Infection Thread Growth Research Articles

The Regulation of Pea (Pisum sativum L.) Symbiotic Nodule Infection and Defense Responses by Glutathione, Homoglutathione, and Their Ratio.

In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, “locked” infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, “locked” infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.

Structure and Development of the Legume-Rhizobial Symbiotic Interface in Infection Threads.

The intracellular infection thread initiated in a root hair cell is a unique structure associated with Rhizobium-legume symbiosis. It is characterized by inverted tip growth of the plant cell wall, resulting in a tunnel that allows invasion of host cells by bacteria during the formation of the nitrogen-fixing root nodule. Regulation of the plant-microbial interface is essential for infection thread growth. This involves targeted deposition of the cell wall and extracellular matrix and tight control of cell wall remodeling. This review describes the potential role of different actors such as transcription factors, receptors, and enzymes in the rearrangement of the plant-microbial interface and control of polar infection thread growth. It also focuses on the composition of the main polymers of the infection thread wall and matrix and the participation of reactive oxygen species (ROS) in the development of the infection thread. Mutant analysis has helped to gain insight into the development of host defense reactions. The available data raise many new questions about the structure, function, and development of infection threads.

Symbiotic Regulatory Genes Controlling Nodule Development in Pisum sativum L.

Analyses of natural variation and the use of mutagenesis and molecular-biological approaches have revealed 50 symbiotic regulatory genes in pea (Pisum sativum L.). Studies of genomic synteny using model legumes, such as Medicago truncatula Gaertn. and Lotus japonicus (Regel) K. Larsen, have identified the sequences of 15 symbiotic regulatory genes in pea. These genes encode receptor kinases, an ion channel, a calcium/calmodulin-dependent protein kinase, transcription factors, a metal transporter, and an enzyme. This review summarizes and describes mutant alleles, their phenotypic manifestations, and the functions of all identified symbiotic regulatory genes in pea. Some examples of gene interactions are also given. In the review, all mutant alleles in genes with identified sequences are designated and still-unidentified symbiotic regulatory genes of great interest are considered. The identification of these genes will help elucidate additional components involved in infection thread growth, nodule primordium development, bacteroid differentiation and maintenance, and the autoregulation of nodulation. The significance of symbiotic mutants of pea as extremely fruitful genetic models for studying nodule development and for comparative cell biology studies of legume nodules is clearly demonstrated. Finally, it is noted that many more sequences of symbiotic regulatory genes remain to be identified. Transcriptomics approaches and genome-wide sequencing could help address this challenge.

Analysis of epitope distribution of arabinogalactan protein-extensins in pea (Pisum Sativum) nodules of wild-type and mutants impaired in infection thread growth

Background. During the colonization of root and nodule tissues of legumes by rhizobia, bacterial cells are immersed in a plant extracellular matrix which includes arabinogalactan protein-extensins (AGPE).
 Materials and methods. Immunogold electron microscopy with monoclonal antibodies MAC204 and MAC236 was used to analyse the distribution and abundance of epitopes of AGPE in wild-type and symbiotically defective pea mutants.
 Results. In the nodules of the wild-type line SGE, both AGPE epitopes were detected to the same extent in the matrix of infection threads and infection droplets. In the nodules of the mutant line SGEFix-1 (sym40), the level of labelling by MAC204 was significantly higher than with SGE in both infection threads and infection droplets, but the level of labelling by MAC236 was only increased in the infection droplets. In the mutant line SGEFix-2 (sym33-3), a relatively high level of both epitopes was observed among all analysed genotypes. The double mutant line RBT3 (sym33-3, sym40) showed an intermediate level of labelling for both epitopes in infection threads compared with the parental mutants. In SGEFix-1, an abnormal distribution of both epitopes was observed in the intercellular space matrix. The MAC204 epitope was found in the cell walls of SGEFix-1 and in the infection thread walls of SGEFix-2, whereas in RBT3 this epitope was detected in both types of walls.
 Conclusions. The sym33-3 and sym40 mutations have different effects on the accumulation of AGPE epitopes recognised by MAC204 and MAC236. This indicates that both the Sym33 and the Sym40 genes affect the composition of AGPE in the matrix of infection threads and infection droplets.

