Sucrose-induced auxin conjugate hydrolase restores symbiosis in a Medicago cytokinin perception mutant.

Rhizobia-legume interactions recruit cytokinin for the induction of nodule primordia in the cortex. Cytokinin signaling regulates auxin transport and biosynthesis, causing local auxin accumulation, which triggers cortical cell division. Since sugar signaling can trigger auxin responses, we explored whether sugar treatments could rescue symbiosis in the Medicago truncatula cytokinin response 1 (cre1) mutant. Herein, we demonstrate that sucrose and its nonmetabolizable isomer turanose can trigger auxin response and recover functional symbiosis in cre1, indicating sucrose signaling to be necessary for the restoration of symbiosis. In both M. truncatula A17 (wild type) and cre1, sucrose signaling significantly upregulated IAA-Ala Resistant 3 (IAR33), encoding an auxin conjugate hydrolase, in rhizobia-infected as well as in uninfected roots. Knockdown of IAR33 (IAR33-KD) significantly reduced nodulation in A17, highlighting the importance of deconjugation-mediated auxin accumulation during nodule inception. In cre1, IAR33-KD restricted the sucrose-mediated restoration of functional symbiosis, suggesting that deconjugation-mediated auxin accumulation plays a key role in the absence of CRE1-mediated auxin biosynthesis and transport control. Overexpression of IAR33 also restored functional symbiosis in cre1, further suggesting that IAR33 mediates auxin accumulation in response to sucrose signaling. Since all the observed sucrose-mediated responses were common to A17 and cre1, deconjugation-mediated auxin response appeared to be independent of CRE1, which normally governs local auxin accumulation in the presence of rhizobia. We propose that sucrose-dependent restoration of symbiosis in cre1 occurs by the activation of IAR33-mediated auxin deconjugation.

Nod factor-independent 'crack-entry' symbiosis in dalbergoid legume Arachis hypogaea.

Dalbergoids are typified by crack-entry symbiosis which is evidenced to be Nod Factor (NF)-independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF-producing, prompting us to check whether symbiosis in this legume could also be NF-independent. For this, we followed the symbiosis with two NF-containing bradyrhizobial strains - SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF-deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis-Oryza intercropped field. While SEMIA6144ΔnodC was non-nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF-deficient strains - BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF-independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF-dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF-dependent legume, Arachis does retain a vestigial, less-efficient form of NF-independent symbiosis.

The Natural Antisense Transcript DONE40 Derived from the lncRNA ENOD40 Locus Interacts with SET Domain Protein ASHR3 During Inception of Symbiosis in Arachis hypogaea.

The long noncoding RNA ENOD40 is required for cortical cell division during root nodule symbiosis (RNS) of legumes, though it is not essential for actinorhizal RNS. Our objective was to understand whether ENOD40 was required for aeschynomenoid nodule formation in Arachis hypogaea. AhENOD40 express from chromosome 5 (chr5) (AhENOD40-1) and chr15 (AhENOD40-2) during symbiosis, and RNA interference of these transcripts drastically affected nodulation, indicating the importance of ENOD40 in A. hypogaea. Furthermore, we demonstrated several distinct characteristics of ENOD40. (i) Natural antisense transcript (NAT) of ENOD40 was detected from the AhENOD40-1 locus (designated as NAT-AhDONE40). (ii) Both AhENOD40-1 and AhENOD40-2 had two exons, whereas NAT-AhDONE40 was monoexonic. Reverse-transcription quantitative PCR analysis indicated both sense and antisense transcripts to be present in both cytoplasm and nucleus, and their expression increased with the progress of symbiosis. (iii) RNA pull-down from whole cell extracts of infected roots at 4 days postinfection indicated NAT-AhDONE40 to interact with the SET (Su(var)3-9, enhancer of Zeste and Trithorax) domain containing absent small homeotic disc (ASH) family protein AhASHR3 and this interaction was further validated using RNA immunoprecipitation and electrophoretic mobility shift assay. (iv) Chromatin immunoprecipitation assays indicate deposition of ASHR3-specific histone marks H3K36me3 and H3K4me3 in both of the ENOD40 loci during the progress of symbiosis. ASHR3 is known for its role in optimizing cell proliferation and reprogramming. Because both ASHR3 and ENOD40 from legumes cluster away from those in actinorhizal plants and other nonlegumes in phylogenetic distance trees, we hypothesize that the interaction of DONE40 with ASHR3 could have evolved for adapting the nodule organogenesis program for legumes.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Involvement of Arachis hypogaea Jasmonate ZIM domain/TIFY proteins in root nodule symbiosis.

