studied growth and yield parameters and the effectiveness of the symbiotic interactions under NB and NB + AMF inoculation. Double inoculation had different effects on Le+ (normal) and le− (dwarf) plants with regard to nitrogen and phosphorus content in seeds. Regardless of the Le-status of plants, allelic states of the symbiotic gene LykX encoding the putative receptor of Nod factors (bacterial signal molecules) were shown to be associated with seed number, thousand-seed weight, and pod number at the level of FDR < 0.001, whereas associations of allelic states of the other studied symbiotic genes were less significant

LysM Receptor-Like Kinase LYK9 of <i>Pisum Sativum</i> L. May Regulate Plant Responses to Chitooligosaccharides Differing in Structure

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

LysM Receptor-Like Kinase LYK9 of Pisum Sativum L. May Regulate Plant Responses to Chitooligosaccharides Differing in Structure

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&ndash;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