A protein complex required for polar growth of rhizobial infection threads

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads.

Structural Variations in LysM Domains of LysM-RLK PsK1 May Result in a Different Effect on Pea⁻Rhizobial Symbiosis Development.

Lysin-motif receptor-like kinase PsK1 is involved in symbiosis initiation and the maintenance of infection thread (IT) growth and bacterial release in pea. We verified PsK1 specificity in relation to the Nod factor structure using k1 and rhizobial mutants. Inoculation with nodO and nodE nodO mutants significantly reduced root hair deformations, curling, and the number of ITs in k1-1 and k1-2 mutants. These results indicated that PsK1 function may depend on Nod factor structures. PsK1 with replacement in kinase domain and PsSYM10 co-production in Nicotiana benthamiana leaves did not induce a hypersensitive response (HR) because of the impossibility of signal transduction into the cell. Replacement of P169S in LysM3 domain of PsK1 disturbed the extracellular domain (ECD) interaction with PsSYM10′s ECD in Y2H system and reduced HR during the co-production of full-length PsK1 and PsSYM0 in N. benthamiana. Lastly, we explored the role of PsK1 in symbiosis with arbuscular mycorrhizal (AM) fungi; no significant differences between wild-type plants and k1 mutants were found, suggesting a specific role of PsK1 in legume–rhizobial symbiosis. However, increased sensitivity to a highly aggressive Fusarium culmorum strain was found in k1 mutants compared with the wild type, which requires the further study of the role of PsK1 in immune response regulation.

An Autophagy-Related Kinase Is Essential for the Symbiotic Relationship between Phaseolus vulgaris and Both Rhizobia and Arbuscular Mycorrhizal Fungi.

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.

Legume nodulation

Legume nodulation

Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress

Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50μM AlCl3, for 2 and 24h) Al stress.The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity.These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed.

A novel GRAS protein gene MtSymSCL1 plays a role in regulating nodule number in Medicago truncatula

Legume-rhizobium symbiosis requires the expression of a series of plant genes playing roles in nodulation signaling that leads to the formation of functional nodules. A few plant-specific GRAS proteins have been shown to be involved in early nodulation signaling. Here, we isolated a novel SCARECROW-like GRAS protein gene, MtSymSCL1, from the model legume Medicago truncatula. MtSymSCL1 has unique gene and protein domain structures, and its homologs are identified from various plant species. Phylogenetic analysis showed that MtSymSCL1 could be classified into a novel subclass in the GRAS protein family. MtSymSCL1 transcript accumulated highly in nodulated roots at 14 days after inoculation of Sinorhizobium meliloti. Translationally fused MtSymSCL1-GFP expressed from a viral or the native promoter was co-localized with nuclear localization signal-fused RFP in leaf mesophyll protoplasts, Agrobacterium rhizogenes-transformed hairy root cells, and epidermal cells of nodules that developed on the transformed roots. Unlike SCARECROW of Arabidopsis, however, RNA silencing of MtsymSCL1 did not affect root growth, nor root hair curling and infection thread growth. Nevertheless, MtSymSCL1-silenced roots developed significantly fewer nodules than GUS-silenced roots, as shown by replicated nodulation assay with pot- or agar plate-grown plants. The structure of MtSymSCL1-silenced root nodules appeared similar to that of the wild type, as shown by sectioned nodules stained with green fluorescent dye. Together, these results suggest that MtSymSCL1 plays a role in regulating nodule number during legume-rhizobium symbiosis.