Jasmonate ZIM domain (JAZ) proteins are the key negative regulators of jasmonate signaling, an important integrator of plant-microbe relationships. Versatility of jasmonate signaling outcomes are maintained through the multiplicity of JAZ proteins and their definitive functionalities. How jasmonate signaling influences the legume-Rhizobium symbiotic relationship is still unclear. In Arachis hypogaea (peanut), a legume plant, one JAZ sub-family (JAZ1) gene and one TIFY sequence containing protein family member (TIFY8) gene show enhanced expression in the early stage and late stage of root nodule symbiosis (RNS) respectively. In plants, JAZ sub-family proteins belong to a larger TIFY family. Here, this study denotes the first attempt to reveal in planta interactions of downstream jasmonate signaling regulators through proteomics and mass spectrometry to find out the mode of jasmonate signaling participation in the RNS process of A. hypogaea. From 4-day old Bradyrhizobium-infected peanut roots, the JAZ1-protein complex shows its contribution towards the rhizobial entry, nodule development, autoregulation of nodulation and photo-morphogenesis during the early stage of symbiosis. From 30-day old Bradyrhizobium infected roots, the TIFY8-protein complex reveals repressor functionality of TIFY8, suppression of root jasmonate signaling, modulation of root circadian rhythm and nodule development. Cellular localization and expression level of the interaction partners during the nodulation process further substantiate the in planta interaction pairs. This study provides a comprehensive insight into the jasmonate functionality in RNS through modulation of nodule number and development, during the early stage and root circadian rhythm during the late stage of nodulation, through the protein complexes of JAZ1 and TIFY8 respectively in A. hypogaea.

Gatekeeper-Activation Loop Cross-Talk Determines Distinct Autoactivation States of Symbiosis Receptor Kinase.

Plant receptor-like kinases (RLKs) have a Tyr in the "gatekeeper" position adjacent to the hinge region. The gatekeeper is phosphorylated in several RLKs, including symbiosis receptor kinase (SYMRK), but the significance of this remains unknown. Gatekeeper substitution did not inactivate Arachis hypogaea SYMRK but affected autophosphorylation at selected sites. Herein, we show that nonphosphorylatable gatekeepers (Y670F and Y670A) restrict SYMRK to be a Ser/Thr kinase with a basal level of phosphorylation (∼5 P/polypeptide, termed state I) whereas phosphorylatable gatekeepers (Y670 and Y670T) allowed SYMRK to be dual specific (Ser/Thr/Tyr) with a maximal level of phosphorylation (∼10 P/polypeptide, termed state II). State II SYMRKs were phosphorylated on gatekeeper residues, and the phosphocode in their activation segment was distinct from state I. The kcat/ Km for substrate phosphorylation was ∼10-fold higher for state II, though for autophosphorylation, it was comparable with those of state I SYMRKs. To identify other determinants of state I features, we mutagenized all nine sites where phosphorylation was affected by nonphosphorylatable gatekeepers (Y670F and Y670A). Only two such mutants, S754A and S757A, located on the activation loop failed to phosphorylate gatekeeper Tyr and restricted SYMRK in state I. Double mutants like Y670F/S754A retained the features of state I, but Y670F/S757A was significantly inactivated, indicating a nonphosphorylatable gatekeeper can bypass phosphorylation of S754 but not S757 in the activation segment. We propose a working model for the hierarchical phosphorylation of SYMRK on gatekeeper and activation segments for its pS757-mediated activation as a Ser/Thr kinase in selfie mode (autophosphorylation) to a pS754/pY670-mediated activation as a Ser/Thr/Tyr kinase that functions in dual mode (both autophosphorylation and substrate phosphorylation).

