Root Hair Infection Research Articles

A plant chitinase controls cortical infection thread progression and nitrogen-fixing symbiosis.

Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.

Jasmonic Acid-Mediated Aliphatic Glucosinolate Metabolism Is Involved in Clubroot Disease Development in Brassica napus L.

Glucosinolate (GSL) is associated with clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae. Due to the complicated composition of GSLs, their exact role in clubroot disease development remains unclear. By investigating clubroot disease resistance in cruciferous plants and characterizing the GSL content in seeds, we can determine if clubroot disease development is related to the components of GSLs. The difference in the infection process between Matthiola incana L. (resistant) and Brassica napus L. (susceptible) was determined. Root hair infection was definitely observed in both resistant and susceptible hosts, but no infection was observed during the cortical infection stage in resistant roots; this finding was verified by molecular detection of P. brassicae via PCR amplification at various times after inoculation. Based on the time course detection of the contents and compositions of GSLs after P. brassicae inoculation, susceptible roots exhibited increased accumulation of aliphatic, indolic, and aromatic GSLs in B. napus, but only aromatic GSLs were significantly increased in M. incana. Gluconapin, which was the main aliphatic GSL in B. napus and present only in B. napus, was significantly increased during the secondary infection stage. Quantification of the internal jasmonic acid (JA) concentration showed that both resistant and susceptible plants exhibited an enhanced level of JA, particularly in susceptible roots. The exogenous JA treatment induced aliphatic GSLs in B. napus and aromatic GSLs in M. incana. JA-induced aromatic GSLs may be involved in the defense against P. brassicae, whereas aliphatic GSLs induced by JA in B. napus likely play a role during the secondary infection stage. Three candidate MYB28 genes regulate the content of aliphatic GSLs identified in B. napus; one such gene was BnMYB28.1, which was significantly increased following both the treatment with exogenous JA and P. brassicae inoculation. In summary, the increased content of JA during the secondary infection stage may induce the expression of BnMYB28.1, which caused the accumulation of aliphatic GSLs in clubroot disease development.

Epidermal auxin biosynthesis facilitates rhizobial infection in Lotus japonicus.

Symbiotic nitrogen fixation in legumes requires nodule organogenesis to be coordinated with infection by rhizobia. The plant hormone auxin influences symbiotic infection, but the precise timing of auxin accumulation and the genetic network governing it remain unclear. We used a Lotus japonicus optimised variant of the DII-based auxin accumulation sensor and identified a rapid accumulation of auxin in the epidermis, specifically in the root hair cells. This auxin accumulation occurs in the infected root hairs during rhizobia invasion, while Nod factor application induces this response across a broader range of root hairs. Using the DR5 auxin responsive promoter, we demonstrate that activation of auxin signalling also occurs specifically in infected root hairs. Analysis of root hair transcriptome data identified induction of an auxin biosynthesis gene of the Tryptophan Amino-transferase Related (LjTar1) family following both bacteria inoculation and Nod factor treatment. Genetic analysis showed that both expression of the LjTar1 biosynthesis gene and the auxin response requires Nod factor perception, while common symbiotic pathway transcription factors are only partially required or act redundantly to initiate auxin accumulation. Using a chemical genetics approach, we confirmed that auxin biosynthesis has a functional role in promoting symbiotic infection events in the epidermis.

Role of the Nod Factor Hydrolase MtNFH1 in Regulating Nod Factor Levels during Rhizobial Infection and in Mature Nodules of Medicago truncatula.

Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.

In BPS1 Downregulated Roots, the BYPASS1 Signal Disrupts the Induction of Cortical Cell Divisions in Bean-Rhizobium Symbiosis.