Monoxenic nodulation process of Acacia mangium (Mimosoideae, Phyllodineae) by Bradyrhizobium sp

Acacia mangium Willd., a native tree in Australia and Papua New Guinea, has been introduced to countries in Asia and South America where plantations have been established that cover several hundred thousand ha. The present study investigated the early stages of the nodulation process in A. mangium using an homologous Australian Bradyrhizobium strain. After optimizing the axenic nodulation, histological and cytological studies were conducted using light and electron microscopy. These documented the proliferation of Bradyrhizobium, the lysis of mucilage at the root surface, root hair deformation and initiation, as well as the development and growth of multiple infection threads. A belt of tannin-filled cells was shown to surround the central nodular fixation zone. The nodules were of the indeterminate type and the bacteroids had a rod shape, without size modification and with few polyhydroxybutyrate (PHB) granules. Several bacteroids can share the same symbiosome. A. mangium exhibits both classical and novel features in its nodulation.

The Small GTPase ROP6 Interacts with NFR5 and Is Involved in Nodule Formation inLotus japonicus

Nod Factor Receptor5 (NFR5) is an atypical receptor-like kinase, having no activation loop in the protein kinase domain. It forms a heterodimer with NFR1 and is required for the early plant responses to Rhizobium infection. A Rho-like small GTPase from Lotus japonicus was identified as an NFR5-interacting protein. The amino acid sequence of this Rho-like GTPase is closest to the Arabidopsis (Arabidopsis thaliana) ROP6 and Medicago truncatula ROP6 and was designated as LjROP6. The interaction between Rop6 and NFR5 occurred both in vitro and in planta. No interaction between Rop6 and NFR1 was observed. Green fluorescent protein-tagged ROP6 was localized at the plasma membrane and cytoplasm. The interaction between ROP6 and NFR5 appeared to take place at the plasma membrane. The expression of the ROP6 gene could be detected in vascular tissues of Lotus roots. After inoculation with Mesorhizobium loti, elevated levels of ROP6 expression were found in the root hairs, root tips, vascular bundles of roots, nodule primordia, and young nodules. In transgenic hairy roots expressing ROP6 RNA interference constructs, Rhizobium entry into the root hairs did not appear to be affected, but infection thread growth through the root cortex were severely inhibited, resulting in the development of fewer nodules per plant. These data demonstrate a role of ROP6 as a positive regulator of infection thread formation and nodulation in L. japonicus.

IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago Spp.

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.

Localizatlon of expansin-like protein in apoplast of pea (Pisum sativum L.) root nodules during interaction with Rhizobium leguminosarum bv. viciae

During nodule development on pea roots, apoplast undergoes changes in activity of plant cell wall proteins such as expansins (EXPs). Because the accumulation of EXP protein has been correlated with the growth of various plant organs, we investigated using Western Blot and immunolocalization studies with antibody against PsEXP1, whether this protein was accumulated in the expanding cells of nodule. Immunoblot results indicated the presence of a 30-kDa band specific for pea root nodules. The EXP proteins content rose during growth of pea root nodules. Expansin(s) protein was localized in nodule apoplast as well as in the infection thread walls. The enhanced amount of expansin-like proteins in meristematic part of nodule, root and shoot was shown. The localization of this protein in the meristematic cell walls can be related to the loosening of plant cell wall before cell enlargement. Both, plant cell enlargement and infection thread growth require activity of expansin(s). Possible involvement of EXPs in the process of pea root nodule development is also discussed.

Conservation in Function of a SCAR/WAVE Component During Infection Thread and Root Hair Growth in Medicago truncatula

Nitrogen-fixing symbioses of plants are often associated with bacterially infected nodules where nitrogen fixation occurs. The plant host facilitates bacterial infection with the formation of infection threads, unique structures associated with these symbioses, which are invaginations of the host cell with the capability of traversing cellular junctions. Here, we show that the infection thread shares mechanistic similarities to polar-growing cells, because the required for infection thread (RIT) locus of Medicago truncatula has roles in root-hair, trichome, and infection-thread growth. We show that RIT encodes the M. truncatula ortholog of NAP1, a component of the SCAR/WAVE (suppressor of cAMP receptor/WASP-family verprolin homologous protein) complex that regulates actin polymerization, through the activation of ARP2/3. NAP1 of Arabidopsis thaliana functions equivalently to the M. truncatula gene, indicating that the mode of action of NAP1 is functionally conserved across species and that legumes have not evolved a unique functionality for NAP1 during rhizobial colonization. This work highlights the surprising commonality between polar-growing cells and a polar-growing cellular intrusion and reveals important insights into the formation and maintenance of infection-thread development.

