Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.     

A white clover nodulin gene,dd23b,encoding a cysteine cluster protein,is expressed in roots during the very early stages of interaction with Rhizobium leguminosarumbiovar trifolii and after treatment with chitolipooligosaccharide Nod factors

An early nodulin cDNA, dd23b, was isolated from white clover root tissue by differential display RT-PCR. Its full-length sequence of 340 nucleotides encodes a predicted 72-amino-acid protein of molecular mass 8.3 kDa, with a polypeptide region containing cysteine pairs spaced in the manner of a cysteine cluster protein. This feature, which is shared by some other late and early nodulins from pea and broad bean, suggests a role in metal ion binding and membrane transport. Temporal and spatial expression patterns were determined during infection and nodulation by the homologous microsymbiont. No expression was found in unchallenged root tissue over a 7-day sampling period. Expression was first detectable in roots by RT-PCR 6 h post-inoculation with Rhizobium leguminosarum biovar trifolii, placing dd23b among the earliest nodulins to be detected to date. In root nodules, expression occurred primarily in the central symbiotic zone, but also in some host cells within the infection zone. Addition of purified wild-type chitolipooligosaccharide Nod factor to axenic white clover roots induced dd23b expression, providing further evidence for the role of this gene in the early plant response to infection by rhizobia. Electronic Publication     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

Structural determination of the Nod factors produced by Rhizobium gallicum bv. gallicum R602

Rhizobium gallicum is a fast-growing bacterium found in European, Australian and African soils; it was first isolated in France. It is a microsymbiont which is able to nodulate plants of the genus Phaseolus. Rhizobium gallicum bv. gallicum R602 produces four extracellular signal molecules consisting of a linear backbone of N-acetyl glucosamine, bearing on the nonreducing terminal residue an N-methyl group and different N-acyl substituents. The four acyloligosaccharides terminate with a sulfated N-acetylglucosaminitol. This unit may be also acetylated. These structures were determined using carbohydrate and methylation analysis, mass spectrometric analysis and one-dimensional- and two-dimensional-nuclear magnetic resonance experiments. This work establishes the common structure that a lipochito-oligosaccharide must have so that the Rhizobium that produces and excretes it is able to nodulate plants of Phaseolus vulgaris. The substituents common to all the molecules are an N-methyl group and a C(18:1) fatty acid on the nonreducing terminal residue.     

Early responses to Nod factors and mycorrhizal colonization in a non-nodulating Phaseolus vulgaris mutant

Legumes can acquire nitrogen through a symbiotic interaction with rhizobial bacteria. The initiation of this process is determined by a molecular dialogue between the two partners. Legume roots exude flavonoids that induce the expression of the bacterial nodulation genes, which encode proteins involved in the synthesis and secretion of signals called Nod factors (NFs). NFs signal back to the plant root and trigger several responses, leading to bacterial invasion and nodule formation. Here, we describe the molecular and cellular characterization of a Phaseolus vulgaris non-nodulating mutant (NN-mutant). Root hair cells of the NN-mutant plant respond with swelling and branching when inoculated with Rhizobium etli, albeit without curling induction. Furthermore, neither initiation of cell division in the outer cortex, nor entrapment of bacteria nor infection thread formation was observed. Both the bean wild-type and the NN-mutant responded with elevated intracellular calcium changes in the root hairs. Although the NN-mutant is deficient in early nodulin gene expression when inoculated with R. etli, it can be effectively colonized by arbuscular mycorrhizal fungi (Glomus intraradices). Our data indicate that the P. vulgaris NN-mutant is not blocked at the NFs early perception stage, but at later downstream stages between Ca²⁺ signaling and early nodulin induction. This supports the idea that both microsymbionts are perceived and trigger different downstream pathways in the host plant.     

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-kappaB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-kappaB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IkappaB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-kappaB activation.     

