Reduction In Number Of Nodules Research Articles

A lateral organ boundaries domain transcription factor acts downstream of the auxin response factor 2 to control nodulation and root architecture in Medicago truncatula.

Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans-Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes. Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP-PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2. Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

GmTNRP1, associated with rhizobial type‐III effector NopT, regulates nitrogenase activity in the nodules of soybean (Glycine max)

AbstractSoybean is a rich source of protein and oil for humans. It forms a symbiotic association with rhizobia to fix atmospheric nitrogen for its growth and development. Rhizobial type‐III effectors act as important signaling molecules in symbiosis. However, they are poorly studied. In this study, mutation of TtsI in Sinorhizobium fredii HH103 reduced nodule numbers and dry weights in SN14 (cultivated soybean) and ZYD00006 (wild soybean). Moreover, it reduced nitrogenase activity in SN14 but not in ZYD00006. Based on the differences in nitrogenase activity between SN14 and ZYD00006 inoculated with HH103 and the TtsI mutant, two conditional quantitative trait loci associated with nitrogenase activity were identified in chromosome segment substitution lines derived from SN14 and ZYD00006. Using transcriptome analysis, single‐nucleotide polymorphism (SNP) analysis, and qRT‐PCR, Glyma.13G150000 (GmTNRP1) was identified as a candidate gene in soybean that was associated with nitrogenase activity, along with NopT (a type‐III effector). In addition, GmTNRP1 (an LRR‐RK family protein located on the cell membrane) was observed to be induced by NopT to negatively regulate nitrogenase activity. In transgenic hairy roots with GmTNRP1 silencing, no significant difference was observed in terms of nitrogenase activity between nodules inoculated with the NopT mutant and those inoculated with HH103 (wild type). However, the nitrogenase activity in the nodules decreased after overexpression of GmTNRP1. SNP located in the GmTNRP1 promoter affected its expression in response to NopT. Haplotype analysis revealed that SN14 belonged to Hap1 and ZYD00006 belonged to Hap2, which explained the different results in the two varieties. To the best of our knowledge, this is the first study to identify a soybean gene associated with a rhizobial type‐III effector in the nodules. GmTNRP1 can negatively regulate nitrogenase activity in the nodules. This study enhanced the understanding of the mechanism underlying symbiotic association between soybean and rhizobia.

GmNLP7a inhibits soybean nodulation by interacting with GmNIN1a

Nitrogen (N) is an essential macronutrient for plant growth and productivity. Leguminous plants establish symbiotic relationships with nitrogen-fixing rhizobial bacteria to use atmospheric dinitrogen gas to meet high N demand under low-N conditions. Nodule formation and N fixation are energy-consuming processes and are inhibited by nitrate present in the environment. Previous studies in model leguminous plants characterized NIN-LIKE PROTEIN (NLP) proteins that mediate nitrate control of root nodule symbiosis, but the mechanism by which nitrate regulates soybean root nodules via NLP remains unclear. In the soybean genome we found four homologs of AtNLP7, named GmNLP7a–GmNLP7d. We showed that the expression of GmNLP7s is responsive to nitrate but not to rhizobial infection and localized GmNLP7a to the nucleus. Downregulation of GmNLP7s increased nodule number, and overexpression of GmNLP7a (GmNLP7aOE) reduced nodule number regardless of nitrate availability, suggesting a negative role for GmNLP7s in nodulation. Nitrogenase activity in the GmNLP7aOE line was comparable to that of the wild type, indicating that GmNLP7a does not affect mature nodule activity. Overexpression of GmNLP7a downregulated the expression of GmNIN1a and GmENOD40-1. GmNLP7a interacted with GmNIN1a via the PB1 domain. Our results reveal a new regulator of GmNLP7 in nodulation and a molecular mechanism by which nitrate affects nodule number in soybean.

Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula

BackgroundSymbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules.ResultsWe profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia.ConclusionsOur transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

Suitability of selected legume (Vachellia spp.) tree species for forest restoration in the Central Ethiopian highlands

Abstract. Asmelash F, Getachew E. 2022. Suitability of selected legume (Vachellia spp.) tree species for forest restoration in the Central Ethiopian highlands. Nusantara Bioscience 14: 195-202. This study aimed to evaluate the comparative suitability of the legume tree species viz., Vachellia abyssinica (Hochst. ex. Benth.) Kyal. & Boatwr., Vachellia etbaica (Schweinf.) Kyal. & Boatwr., Vachellia lahai (Steud. & Hochst. ex Benth.) Kyal. & Boatwr., and Vachellia seyal (Delile) P.J.H.Hurter for restoring forests in Central Ethiopian highlands. The suitability of three accessions of V. seyal was also compared. The correlation between root nodule number and root Arbuscular Mycorrhizal Fungi (AMF) colonization (RC) and seedlings’ growth variables were computed, and the effect of seedling age on nodule number and RC was determined. Seedlings were grown for nine months on degraded local soil in a mesh house in central Ethiopia. We measured shoot height, shoot fresh weight, rooting depth, root nodule number, and RC in the third and ninth months. The one-way ANOVA results indicated that tree species and/or accession (for V. seyal) had a significant (p<0.05) effect on all the measured variables except shoot height and rooting depth at the ninth month. Generally, nodule number and RC increased with seedling age. However, according to the independent t-test results, significant (p<0.05) differences were recorded for V. abyssinica, with a 57.16% reduction in nodule number, and V. seyal accession-1, with a 418.52% increase in RC. The Spearman’s rank correlation results indicated that the correlation between nodule number and RC was weak and non-significant (p>0.05) both in the third and ninth months. Based on the measured growth variables, nodule number (N-fixation potential), and RC, V. etbaica was the least suitable species for forest restoration in central Ethiopian highlands. The remaining species/accessions are comparably suitable. The V. abyssinica lost its comparative fitness with seedling age, maybe because it is a provenance far away from central Ethiopia. However, V. seyal accession-3, the furthest provenance, has performed much better. The legume trees of Ethiopia are less studied. Their role as environmental engineers could be better understood by knowing more about their root traits. Therefore, this study could motivate future research in this regard. Long-term experiments are required to consider more legume tree species and provenances in the future.

Genome-wide identification, expression, and characterization of CaLysM-RLKs in chickpea root nodule symbiosis

Legumes establish a nitrogen-fixing symbiosis with rhizobia which depends on the exchange of chemical signals between the two partners. Successful interaction between legumes and rhizobia depends on the perception of Nod factors (NFs) by the receptor-like kinases. A few cell surface receptors have been identified that perceive the rhizobial NFs and activate the downstream signaling pathway for nodulation in model legumes like Lotus japonicus and Medicago truncatula . However, crop legume such as chickpea lacks detailed analysis of LysM-RLKs genes for NFs perception. Here, we identified and characterized CaLysM-RLK genes and analyzed their gene structure, motif composition, chromosomal localization, phylogenetic relationship with Arabidopsis, Oryza sativa , and Medicago truncatula . To understand the spatiotemporal expression patterns, we performed transcript profiling of Ca LysM-RLKs genes at a different time interval of the chickpea-rhizobia symbiotic interaction that revealed CaNFP and CaLYK3 can act as candidate genes for NF perception. Down-regulation of CaNFP and CaLYK3 in chickpea by agrobacterium rhizogenes mediated hairy root sysytem leads to signifificant reduction in nodule numbers. This study will be useful for improving root nodule symbiosis in chickpea. • Seventeen CaLysM-RLKs genes were identified in chickpea. • Based on the structure CaLysM-RLKs family members were classified into three main groups LYS-I, LYS-II, and LYS-III. • Analysis of paralogous gene pair (PGP) leads to the identification of a total of 6 PGPs. • CaLYK3 , and CaNFP regulate root nodule symbiosis in chickpea.

Genetic variation in GmCRP contributes to nodulation in soybean (Glycine max Merr.)