BYPASS1 (BPS1), which is a well-conserved gene in plants, is required for normal root and shoot development. In the absence of BPS1 gene function, Arabidopsis overproduces a mobile signalling compound (the BPS1 signal) in roots, and this transmissible signal arrests shoot growth and causes abnormal root development. In addition to the shoot and root meristem activities, the legumes also possess transient meristematic activity in root cortical cells during Rhizobium symbiosis. We explored the role of Phaseolus vulgaris BPS1 during nodule primordium development using an RNA-interference (RNAi) silencing approach. Our results show that upon Rhizobium infection, the PvBPS1-RNAi transgenic roots failed to induce cortical cell divisions without affecting the rhizobia-induced root hair curling and infection thread formation. The transcript accumulation of early nodulin genes, cell cyclins, and cyclin-dependent kinase genes was affected in RNAi lines. Interestingly, the PvBPS1-RNAi root nodule phenotype was partially rescued by exogenous application of fluridone, a carotenoid biosynthesis inhibitor, which was used because the carotenoids are precursors of BPS1 signalling molecules. Furthermore, we show that the PvBPS1 promoter was active in the nodule primordia. Together, our data show that PvBPS1 plays a vital role in the induction of meristematic activity in root cortical cells and in the establishment of nodule primordia during Phaseolus-Rhizobium symbiosis.

Regulation of Nod factor biosynthesis by alternative NodD proteins at distinct stages of symbiosis provides additional compatibility scrutiny.

The Lotus japonicus symbiont Mesorhizobium loti R7A encodes two copies of nodD and here we identify striking differences in Nod factor biosynthesis gene induction by NodD1 and NodD2 both in vitro and in planta. We demonstrate that induction of Nod factor biosynthesis genes is preferentially controlled by NodD1 and NodD2 at specific stages of symbiotic infection. NodD2 is primarily responsible for induction in the rhizosphere and within nodules, while NodD1 is primarily responsible for induction within root hair infection threads. nodD1 and nodD2 mutants showed significant symbiotic phenotypes and competition studies establish that nodD1 and nodD2 mutants were severely outcompeted by wild-type R7A, indicating that both proteins are required for proficient symbiotic infection. These results suggest preferential activation of NodD1 and NodD2 by different inducing compounds produced at defined stages of symbiotic infection. We identified Lotus chalcone isomerase CHI4 as a root hair induced candidate involved in the biosynthesis of an inducer compound that may be preferentially recognized by NodD1 within root hair infection threads. We propose an alternative explanation for the function of multiple copies of nodD that provides the host plant with another level of compatibility scrutiny at the stage of infection thread development.

Effects of rate and application method on the efficacy of metam sodium to reduce clubroot (Plasmodiophora brassicae) of canola

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted to evaluate the effect of rate of metam sodium fumigant (dithiocarbamate; sodium N-methyldithiocarbamate; trade name Vapam) and application methods including watering, soil surface covering, and soil incorporation on clubroot of canola. At higher rates (0.4–1.6 mL−1 L soil) metam sodium increased canola seedling emergence and plant health, and reduced root hair infection, gall weight and clubroot severity under greenhouse conditions. Metam sodium application improved subsequent plant growth and reduced clubroot severity, but land preparation and volume of water applied did not affect efficacy. The incorporation of metam sodium into the soil and plastic covering after application improved fumigant efficacy. The study showed that soil fumigation with metam sodium can reduce clubroot severity and improve plant health in the subsequent canola crop.

Virulence and inoculum density‐dependent interactions between clubroot resistant canola (Brassica napus) and Plasmodiophora brassicae

To mitigate the impact and dissemination of clubroot in western Canada, canola (Brassica napus) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae, emerged that are virulent on most clubroot‐resistant (CR) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae, the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 103 spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 105 spores mL−1. At a spore density of 106 spores mL−1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.

The Ethylene Responsive Factor Required for Nodulation 1 (ERN1) Transcription Factor Is Required for Infection-Thread Formation in Lotus japonicus.

Several hundred genes are transcriptionally regulated during infection-thread formation and development of nitrogen-fixing root nodules. We have characterized a set of Lotus japonicus mutants impaired in root-nodule formation and found that the causative gene, Ern1, encodes a protein with a characteristic APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription-factor domain. Phenotypic characterization of four ern1 alleles shows that infection pockets are formed but root-hair infection threads are absent. Formation of root-nodule primordia is delayed and no normal transcellular infection threads are found in the infected nodules. Corroborating the role of ERN1 (ERF Required for Nodulation1) in nodule organogenesis, spontaneous nodulation induced by an autoactive CCaMK and cytokinin-induced nodule primordia were not observed in ern1 mutants. Expression of Ern1 is induced in the susceptible zone by Nod factor treatment or rhizobial inoculation. At the cellular level, the pErn1:GUS reporter is highly expressed in root epidermal cells of the susceptible zone and in the cortical cells that form nodule primordia. The genetic regulation of this cellular expression pattern was further investigated in symbiotic mutants. Nod factor induction of Ern1 in epidermal cells was found to depend on Nfr1, Cyclops, and Nsp2 but was independent of Nin and Nf-ya1. These results suggest that ERN1 functions as a transcriptional regulator involved in the formation of infection threads and development of nodule primordia and may coordinate these two processes.

Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads.

Legume-rhizobium symbiosis is achieved by two major events evolutionarily acquired: root hair infection and organogenesis. Infection thread (IT) development is a distinct element for rhizobial infection. Through ITs, rhizobia are efficiently transported from infection foci on root hairs to dividing meristematic cortical cells. To unveil this process, we performed genetic screening using Lotus japonicus MG-20 and isolated symbiotic mutant lines affecting nodulation, root hair morphology, and IT development. Map-based cloning identified an AP2/ERF transcription factor gene orthologous to Medicago truncatula ERN1. LjERN1 was activated in response to rhizobial infection and depended on CYCLOPS and NSP2. Legumes conserve an ERN1 homolog, ERN2, that functions redundantly with ERN1 in M. truncatula. Phylogenetic analysis showed that the lineages of ERN1 and ERN2 genes originated from a gene duplication event in the common ancestor of legume plants. However, genomic analysis suggested the lack of ERN2 gene in the L. japonicus genome, consistent with Ljern1 mutants exhibited a root hair phenotype that is observed in ern1/ern2 double mutants in M. truncatula. Molecular evolutionary analysis suggested that the nonsynonymous/synonymous rate ratios of legume ERN1 genes was almost identical to that of non-legume plants, whereas the ERN2 genes experienced a relaxed selective constraint.

Migration of endophytic diazotroph Azorhizobium caulinodans ORS571 inside wheat (Triticum aestivum L) and its effect on microRNAs.

Azorhizobium caulinodans ORS571, a novel rhizobium, forms endosymbionts with its nature host Sesbania rostrata, a semi-aquatic leguminous tree. Recent studies showed that A. caulinodans ORS571, as endophytic rhizobium, disseminated and colonized inside of cereal plants. However, how this rhizobium infects monocot plants and the regulatory mechanism remains unknown. MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression at the post-transcriptional levels. In this study, we employed laser scanning confocal microscope to monitor the pathway that rhizobium invade wheat; we also investigated the potential role of miRNAs during A. caulinodans ORS571 infecting wheat. Our results showed that gfp-labeled A. caulinodans ORS571 infected wheat root hairs and emerged lateral roots, then disseminated and colonized within roots and migrated to other plant tissues, such as stems and leaves. Endophytic rhizobium induced the aberrant expression of miRNAs in wheat with a tissue- and time-dependent manner with a peak at 12-24h after rhizobium infection. Some miRNAs, such as miR167 and miR393 responded more in roots than that in shoots. In contrast, miR171 responded higher in shoots than that in roots. These results suggested that miRNAs could be responsive to A. caulinodans ORS571 infection and played important role in plant growth, nutrient metabolisms, and wheat-rhizobium interactions.

Indole-3-acetic acid production via the indole-3-pyruvate pathway by plant growth promoter Rhizobium tropici CIAT 899 is strongly inhibited by ammonium

Like many rhizobia, Rhizobium tropici produces indole-3-acetic acid (IAA), an important signal molecule required for root hair infection in rhizobia-legume symbioses. However, the IAA biosynthesis pathway and its regulation by R. tropici are still poorly understood. In this study, IAA synthesis and the effects of mineral N in IAA production by R. tropici CIAT 899 were verified by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). Furthermore, expression of genes related to IAA biosynthesis and metabolism were evaluated by RT-qPCR. Results indicated that IAA production by CIAT 899 was 12 times lower in the presence of NH4+. Moreover, it was found that indole-3-pyruvate (IPyA) is the major IAA biosynthesis intermediate. Genes y4wE, lao and iorA were identified by analysis of R. tropici genome in silico and were upregulated by tryptophan, indicating a possible role of these genes in IAA biosynthesis by CIAT 899. In conclusion, we show that IPyA is the major pathway for IAA biosynthesis in CIAT 899 and that its production is strongly inhibited by NH4+. Although present results arose from in vitro experiments, they provide new insight into the role of nitrogen in early events related to legume nodulation.