Signal molecules in the peanut–bradyrhizobia interaction

Main nodulation signal molecules in the peanut–bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed. Accession numbers: AY427207, EF202193, EF158295 (16S rRNA gene of strains NLH25, NOD31 and NDEHE, respectively); DQ295199, DQ295200, DQ295201 (Partial nifD gene sequences of strains NLH25, NOD31 and NDEHE, respectively).     

昆明西山滇青冈林内滇青冈种子库动态的研究

通过对昆明西山滇青冈林内滇青冈种子库的跟踪取样调查和种子埋藏试验,对滇青冈种子库的动态进行了研究。昆虫在种子成熟前侵入种子,经种子雨进入种子库时已有71.8%的种子失去萌发能力。种子雨输入种子库的绝大部分种子停留在表面种子库,其中48.55%的种子被虫害,25.36%被某些非生物或生物搬运,17.39%的腐烂,8.7%的被动物当场取食,没有种子萌发,影响种子库动态的各种因子的作用大小在时间上是变化。被搬运的种子中,有4.9%的由表面种子库转移到埋藏种子库。土层是滇青冈种子的安全生境,土壤种子库的存在时间超过250天。埋入土壤的试验种子一直处于静止状态,到6月雨季后有80%种子萌发,20%的腐烂。萌发种子数是当年产种子的0.26%。滇青冈林内的滇青冈种子库是季节性的,种子库对种群个体的补充作用是有限的。     

Comparison between parental and variant soybeanBradyrhizobium strains with regard to the production of lipo-chitin nodulation signals,early stages of root infection,nodule occupancy,and N2 fixation rates

Soybean is the most important leguminous crop in Brazil and the nitrogen required for plant growth is supplied byBradyrhizobium bacteria through the symbiotic relation established by the inoculation process. Since 1992, two new strains, CPAC 7 and CPAC 15, which have been shown to increase yields in several field experiments, have been recommended in Brazilian commercial inoculants. CPAC 15 is a natural variant of theB. elkanii SEMIA 566 strain, and was isolated after several years of adaptation to a Brazilian Cerrado soil, while CPAC 7 is a variant ofB. japonicum strain CB 1809, selected under laboratory conditions for higher nodulation and yield. The comparison between parental and variant strains, under greenhouse conditions, showed that both CPAC 15 and CPAC 7 increased N₂ fixation rates in relation to the parental strains. The better performance of CPAC 15 was related to an increase in nodule efficiency (mg N₂ fixed mg^-1 nodule) while with CPAC 7 the higher N₂ fixation rates were due to increased nodulation. Both CPAC 15 and CPAC 7 increased nodule occupancy, when co-inoculated at a ratio of 1:1 withB. elkanii 29w, in relation to their parental strains. Variant strains also differed from parental in their ability to increase numbers of root hairs (Hai phenotype) either when inoculated onto plants, or when supernatants of bacteria exposed to seed exudates were used as inoculants. This results lead to the hypothesis that a modification in some of the “common” nodulation genes had occurred. However, the increase in Hai phenotype with CPAC 7 was dependent on the soybean cultivar, indicating a possible alteration in some genotypic specific nodulation gene. Apparently, there were no differences in Nod metabolites produced by strains CPAC 15 and SEMIA 566, but a more detailed chemical analysis would be required to rule out subtle differences. On the contrary, significant differences were found between CPAC 7 and the parental strain CP 1809, in the profile of Nod metabolites. Consequently, it may be possible that diffusable molecules, responsible for Hai phenotype, would be related to nodulation ability, competiviveness, and N₂ fixation, resulting in the higher yields that have been associated with CPAC 7 and CPAC 15. For the CPAC 7 strain, the increase in Hai phenotype could be atributed to the differences found in the Nod molecules. Consequently, a high degree of physiological and genetic variability can result from the adaptation of rhizobial strains to the soil. Also, this variability can be found under laboratory conditions, when searching single colonies with specific properties. ei]Section editor: R O D Dixon