Transcriptomic Analysis With the Progress of Symbiosis in 'Crack-Entry' Legume Arachis hypogaea Highlights Its Contrast With 'Infection Thread' Adapted Legumes.

In root-nodule symbiosis, rhizobial invasion and nodule organogenesis is host controlled. In most legumes, rhizobia enter through infection threads and nodule primordium in the cortex is induced from a distance. But in dalbergoid legumes like Arachis hypogaea, rhizobia directly invade cortical cells through epidermal cracks to generate the primordia. Herein, we report the transcriptional dynamics with the progress of symbiosis in A. hypogaea at 1 day postinfection (dpi) (invasion), 4 dpi (nodule primordia), 8 dpi (spread of infection in nodule-like structure), 12 dpi (immature nodules containing rod-shaped rhizobia), and 21 dpi (mature nodules with spherical symbiosomes). Expression of putative ortholog of symbiotic genes in 'crack entry' legume A. hypogaea was compared with infection thread-adapted model legumes. The contrasting features were i) higher expression of receptors like LYR3 and EPR3 as compared with canonical Nod factor receptors, ii) late induction of transcription factors like NIN and NSP2 and constitutive high expression of ERF1, EIN2, bHLH476, and iii) induction of divergent pathogenesis-responsive PR-1 genes. Additionally, symbiotic orthologs of SymCRK, ROP6, RR9, SEN1, and DNF2 were not detectable and microsynteny analysis indicated the absence of a RPG homolog in diploid parental genomes of A. hypogaea. The implications are discussed and a molecular framework that guides crack-entry symbiosis in A. hypogaea is proposed.

p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

Silencing of Putative Cytokinin Receptor Histidine Kinase1 Inhibits Both Inception and Differentiation of Root Nodules in Arachis hypogaea.

Rhizobia-legume interaction activates the SYM pathway that recruits cytokinin signaling for induction of nodule primordia in the cortex. In Arachis hypogaea, bradyrhizobia invade through natural cracks developed in the lateral root base and are directly endocytosed in the cortical cells to generate the nodule primordia. To unravel the role of cytokinin signaling in A. hypogaea, RNA-interference (RNAi) of cytokinin receptor histidine-kinase1 (AhHK1) was done. AhHK1-RNAi downregulated the expression of type-A response regulators such as AhRR5 and AhRR3 along with several symbiotic genes, indicating that both cytokinin signaling and the SYM pathway were affected. Accordingly, there was a drastic downregulation of nodulation in AhHK1-RNAi roots and the nodules that developed were ineffective. These nodules were densely packed, with infected cells having a higher nucleo-cytoplasmic ratio and distinctively high mitotic index, where the rod-shaped rhizobia failed to differentiate into bacteroids within spherical symbiosomes. In accordance with the proliferating state, expression of a mitotic-cyclin AhCycB2.1 was higher in AhHK1-RNAi nodules, whereas expression of a retinoblastoma-related (AhRBR) nodule that restrains proliferation was lower. Also, higher expression of the meristem maintenance factor WUSCHEL-RELATED HOMEOBOX5 correlated with the undifferentiated state of AhHK1-RNAi nodules. Our results suggest that AhHK1-mediated cytokinin signaling is important for both inception and differentiation during nodule development in A. hypogaea.

A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses.

BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. RESULTS: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. CONCLUSIONS: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses.

Gatekeeper Tyrosine Phosphorylation of SYMRK Is Essential for Synchronizing the Epidermal and Cortical Responses in Root Nodule Symbiosis.