A rhamnose-deficient lipopolysaccharide mutant of Rhizobium sp. IRBG74 is defective in root colonization and beneficial interactions with its flooding-tolerant hosts Sesbania cannabina and wetland rice.

Rhizobium sp. IRBG74 develops a classical nitrogen-fixing symbiosis with the aquatic legume Sesbania cannabina (Retz.). It also promotes the growth of wetland rice (Oryza sativa L.), but little is known about the rhizobial determinants important for these interactions. In this study, we analyzed the colonization of S. cannabina and rice using a strain of Rhizobium sp. IRBG74 dually marked with β-glucuronidase and the green fluorescent protein. This bacterium colonized S. cannabina by crack entry and through root hair infection under flooded and non-flooded conditions, respectively. Rhizobium sp. IRBG74 colonized the surfaces of wetland rice roots, but also entered them at the base of lateral roots. It became endophytically established within intercellular spaces in the rice cortex, and intracellularly within epidermal and hypodermal cells. A mutant of Rhizobium sp. IRBG74 altered in the synthesis of the rhamnose-containing O-antigen exhibited significant defects, not only in nodulation and symbiotic nitrogen fixation with S. cannabina, but also in rice colonization and plant growth promotion. Supplementation with purified lipopolysaccharides from the wild-type strain, but not from the mutant, restored the beneficial colonization of rice roots, but not fully effective nodulation of S. cannabina Commonalities and differences in the rhizobial colonization of the roots of wetland legume and rice hosts are discussed.

Arabidopsis Mutant bik1 Exhibits Strong Resistance to Plasmodiophora brassicae.

Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1, and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2, and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40–50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 [non-expresser of PR genes that regulate systemic acquired resistance (SAR)] mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms.

MtSWEET11, a Nodule-Specific Sucrose Transporter of Medicago truncatula.

Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable agriculture. Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and depends on a steady supply of carbon to both plant and bacterial cells. Here we report the functional characterization of a nodule-specific Suc transporter, MtSWEET11 from Medicago truncatula MtSWEET11 belongs to a clade of plant SWEET proteins that are capable of transporting Suc and play critical roles in pathogen susceptibility. When expressed in mammalian cells, MtSWEET11 transported sucrose (Suc) but not glucose (Glc). The MtSWEET11 gene was found to be expressed in infected root hair cells, and in the meristem, invasion zone, and vasculature of nodules. Expression of an MtSWEET11-GFP fusion protein in nodules resulted in green fluorescence associated with the plasma membrane of uninfected cells and infection thread and symbiosome membranes of infected cells. Two independent Tnt1-insertion sweet11 mutants were uncompromised in SNF Therefore, although MtSWEET11 appears to be involved in Suc distribution within nodules, it is not crucial for SNF, probably because other Suc transporters can fulfill its role(s).

A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation.

The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M truncatula Cystathionine-β-Synthase-like1 (MtCBS1), using a promoter-β-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca(2+) responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall.

Putative role of IAA during the early response of Brassica napus L. to Plasmodiophora brassicae

This study examined the role of indole-3-acetic acid (IAA) during the early stage of infection of rapeseed (Brassica napus L.) by the obligate biotrophic protist Plasmodiophora brassicae Woron., which causes clubroot disease. Treatment of infected B. napus seedlings with exogenous IAA promoted the development of clubroot disease, including an increase in the number and size of galls, whereas treatment with N-1-naphthylphthalamic acid (NPA) attenuated the disease and altered the location and size of the root nodules, resulting in a reduced disease index. An increased level of free IAA was detected in the roots at 3 and 7 days after inoculation (DAI) compared with the control, suggesting the activation of IAA signaling in the early stage of P. brassicae infection. Moreover, IAA treatment caused no significant change in the germination rate of the resting spores but resulted in enhanced root hair infection. RT–PCR and quantitative real-time PCR (qPCR) were used to analyze the expression of IAA homeostasis-related genes (3, 7, and 10 DAI) to identify gene expression patterns in the roots and leaves of B. napus after inoculation. Five IAA biosynthesis-related genes examined in this study were induced in the roots after inoculation with P. brassicae but not in the leaves. The rapid induction of BnAAO4 expression at 3 DAI may be responsible for the overproduction of IAA during the early stages of infection. This study suggests that IAA acts as a signaling molecule that putatively stimulates root hair infection in the early response of B. napus to P. brassicae infection.