Symbiosis between soybean and rhizobia contributes to soybean yield and quality. Although secreted rhizobial type III effectors are known to regulate infection and promote nitrogen fixation, much about them remains unknown. Mutation of NopC, a type III effector from Sinorhizobium fredii HH103, reduced nodule numbers and dry weights in 310 soybean accessions, and expression of NopC in soybean hairy roots promoted symbiosis. Based on observed differences in nodule traits between Suinong 14 and Zyd 00,006 inoculated with HH103 and the NopC mutant, 11 QTL associated with rhizobia were identified in chromosome segment substitution lines (CSSLs) derived from Suinong 14 and Zyd 00006. Using chromosome fragment insertion, whole-genome sequencing of Suinong 14 and Zyd 00006, and qRT-PCR, Glyma.19G176300 (GmCRP) was identified as a candidate gene associated with NopC, and GmCRP was found to be induced by NopC to positively regulate nodulation. SNPs located in the regulatory regions of GmCRP influenced its expression response to NopC, with SNPs contributing to nodulation having been selected during domestication. Our findings reveal the function of a soybean gene encoding a rhizobial type III effector that contributes to symbiosis, and will facilitate the practical application of symbiotic nitrogen fixation in molecular breeding.

Clade-dependent effects of drought on nitrogen fixation and its components – Number, size, and activity of nodules in legumes

Drought affects the growth of legumes directly, and indirectly, by reducing total nitrogen fixation . Here, we compiled published data to compare the sensitivity to water deficit on plant growth and total nitrogen fixation traits, i.e., the number of nodules per plant, average nodule mass, and nitrogen fixation per unit nodule mass. Hierarchies of phenotypic plasticity have been established for seeds and organelles , whereby variation in number associates with conserved size. By analogy, our first hypothesis is that there is a hierarchy of plasticities between nitrogen fixation traits. Our second hypothesis is that determinate nodules are more sensitive to water deficit than their indeterminate counterparts, because the latter can reactivate meristems when water becomes available. In our sample, onset of stress treatment averaged 28 d after sowing; median duration of stress was 12 d; and intensity of stress (ratio of shoot biomass between stressed and control) averaged 0.65. These drought conditions (i) reduced total nitrogen fixation and average nodule mass more severely than plant shoot mass, (ii) elicited a hierarchy of plasticities whereby number of nodules per plant varied substantially, and average nodule mass and nitrogen fixation per unit nodule mass were relatively conserved, and (iii) affected more severely Milletioids (determinate, ureide exporting nodules) than their IRLC counterparts (indeterminate, amide exporting nodules). • Total N fixation was partitioned as nodule number, average nodule mass, and nitrogen fixation per unit nodule mass. • Early-season drought reduced nodule number more than average nodule mass and N fixation per unit nodule mass. • Drought effect were more severe in Milletioid (determinate, ureide) than in IRLC (indeterminate, amide) nodules.

Spatiotemporal changes in gibberellin content are required for soybean nodulation.

The plant hormone gibberellin (GA) is required at different stages of legume nodule development, with its spatiotemporal distribution tightly regulated. Transcriptomic and bioinformatic analyses established that several key GA biosynthesis and catabolism enzyme encoding genes are critical to soybean (Glycine max) nodule formation. We examined the expression of several GA oxidase genes and used a Förster resonance energy transfer-based GA biosensor to determine the bioactive GA content of roots inoculated with DsRed-labelled Bradyrhizobium diazoefficiens. We manipulated the level of GA by genetically disrupting the expression of GA oxidase genes. Moreover, exogenous treatment of soybean roots with GA3 induced the expression of key nodulation genes and altered infection thread and nodule phenotypes. GmGA20ox1a, GmGA3ox1a, and GmGA2ox1a are upregulated in soybean roots inoculated with compatible B. diazoefficiens. GmGA20ox1a expression is predominately localized to the transient meristem of soybean nodules and coincides with the spatiotemporal distribution of bioactive GA occurring throughout nodule organogenesis. GmGA2ox1a exhibits a nodule vasculature-specific expression pattern, whereas GmGA3ox1a can be detected throughout the nodule and root. Disruptions in the level of GA resulted in aberrant rhizobia infection and reduced nodule numbers. Collectively, our results establish a central role for GAs in root hair infection by symbiotic rhizobia and in nodule organogenesis.

Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis

Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.

Asymmetric redundancy of soybean Nodule Inception (NIN) genes in root nodule symbiosis.

Nodule Inception (NIN) is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legumes. Unlike most legumes with a sole NIN gene, there are four putative orthologous NIN genes in soybean (Glycine max). Whether and how these NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that the Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced fewer nodules but displayed a hyper infection phenotype compared to wild type (WT), while the Gmnin1a nin1b double mutant nodulated similar to WT. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule-like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a, and GmNIN2b play functionally redundant yet complicated roles in soybean nodulation. GmNIN1b, although expressed at a comparable level with the other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of the asymmetrically redundant function of GmNIN genes in soybean.

NSP1 allies with GSK3 to inhibit nodule symbiosis.

Salt stress reduces N2 fixation by causing a reduction in nodule number, nodule weight, and nitrogenase activity in legumes. Emerging evidence from He et al. now suggests that glycogen synthase kinase 3 (GSK3) phosphorylates nodulation signaling pathway 1 (NSP1) in response to salt stress, reducing its DNA-binding activity, and thereby causing a reduction in nodulation.

Shoot Extracts from Two Low Nodulation Mutants Significantly Reduce Nodule Number in Pea.

E107 and E132 are pea mutants that nodulate poorly. Because they have a shoot-controlled nodulation phenotype, we asked if their mutated genes were implicated in the autoregulation of nodulation (AON), a mechanism which consists of two systemic circuits, the positive CEP/CRA2 and the negative CLE/SUNN, coordinated via NIN and miR2111. We further characterized the mutants’ phenotype by studying nodule distribution and nodulation efficiency. E107 was similar to wild-type (WT) in its nodule distribution, but E132 had an extended nodulation zone with nodules forming distally on its lateral roots. Moreover, we tested whether their shoots produced a compound inhibitory to nodulation. We made ethyl-acetate extracts of roots and shoots of both mutants and WT, which we applied to rhizobia-inoculated WT seedlings and to pure rhizobial cultures. Whereas free-living bacteria were unaffected by any of the extracts, WT treated with shoot extracts from either inoculated mutant had fewer nodules than that of control. E107 and E132 shoot extracts led to a 50% and a 35% reduction in nodule number, respectively. We propose that E107 and E132 belong to a new sub-class of AON mutants, i.e., hypo-nodulators, and that their respective gene products are acting in the AON descending branch, upstream of TML signaling.

Isoflavone malonyl-CoA acyltransferase GmMaT2 is involved in nodulation of soybean by modifying synthesis and secretion of isoflavones.

Malonyl-CoA:flavonoid acyltransferases (MaTs) modify isoflavones, but only a few have been characterized for activity and assigned to specific physiological processes. Legume roots exude isoflavone malonates into the rhizosphere, where they are hydrolyzed into isoflavone aglycones. Soybean GmMaT2 was highly expressed in seeds, root hairs, and nodules. GmMaT2 and GmMaT4 recombinant enzymes used isoflavone 7-O-glucosides as acceptors and malonyl-CoA as an acyl donor to generate isoflavone glucoside malonates. GmMaT2 had higher activity towards isoflavone glucosides than GmMaT4. Overexpression in hairy roots of GmMaT2 and GmMaT4 produced more malonyldaidzin, malonylgenistin, and malonylglycitin, and resulted in more nodules than control. However, only GmMaT2 knockdown (KD) hairy roots showed reduced levels of malonyldaidzin, malonylgenistin, and malonylglycitin, and, likewise, reduced nodule numbers. These were consistent with the up-regulation of only GmMaT2 by rhizobial infection, and higher expression levels of early nodulation genes in GmMaT2- and GmMaT4-overexpressing roots, but lower only in GmMaT2-KD roots compared with control roots. Higher malonyl isoflavonoid levels in transgenic hairy roots were associated with higher levels of isoflavones in root exudates and more nodules, and vice versa. We suggest that GmMaT2 participates in soybean nodulation by catalyzing isoflavone malonylation and affecting malonyl isoflavone secretion for activation of Nod factor and nodulation.

