Initiation of a legume nodule with an indeterminate meristem involves proliferating host cells that harbour infection threads.

A comparative analysis of nodule morphogenesis was carried out for three symbiotically defective pea (Pisum sativum) mutants that show abnormalities in nodule development.In the wild-type lines, resumption of cell proliferation in the pericycle and inner cortex results in the development of a nodule primordium, within which are found proliferating cells that harbour infection threads. However, this class of cell is not observed in the mutants RisFixA (sym41) and SGEFix­-2 (sym33) where nodule development is arrested at the point of formation of the apical nodule meristem. Itis proposed that the presence of proliferating cells harbouring infection threads is a prerequisite for normal formation of the nodule meristem.In mutant SGEFix­-1 (sym40), nodule development does not differ from that of wild-type plants in the early stages but is blocked at the stage after nodule meristem persistence. A scheme is proposed for the sequential functioning of pea symbiotic genes Sym33, Sym40 and Sym41 in the programme of nodule development.

Defects in rhizobial cyclic glucan and lipopolysaccharide synthesis alter legume gene expression during nodule development.

cDNA array technology was used to compare transcriptome profiles of Lotus japonicus roots inoculated with a Mesorhizobium loti wild-type and two mutant strains affected in cyclic beta(1-2) glucan synthesis (cgs) and in lipopolysaccharide synthesis (lpsbeta2). Expression of genes associated with the development of a fully functional nodule was significantly affected in plants inoculated with the cgs mutant. Array results also revealed that induction of marker genes for nodule development was delayed when plants were inoculated with the lpsbeta2 mutant. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to quantify gene expression of a subset of genes involved in plant defense response, redox metabolism, or genes that encode for nodulins. The majority of the genes analyzed in this study were more highly expressed in roots inoculated with the wild type compared with those inoculated with the cgs mutant strain. Some of the genes exhibited a transient increase in transcript levels during intermediate steps of normal nodule development while others displayed induced expression during the final steps of nodule development. Ineffective nodules induced by the glucan mutant showed higher expression of phenylalanine ammonia lyase than wild-type nodules. Differences in expression pattern of genes involved in early recognition and signaling were observed in plants inoculated with the M. loti mutant strain affected in the synthesis of cyclic glucan.

Dissection of symbiosis and organ development by integrated transcriptome analysis of lotus japonicus mutant and wild-type plants.

Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set.

The Sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus.

Comparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for the PsSym35 and LjNin genes in early stages of root nodule formation. Both the pea and L. japonicus mutants are non-nodulating but normal in their arbuscular mycorrhizal association. Both are characterized by excessive root hair curling in response to the bacterial microsymbiont, lack of infection thread initiation, and absence of cortical cell divisions. To investigate the molecular basis for the similarity, we cloned and sequenced the PsNin gene, taking advantage of sequence information from the previously cloned LjNin gene. An RFLP analysis on recombinant inbred lines mapped PsNin to the same chromosome arm as the PsSym35 locus and direct evidence demonstrating that PsNin is the PsSym35 gene was subsequently obtained by cosegregation analysis and sequencing of three independent Pssym35 mutant alleles. L. japonicus and pea root nodules develop through different organogenic pathways, so it was of interest to compare the expression of the two orthologous genes during nodule formation. Overall, a similar developmental regulation of the PsNin and LjNin genes was shown by the transcriptional activation in root nodules of L. japonicus and pea. In the indeterminate pea nodules, PsNin is highly expressed in the meristematic cells of zone I and in the cells of infection zone II, corroborating expression of LjNin in determinate nodule primordia. At the protein level, seven domains, including the putative DNA binding/dimerization RWP-RK motif and the PB1 heterodimerization domain, are conserved between the LjNIN and PsNIN proteins.