Stimulation of nitrogen fixation and trehalose biosynthesis by naringenin (Nar) and arbuscular mycorrhiza (AM) in chickpea under salinity stress

Legumes are extremely susceptible to soil salinity because of high sensitivity of nitrogen‐fixing nodules, where root hair infection and nodulation are particularly salt sensitive. Arbuscular mycorrhiza (AM) inoculation is a promising approach to improve rhizobial symbiosis and hence growth. Flavonoids such as naringenin (Nar), play an imperative role in tripartite symbiosis of rhizobia and AM with legumes by signalling both symbioses. Modulation in flavonoids content is one of the important factors limiting nodulation and mycorrhization in salt stressed plants. Green house study investigated the potential of Nar (4 µM) and mycorrhiza (Funneliformis mosseae) in enhancing nodulation and nitrogen fixation in Cicer arietinum L. genotypes (PBG 5, DCP 92-3) under NaCl (0–100 mM). High sodium concentration in the nodules deleteriously affected nodulation, rate of nitrogen fixation, endogenous Nar and nutrient status, with higher negative effects in DCP 92-3 than PBG 5, which could be directly correlated with higher mycorrhizal dependency and lower colonization. Exogenous Nar partly restored nodulation and mycorrhization indicating its involvement as signal molecule in symbiosis. Mycorrhization and Nar enhanced salt induced trehalose 6-P-synthase and phosphatase and reduced trehalase activity, ensuing higher trehalose biosynthesis in nodules. Relative assessment of AM and Nar indicated a more prominent contribution of AM in reducing Na+ uptake and improving phosphorus, with nodulation and trehalose synthesis exhibiting higher dependency on Nar. Complete amelioration of negative effects of salinity were observed with +Nar+AM, thereby suggesting complementation of Nar and AM in improving symbiotic efficiency of chickpea under salt stress.

Rhizobial Diversity and Nodulation Characteristics of the Extremely Promiscuous Legume Sophora flavescens.

In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class α-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class β-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-β-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.

SCARN a Novel Class of SCAR Protein That Is Required for Root-Hair Infection during Legume Nodulation.

Rhizobial infection of legume root hairs requires a rearrangement of the actin cytoskeleton to enable the establishment of plant-made infection structures called infection threads. In the SCAR/WAVE (Suppressor of cAMP receptor defect/WASP family verpolin homologous protein) actin regulatory complex, the conserved N-terminal domains of SCAR proteins interact with other components of the SCAR/WAVE complex. The conserved C-terminal domains of SCAR proteins bind to and activate the actin-related protein 2/3 (ARP2/3) complex, which can bind to actin filaments catalyzing new actin filament formation by nucleating actin branching. We have identified, SCARN (SCAR-Nodulation), a gene required for root hair infection of Lotus japonicus by Mesorhizobium loti. Although the SCARN protein is related to Arabidopsis thaliana SCAR2 and SCAR4, it belongs to a distinct legume-sub clade. We identified other SCARN-like proteins in legumes and phylogeny analyses suggested that SCARN may have arisen from a gene duplication and acquired specialized functions in root nodule symbiosis. Mutation of SCARN reduced formation of infection-threads and their extension into the root cortex and slightly reduced root-hair length. Surprisingly two of the scarn mutants showed constitutive branching of root hairs in uninoculated plants. However we observed no effect of scarn mutations on trichome development or on the early actin cytoskeletal accumulation that is normally seen in root hair tips shortly after M. loti inoculation, distinguishing them from other symbiosis mutations affecting actin nucleation. The C-terminal domain of SCARN binds to ARPC3 and ectopic expression of the N-terminal SCAR-homology domain (but not the full length protein) inhibited nodulation. In addition, we found that SCARN expression is enhanced by M. loti in epidermal cells and that this is directly regulated by the NODULE INCEPTION (NIN) transcription factor.