The quantity of nitrogen derived from symbiotic N fixation but not the relative contribution of N fixation to total N uptake increased with breeding for greater soybean yields

• Long-term soybean breeding for yield has increased plant N demands. • Total shoot N and N from BNF increased with cultivar year of release. • Sensitivity of BNF to soil mineral N has not changed over years of soybean breeding. Long-term soybean [ Glycine max (L) Merr.] breeding for yield has increased plant nitrogen (N) demands. On one hand, because N fertilizer application in soybean production systems continues to be insignificant, increased plant N demands over time may have been satisfied from greater biological N fixation (BNF). On the other hand, increased soil residual N over time may have affected the sensitivity of nodulation and BNF. To understand the impact of breeding for greater yield and the effect of soil residual N on nodulation and BNF, two field and a greenhouse study were conducted. Field studies were conducted with maturity group IV soybean cultivars released from 1930 to 2005 and included experiments in four environments. Total shoot N and N from BNF increased with cultivar year of release in two of the four environments. Simulation of different levels of residual soil mineral N by application of 0, 34, 67, and 101 kg N ha −1 shortly after planting resulted in linear increases in shoot N content and δ 15 N, and linear decreases in nodule number and nodule dry matter in the field. Consistent with these results, fertigation of greenhouse-grown soybean cultivars with different levels of NH 4 NO 3 led to a reduction in nodule number, dry matter, and size. Overall, results from these studies indicate that increases in seed yields with cultivar year of release were associated with greater amounts of N derived from BNF as well as greater total shoot N accumulation, but the relative contribution of BNF to total shoot N did not change over time. Analysis also suggest that the sensitivity of nodulation and BNF to soil mineral N has not been altered over the course of soybean breeding.

Infection of Medicago truncatula by the Root-Knot Nematode Meloidogyne javanica Does Not Require Early Nodulation Genes.

Because of the developmental similarities between root nodules induced by symbiotic rhizobia and root galls formed by parasitic nematodes, we investigated the involvement of nodulation genes in the infection of Medicago truncatula by the root knot nematode (RKN), Meloidogyne javanica. We found that gall formation, including giant cell formation, pericycle and cortical cell division, as well as egg laying, occurred successfully in the non-nodulating mutants nfp1 (nod factor perception1), nin1 (nodule inception1) and nsp2 (nodulation signaling pathway2) and the cytokinin perception mutant cre1 (cytokinin receptor1). Gall and egg formation were significantly reduced in the ethylene insensitive, hypernodulating mutant skl (sickle), and to a lesser extent, in the low nodulation, abscisic acid insensitive mutant latd/nip (lateral root-organ defective/numerous infections and polyphenolics). Despite its supernodulation phenotype, the sunn4 (super numeric nodules4) mutant, which has lost the ability to autoregulate nodule numbers, did not form excessive numbers of galls. Co-inoculation of roots with nematodes and rhizobia significantly reduced nodule numbers compared to rhizobia-only inoculated roots, but only in the hypernodulation mutant skl. Thus, this effect is likely to be influenced by ethylene signaling, but is not likely explained by resource competition between galls and nodules. Co-inoculation with rhizobia also reduced gall numbers compared to nematode-only infected roots, but only in the wild type. Therefore, the protective effect of rhizobia on nematode infection does not clearly depend on nodule number or on Nod factor signaling. Our study demonstrates that early nodulation genes that are essential for successful nodule development are not necessary for nematode-induced gall formation, that gall formation is not under autoregulation of nodulation control, and that ethylene signaling plays a positive role in successful RKN parasitism in M. truncatula.

Inhibition of legume nodulation by Pi deficiency is dependent on the autoregulation of nodulation (AON) pathway.

