Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

Autoregulation and Nitrate Inhibition of Nodule Formation in Soybean cv. Enrei and its Nodulation Mutants

The regulation and nitrate inhibition of nodule formation insoybean, Glycine max (L.) Merr., was further examined usingthe nodulation mutants of cv. Enrei. The non-nodulating mutantsEn115, Enl282, and En1314 produced extremely few markedly-curledroot hairs which were all devoid of infection threads, and invariablyfailed to initiate sub-epidermal cell divisions (SCDs) in theroot cortex. A considerable number of arrested SCDs was foundbefore nodule emergence in Enrei, but not in En6500 which hadsignificantly more SCDs that progressively increased at moreadvanced stages of nodule ontogeny. These observations indicatethat autoregulation acts by blocking the developmental stagebefore nodule emergence. In both Enrei and En65OO, the maturationof emerged nodules was restricted by a late-acting nodulationcontrol mechanism that is apparently unrelated to autoregulation.Reciprocal wedge-grafts of plants inoculated at sowing showedthat the control of the supernodulating phenotype resides inthe shoot, while the non-nodulating phenotype is strictly root-controlled.The nodulation phenotype of the current non-nodulating mutantsresults not from an alteration of the autoregulatory mechanism,but from mutation that exerts a root-localized effect that blocksSCDs which trigger the autoregulatory mechanism. Reciprocalgrafting experiments on Enrei and En6500 seedlings grown undervarious nitrate levels suggest that nitrate inhibition of nodulation,like autoregulation, is shoot-controlled. Since these two processesare invariably expressed together, they are probably causallyrelated, acting synergistically to regulate nodule formationin soybean. These results indicate that the regulation and nitrateinhibition of nodulation in the nodulation mutants of cv. Enreiare similar to those of cv. Bragg nodulation mutants. Key words: Autoregulation, nitrate-tolerant symbosis, non-nodulating mutants, soybean, supernodulating mutant     

Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti

We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.     

Key genes and proteins involved in CTCM-reducing microvascular endothelial cell permeability induced by SLT-IIv using gene chips and DIGE

An Affymetrix mouse genome array and differential in-gel electrophoresis (DIGE) techniques were used to investigate the pharmacological mechanisms of a mixture of herbs, designated CTCM, a compound of traditional Chinese medicine, for the treatment of increased permeability in mouse intestinal microvascular endothelial cells (MIMECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMECs were challenged with 10 μg/ml SLT-IIv for 12 h and then treated with CTCM at a concentration of 200 μg/ml for 12 h. Total RNA and proteins from each treatment group were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip® Mouse Genome 430 2.0 microarray and DIGE. The results obtained demonstrated that there were one genes downregulated and one genes upregulated, one protein downregulated and four proteins upregulated in the SLT-IIv group compared to the control group. In the CTCM group, four genes were upregulated, three genes were downregulated, a single protein was downregulated and a single protein was upregulated when compared to the control group. When the CTCM-treated group was compared to the SLT-IIv group, expression of one gene was found to be increased, and all other genes were decreased, with five proteins downregulated. Analysis of the data suggested that CTCM specifically and effectively reduced microvascular endothelial cell permeability to SLT-IIv in the treatment of pig edema disease. In the CTCM-treated group, hspa9 expression was increased in both gene chip and DIGE analysis, so it may be a key protein in reducing cell permeability and utilized in medical treatments.     

Shoot apex removal does not alter autoregulation of Modulation in soybean

The suppression of new nodule development in soybean (Glycine max (L.) Merr.) has been previously demonstrated to involve the shoot through reciprocal grafts between the wild-type cultivar Bragg and its supernodulating mutant nts382. Using the same grafting technique, but modified through the excision of the shoot apex region and emerging lateral shoots, we show here that autoregulation of nodule number still existed despite apex removal. This radical treatment lowered total nodule number per plant as well as root, shoot and nodule dry weight. Bragg shoots grafted onto nts382 roots gave wild-type nodulation (26 nodules, 15mg total nodule mass) as compared to nts382 shoots grafted onto Bragg roots (340 nodules, 277 mg total nodule mass). Specific nodule mass differed between supernodulating (about 0·5-1·0mg per nodule) and wild-type nodulating (2·3 mg per nodule) plants. In contrast to other growth characteristics, apex removal did not affect specific nodule size, except in plants with wild-type shoots and nts382 (supernodulation) roots. Apex removal only slightly affected the percentage of nodule weight per total root weight in nts382, but had a severe effect in wild type. Growth reductions varied between the normal and supernodulating plants. The fact that autoregulation of nodulation still functions in plants devoid of functional shoot apices suggests that the autoregulation signal may not be derived from the apex regions and that the leaf may be a likely source.     