Symbiosis receptor kinase (SYMRK) is indispensable for activation of root nodule symbiosis (RNS) at both epidermal and cortical levels and is functionally conserved in legumes. Previously, we reported SYMRK to be phosphorylated on "gatekeeper" Tyr both in vitro as well as in planta. Since gatekeeper phosphorylation was not necessary for activity, the significance remained elusive. Herein, we show that substituting gatekeeper with nonphosphorylatable residues like Phe or Ala significantly affected autophosphorylation on selected targets on activation segment/αEF and ß3-αC loop of SYMRK. In addition, the same gatekeeper mutants failed to restore proper symbiotic features in a symrk null mutant where rhizobial invasion of the epidermis and nodule organogenesis was unaffected but rhizobia remain restricted to the epidermis in infection threads migrating parallel to the longitudinal axis of the root, resulting in extensive infection patches at the nodule apex. Thus, gatekeeper phosphorylation is critical for synchronizing epidermal/cortical responses in RNS.

Segregation of nod-containing and nod-deficient bradyrhizobia as endosymbionts of Arachis hypogaea and as endophytes of Oryza sativa in intercropped fields of Bengal Basin, India.

Bradyrhizobial invasion in dalbergoid legumes like Arachis hypogaea and endophytic bacterial invasions in non-legumes like Oryza sativa occur through epidermal cracks. Here, we show that there is no overlap between the bradyrhizobial consortia that endosymbiotically and endophytically colonise these plants. To minimise contrast due to phylogeographic isolation, strains were collected from Arachis/Oryza intercropped fields and a total of 17 bradyrhizobia from Arachis (WBAH) and 13 from Oryza (WBOS) were investigated. 16SrRNA and concatenated dnaK-glnII-recA phylogeny clustered the nodABC-positive WBAH and nodABC-deficient WBOS strains in two distinct clades. The in-field segregation is reproducible under controlled conditions which limits the factors that influence their competitive exclusion. While WBAH renodulated Arachis successfully, WBOS nodulated in an inefficient manner. Thus, Arachis, like other Aeschynomene legumes support nod-independent symbiosis that was ineffectual in natural fields. In Oryza, WBOS recolonised endophytically and promoted its growth. WBAH however caused severe chlorosis that was completely overcome when coinfected with WBOS. This explains the exclusive recovery of WBOS in Oryza in natural fields and suggests Nod-factors to have a role in counterselection of WBAH. Finally, canonical soxY1 and thiosulphate oxidation could only be detected in WBOS indicating loss of metabolic traits in WBAH with adaptation of symbiotic lifestyle.

Intracellular catalytic domain of symbiosis receptor kinase hyperactivates spontaneous nodulation in absence of rhizobia.

Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.

Trans-thylakoid ∆pH dependent oscillation of F(PSI)/F(PSII) under continuous irradiance in isolated thylakoids.

Energy distribution between photosystems (PSI & PSII) under prolonged and continuous white light irradiance was assessed by monitoring the progress of their fluorescence emission (FPSI/FPSII) at 77 K. Our observations indicate FPSI/FPSII to oscillate with the progress of irradiance treatments at all intensities tested (100, 200, 500, and 800 µE m(-2) S(-1)). The amplitude of the oscillation increased with the progress, whereas the periodicity of the oscillation increased with the intensity of the incident irradiance. Spectral analysis indicated fluctuation of FPSI to be the major determinant of the observed oscillation. The first rise and fall of FPSI/FPSII overlapped with phosphorylation and dephosphorylation of LHCII, but oscillation of FPSI/FPSII continued for several cycles without any further phosphorylation of LHCII. Moreover, in presence of DCMU where linear electron flow (LEF) is suppressed and LHCII phosphorylation is completely abolished, the oscillation of FPSI/FPSII was not abolished. These data indicated that LHCII phosphorylation was not essential for the observed oscillation of energy distribution between the photosystems. In contrast, in the presence of inhibitors of cyclic electron flow (CEF) like Antimycin A (AA) and rotenone, the oscillation of FPSI/FPSII was either abolished or severely dampened. Additionally, the oscillation was also abolished in presence of uncouplers like NH4Cl and nigericin that cancels the trans-thylakoid ∆pH. Thus, trans-thylakoid ∆pH, generated through CEF, appear to be an important determinant of oscillation of FPSI/FPSII in isolated thylakoids. The phenomenon of oscillation could be associated with a CEF mediated chromatic adaptation of PSI in presence of excess irradiance.