Borate promotes the formation of a complex between legume AGP-extensin and Rhamnogalacturonan II and enhances production of Rhizobium capsular polysaccharide during infection thread development in Pisum sativum symbiotic root nodules.

The capacity to bind to biomolecules is considered to be the basis for any physiological role of boron (B). Legume arabinogalactan protein-extensin (AGPE), a major component of the infection thread matrix of legume nodules is a potential B-ligand. Therefore, its role in infection threads development was investigated in Pisum sativum grown under B deficiency. Using the AGPE-specific antibody MAC265, immunochemical analysis revealed that a 175 kDa MAC265 antigen was abundant in +B but much weaker in -B nodule extracts. A B-dependent complex involving AGPE and rhamnogalacturonan II (RGII) could be co-purified using anti-RGII antiserum. Following fractionation of -B nodules, MAC265 antigens were mostly associated with the bacterial pellet. Immunogold staining confirmed that AGPE was closely associated with the surface of rhizobia in the lumen of threads in -B nodules whereas in +B nodules, AGPE was separated from the bacterial surface by a sheath of capsular polysaccharide. Interestingly, colonies of rhizobia grown in free-living culture without B developed low capsule production. Therefore, we propose that B could be important for apical growth of infection threads by strengthening thread wall through a B-dependent AGPE-RGII interaction and by promoting bacterial advance through a B-dependent production of a stable rhizobial capsule that prevents AGPE attachment.

Plasmodesmata distribution and sugar partitioning in nitrogen-fixing root nodules of Datisca glomerata

To understand carbon partitioning in roots and nodules of Datisca glomerata, activities of sucrose-degrading enzymes and sugar transporter expression patterns were analyzed in both organs, and plasmodesmal connections between nodule cortical cells were examined by transmission electron microscopy. The results indicate that in nodules, the contribution of symplastic transport processes is increased in comparison to roots, specifically in infected cells which develop many secondary plasmodesmata. Invertase activities are dramatically reduced in nodules as compared to roots, indicating that here the main enzyme responsible for the cleavage of sucrose is sucrose synthase. A high-affinity, low-specificity monosaccharide transporter whose expression is induced in infected cells prior to the onset of bacterial nitrogen fixation, and which has an unusually low pH optimum and may be involved in turgor control or hexose retrieval during infection thread growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-010-1285-8) contains supplementary material, which is available to authorized users.

LIN, a Novel Type of U-Box/WD40 Protein, Controls Early Infection by Rhizobia in Legumes

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

Distribution of legume arabinogalactan protein-extensin (AGPE) glycoproteins in symbiotically defective pea mutants with abnormal infection threads

The interface between the host cell and the microsymbiont is an important zone for development and differentiation during successive stages of the rhizobium-legume symbiosis. Legume root nodule extensins, otherwise known as arabinogalactanprotein-extensin (AGPE) are abundant components of the infection thread matrix. We have characterized the origin and distribution of these glycoproteins at the symbiotic interface of root nodules of symbiotically defective mutants of pea (Pisum sativum L.) using immunogold localization with MAC265 an anti-AGPE monoclonal antibody. For mutants with defective growth of infection threads, the AGPE epitope was abundant in the extracellular matrix surrounding infected host cells in the central infected tissue of the nodule as well as being present in the lumen of Rhizobium-induced infection threads. This suggests a mis-targetting of AGPE as a consequence of abnormal growth of the infection threads. Furthermore, mutants in gene sym33 showed reduced labelling with MAC265, and in infection threads and droplets label was completely absent, a phenomenon not observed in wild-type nodules. This suggests an alteration in the composition of the infection thread matrix for sym33 mutants which may be correlated with the absence of endocytosis of rhizobia into the host cytoplasm.

Medicago LYK3, an Entry Receptor in Rhizobial Nodulation Factor Signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.