Inhibition of nodule development is one of the main adverse effects of phosphate (Pi) deficiency in legumes. Despite all of the efforts made over the last decades to understand how root nodules cope with Pi deficiency, the molecular mechanisms leading to the reduction in nodule number under Pi deficiency remain elusive. In the present study, we provide experimental evidence indicating that Pi deficiency activates the autoregulation of nodulation (AON) pathway, leading to a reduction in nodule numbers in both common bean and soybean. A transcriptional profile analysis revealed that the expression of the AON-related genes PvNIN, PvRIC1, PvRIC2, and PvTML is upregulated under Pi deficiency conditions. The downregulation of the MYB transcription factor PvPHR1 in common bean roots significantly reduced the expression of these four AON-related genes. Physiological analyses indicated that Pi deficiency does not affect the establishment of the root nodule symbiosis in the supernodulation mutant lines Pvnark and Gmnark. Reciprocal grafting and split-roots analyses determined that the activation of the AON pathway was required for the inhibitory effect of Pi deficiency. Altogether, these data improve our understanding of the genetic mechanisms controlling the establishment of the root nodule symbiosis under Pi deficiency.

The effect of Frankia and multiple ectomycorrhizal fungil species on Alnus growing in low fertility soil

Plant interactions with single symbionts of nitrogen fixing bacteria or mycorrhizal fungi are well studied. However, less is known about how plants interact with multiple symbiont species. The aim of this study was to assess whether multiple symbionts had a synergistic effect on host plant performance compared with a single symbiont and whether increasing the number of non-specific fungal species would increase plant performance due to niche complementarity. Green alder (Alnus viridis ssp. crispa) were inoculated with non-specific ectomycorrhizal fungi (Lactarius torminosus, Lactarius theiogalus, Hebeloma crustuliniforme) alone or in combination, with and without Frankia, and grown in nutrient-poor soil. Frankia significantly increased plant growth by 48.5%, doubled the proportion of plants forming ectomycorrhizae, as well as increased root extracellular phosphatase activity by 8.8% compared to non-Frankia treatments. However, increasing number of fungal species decreased nodulated plant biomass. Plants that formed a Hartig net had reduced nodule number and nodule biomass allocation, as well as total nodule activity compared with plants with no Hartig net. Our results indicated that Frankia inoculation provided benefit for both host plants and ectomycorrhizal fungi in the nutrient-poor soil, while ectomycorrhizal fungi did not. These negative effects of mycorrhizal fungi are likely dependent on soil nutrient P availability.

Evaluation of Immune Responses Induced by Simultaneous Inoculations of Soybean (Glycine max [L.] Merr.) with Soil Bacteria and Rhizobia.

Legumes form root nodules and fix atmospheric nitrogen by establishing symbiosis with rhizobia. However, excessive root nodules are harmful to plants because of the resulting overconsumption of energy from photosynthates. The delay of an inoculation of the soybean super-nodulation mutant NOD1–3 with Bradyrhizobium diazoefficiens USDA110T by 5 d after an inoculation with several soil bacteria confirmed that one bacterial group significantly decreased root nodules throughout the study period. Moreover, no significant changes were observed in nitrogen fixation by root nodules between an inoculation with USDA 110T only and co-inoculation treatments. To clarify the potential involvement of PR proteins in the restriction of nodule formation in the plants tested, the relative expression levels of PR-1, PR-2, PR-5, and PDF1.2 in NOD1–3 roots were measured using real-time PCR. One group of soil bacteria (Gr.3), which markedly reduced nodule numbers, significantly induced the expression of PR-1, PR-5 and PDF1.2 genes by day 5 after the inoculation. By days 7, 10, and 20 after the inoculation, the expression levels of PR-2 and PR-5 were lower than those with the uninoculated treatment. Inoculations with this group of soil bacteria resulted in lower root nodule numbers than with other tested soil bacteria exerting weak inhibitory effects on nodulation, and were accompanied by the induction of plant defense-related genes. Thus, PR genes appear to play important roles in the mechanisms that suppresses nodule formation on soybean roots.