Static magnetic field (SMF) sensing of the P(723)/P(689) photosynthetic complex.

Moderate intensity SMF have been shown to act as a controller of the protic potential in the coherent milieu of the thylakoid membranes. SMF of the order of 60-500 mT induces memory-like effect in photosystem I (PSI, P723) emission with a correlated oscillation of photosystem II (PSII, P689) fluorescence emission at a temperature of 77 K. The observed magnetic perturbation that affects the thylakoid photon capture circuitry was also found to be associated with the bio-energetic machinery of the thylakoid membranes. At normal pH, SMF causes an enhancement of PSI fluorescence emission intensity (P723/P689 > 1), followed by a slow relaxation on the removal of SMF. The enhancement of the PSI fluorescence intensity also occurs under no-field condition, if either the pH of the medium is lowered, or protonophores, such as carbonyl cyanide chlorophenylhydrazine or nigericin are added (P723/P689≥ 2). If SMF was applied under such a low pH condition or in the presence of protonophore, a reverse effect, particularly, a reduction of the enhanced PSI emission was observed. Because SMF is essentially equivalent to a spin perturbation, the observed effects can be explained in terms of spin re-organization, illustrating a memory effect via membrane re-alignment and assembly. The mimicry of conventional uncouplers by SMF is also notable; the essential difference being the reversibility and manoeuvrability of the latter (SMF). Finally, the effect implies numerous possibilities of externally regulating the photon capture and proton circulation in the thylakoid membranes using controlled SMF.

PRICE (PRotein Interface Conservation and Energetics): a server for the analysis of protein-protein interfaces.

Residues in a protein-protein interface that are important for forming and stabilizing the interaction can usually be identified by looking at patterns of evolutionary conservation in groups of homologous proteins and also by the computational identification of binding hotspots. The PRICE (PRotein Interface Conservation and Energetics) server takes the coordinates of a protein-protein complex, dissects the interface into core and rim regions, and calculates (1) the degree of conservation (measured as the sequence entropy), as well as (2) the change in free energy of binding (∆∆G, due to alanine scanning mutagenesis) of interface residues. Results are displayed as color-coded plots and also made available for download. This enables the computational identification of binding hot spots, based on which further experiments can be designed. The method will aid in protein functional prediction by correct assignment of hot regions involved in binding. Consideration of sequence entropies for residues with large ∆∆G values may provide an indication of the biological relevance of the interface. Finally, the results obtained on a test set of alanine mutants has been compared to those obtained using other servers/methods. The PRICE server is a web application available at http://www.boseinst.ernet.in/resources/bioinfo/stag.html.

Molecular Basis of Root Nodule Symbiosis between Bradyrhizobium and 'Crack-Entry' Legume Groundnut (Arachis hypogaea L.).

Nitrogen is one of the essential plant nutrients and a major factor limiting crop productivity. To meet the requirements of sustainable agriculture, there is a need to maximize biological nitrogen fixation in different crop species. Legumes are able to establish root nodule symbiosis (RNS) with nitrogen-fixing soil bacteria which are collectively called rhizobia. This mutualistic association is highly specific, and each rhizobia species/strain interacts with only a specific group of legumes, and vice versa. Nodulation involves multiple phases of interactions ranging from initial bacterial attachment and infection establishment to late nodule development, characterized by a complex molecular signalling between plants and rhizobia. Characteristically, legumes like groundnut display a bacterial invasion strategy popularly known as "crack-entry'' mechanism, which is reported approximately in 25% of all legumes. This article accommodates critical discussions on the bacterial infection mode, dynamics of nodulation, components of symbiotic signalling pathway, and also the effects of abiotic stresses and phytohormone homeostasis related to the root nodule symbiosis of groundnut and Bradyrhizobium. These parameters can help to understand how groundnut RNS is programmed to recognize and establish symbiotic relationships with rhizobia, adjusting gene expression in response to various regulations. This review further attempts to emphasize the current understanding of advancements regarding RNS research in the groundnut and speculates on prospective improvement possibilities in addition to ways for expanding it to other crops towards achieving sustainable agriculture and overcoming environmental challenges.