Proteomic characterization of the acute-phase response of yellow stingrays Urobatis jamaicensis after injection with a Vibrio anguillarum-ordalii bacterin

Systemic inflammatory responses of mammals and bony fish are primarily driven by coordinated up-regulation and down-regulation of plasma acute-phase proteins. Although this general principle is believed to be universal among vertebrates, it remains relatively unexplored in elasmobranchs. The objective of this study was to characterize acute changes in the plasma proteome of three yellow stingrays Urobatis jamaicensis following intraperitoneal injection with a commercial Vibrio bacterin. Changes in plasma protein levels were analyzed immediately prior to vaccination (time 0) and at 24 and 72 h post-injection by isobaric tags for relative and absolute quantitation (iTRAQ 4-plex) using shotgun-based nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and de novo peptide sequencing. Pooled 2D-LC-MS/MS and de novo sequencing data revealed differential expression of 156 distinct plasma proteins between time 0 and at least one post-vaccination time point. Using 1.5-fold change in expression as physiologically significant, 14/156 (9.0%) proteins were upregulated in at least one stingray through at least one experimental timepoint. Upregulated proteins included complement factors, Mx-protein, hemopexin, factor X and prothrombin. Seventy-six of 156 (48.7%) proteins were downregulated in the acute-phase response, including transferrin, apolipoprotein B, heparin cofactor 2, alpha2-macroglobulin, and various growth factors. Other differentially upregulated or downregulated proteins included intracellular, cell binding and structural proteins, proteins involved in physiologic processes, and unknown/hypothetical proteins. Selected bioactive factors are discussed for their putative roles in the elasmobranchs acute-phase response. These findings contribute to our understanding of disease processes in elasmobranchs, immunologic phylogeny in vertebrates, and begin the search for potential biomarkers of disease in these ecologically important fish.     

Competitive interaction between non-nodulating and nodulating strains for nodulation of cowpea (Vigna unguiculata)

Abstract: We have examined the effect of a non-nodulating mutant (JRW3-Sm^D) on the nodulation ability of cowpea rhizobia ( Bradyrhizobium sp.) strains JRW3 and IRC256. Nodulation of cowpea ( Vigna unguiculata ) by a nodulating Rhizobium strain is suppressed by the presence of a non-nodulating mutant. The msgnitude of suppression for nodule formation by nodulating strains varied between 40% and 80% depending on the strain and the time of inoculation.     

The Auxin Transport Inhibitor N-(1-Naphthyl)phthalamic Acid Elicits Pseudonodules on Nonnodulating Mutants of White Sweetclover

The collection of symbiotic (sym) mutants of white sweetclover (Melilotus alba Desr.) provides a developmental sequence of mutants blocked early in infection or nodule organogenesis. Mutant phenotypes include non-nodulating mutants that exhibit root-hair deformations in response to Rhizobium meliloti, mutants that form ineffective nodules lacking infection threads, and mutants that form infection threads and ineffective nodules. Mutant alleles from both the sym-1 and the sym-3 loci exhibited a non-nodulating phenotype in response to R. meliloti, although one allele in the sym-1 locus formed ineffective nodules at a low frequency. Spot-inoculation experiments on a non-nodulating allele in the sym-3 locus indicated that this mutant lacked cortical cell divisions following inoculation with R. meliloti. The auxin transport inhibitor N-(1-naphthyl)phthalamic acid elicited development of pseudonodules at a high frequency on all of the sweetclover sym mutants, including the non-nodulating mutants, in which the early nodulin ENOD2 was expressed. This suggests that N-(1-naphthyl)phthalamic acid activates cortical cell divisions by circumventing a secondary signal transduction event that is lacking in the non-nodulating sweetclover mutants. The sym-3 locus and possibly the sym-1 locus appear to be essential to early host plant responses essential to nodule organogenesis.     

Effect of the cyclic stretch on the expression of osteogenesis genes in human periodontal ligament cells

Periodontal ligament cells can potentially differentiate into osteoblast-like cells and influence the remodeling of periodontal tissues under mechanical strain conditions. In the present study, Gene chip technology was adopted to investigate the effect of the cyclic stretch on the expression of osteogenic-related genes in human periodontal ligament cells (HPDLCs). Cultured HPDLCs were subjected to 12% elongation cyclic stretch for 24 h using a Flexercell Strain Unit, and then GEArray Q series human osteogenesis gene expression profile chip with 96 spot array numbers was used to conduct parallel analyses on the change of the related gene expression in the osteogenic differentiation of HPDLCs stimulated by cyclic stretch. The results show that after the HPDLCs were stimulated by the cyclic stretch, the expression of 21 osteogenic-related genes was significantly upregulated, including 10 growth factor genes and their associated molecules, 10 extracellular matrix genes and their associated proteins, and 1 cell adhesion molecule. Two genes were significantly downregulated, including one growth factor gene and one cell adhesion molecule. Then the expressions of 10 candidate genes were validated using Real-time RT-PCR. These results indicate that cyclic stretch with 12% deformation can stimulate or inhibit some gene expression which was associated with the process of HPDLCs differentiation.     

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.     

Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots after Inoculation with Rhizobium etli

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.