RNA interference highlights the role of CCaMK in dissemination of endosymbionts in the Aeschynomeneae legume Arachis.

In legume-rhizobia symbiosis, Ca2+/calmodulin-dependent protein kinase (CCaMK) is essential for rhizobial invasion through infection threads in the epidermis and nodule organogenesis in the cortex. Though CCaMK is actively transcribed in the infected zone of nodules, its role in the later stages of nodule development remain elusive because of the epidermal arrest of "loss-of-function" mutants. In Aeschynomeneae legumes such as Arachis hypogea, rhizobia directly access the cortex, where nodule organogenesis as well as endosymbiont dissemination take place by multiplication of infected cortical cells. We characterized CCaMK (GI:195542474) from A. hypogea and downregulated the kinase through RNA interference (RNAi) to understand its role during organogenesis of its characteristic aeschynomenoid nodules. In CCaMK downregulated plants, the inception of nodules was delayed by approximately 4 weeks and nodulation capacity was decreased (>90%). The infected zones of the RNA interference nodules were scattered with uninfected or binucleated cells as opposed to the homogeneous infection zone in empty-vector-transformed nodules. Symbiosomes in RNAi nodules were pleomorphic with diverse geometrical shapes or arrested during division in the final stages of their fission as opposed to uniform-sized, spherical symbiosomes in empty-vector-transformed nodules. Together, our results reveal CCaMK to be essential for development of functional aeschynomenoid nodules, with a critical role in rhizobial dissemination during nodule organogenesis.

Transformed hairy roots of Arachis hypogea: a tool for studying root nodule symbiosis in a non-infection thread legume of the Aeschynomeneae tribe.

Arachis hypogea is a non-"infection thread" (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal "cracks" and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoter-driven green fluorescent protein and beta-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosette-type root hairs and had normal level of expression of symbiosis-related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wild-type nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interactions.

Functional conservation of CYCLOPS in crack entry legume Arachis hypogaea.

Root nodule symbiosis in legumes is established following interaction of compatible rhizobia that activates an array of genes, commonly known as symbiotic-pathway, resulting in nodule development. In model legumes, bacterial entry mainly occurs through infection thread involving the expression of transcription factor CYCLOPS/IPD3. Here we show the functional analysis of AhCYCLOPS in Arachis hypogaea where bacteria invade roots through epidermal cracks. Exploiting significant cross-species domain conservation, trans-complementation experiments involving ectopic expression of AhCYCLOPS in transgenic hairy-roots of Medicago truncatula ipd3 mutants resulted in functional complementation of Medicago nodules. Moreover, native promoter of AhCYCLOPS was sufficient for this cross-species complementation irrespective of the different modes of infection of roots by rhizobia and nodule ontology. To unravel the role of AhCYCLOPS during 'crack-entry' nodulation in A. hypogaea, RNAi of AhCYCLOPS was performed which resulted in delayed nodule inception followed by drastic reduction in nodule number on transgenic hairy-roots. The infection zone of a significant number of RNAi nodules showed presence of infected cells with enlarged nucleus and rod shaped undifferentiated bacteria. Expression analysis showed downregulation of several nodulation responsible effectors endorsing the compromised condition of RNAi roots. Together, the results indicated that AhCYCLOPS plays an important role in A. hypogaea nodule development.