scholarly journals NolX of Sinorhizobium fredii USDA257, a Type III-Secreted Protein Involved in Host Range Determination, Is Localized in the Infection Threads of Cowpea (Vigna unguiculata [L.] Walp) and Soybean (Glycine max [L.] Merr.) Nodules

2002 ◽  
Vol 184 (3) ◽  
pp. 831-839 ◽  
Author(s):  
Hari B. Krishnan
Keyword(s):  
Glycine Max ◽  
Vigna Unguiculata ◽  
Xanthomonas Campestris ◽  
Soybean Cultivar ◽  
Nodule Development ◽  
Sinorhizobium Fredii ◽  
Thin Sections ◽  
Type Iii ◽  
Infection Threads ◽  
Cultivar Specificity

ABSTRACT Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean (Glycine max [L.] Merr.) in a cultivar-specific manner. This strain forms nodules on primitive soybean cultivars but fails to nodulate agronomically improved North American cultivars. Soybean cultivar specificity is regulated by the nolXWBTUV locus, which encodes part of a type III secretion system (TTSS). NolX, a soybean cultivar specificity protein, is secreted by TTSS and shows homology to HrpF of the plant pathogen Xanthomonas campestris pv. vesicatoria. It is not known whether NolX functions at the bacterium-plant interface or acts inside the host cell. Antibodies raised against S. fredii USDA257 NolX were used in immunocytochemical studies to investigate the subcellular localization of this protein. Immunostaining of paraffin-embedded sections of developing soybean and cowpea (Vigna unguiculata [L.] Walp) nodules revealed localization of NolX in the infection threads. Protein A-gold immunocytochemical localization studies utilizing affinity-purified NolX antibodies revealed specific deposition of gold particles in the fibrillar material inside infection threads. Similar immunogold localization studies failed to detect NolX in thin sections of mature soybean and cowpea nodules. The results from this study indicate that NolX is expressed in planta only during the early stages of nodule development.

scholarly journals NopB, a Soybean Cultivar-Specificity Protein from Sinorhizobium fredii USDA257, Is a Type III Secreted Protein

2004 ◽  
Vol 17 (11) ◽  
pp. 1259-1268 ◽  
Author(s):  
Julio C. Lorio ◽  
Won Seok Kim ◽  
Hari B. Krishnan
Keyword(s):  
Deletion Mutant ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Soybean Cultivar ◽  
Affinity Column ◽  
Coding Region ◽  
Sinorhizobium Fredii ◽  
Type Iii ◽  
Specificity Protein ◽  
Cultivar Specificity

The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NolB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.

scholarly journals Translocation of NopP by Sinorhizobium fredii USDA257 into Vigna unguiculata Root Nodules

2010 ◽  
Vol 76 (11) ◽  
pp. 3758-3761 ◽  
Author(s):  
Lisa M. Schechter ◽  
Jeanette Guenther ◽  
Elizabeth A. Olcay ◽  
Sungchan Jang ◽  
Hari B. Krishnan
Keyword(s):  
Vigna Unguiculata ◽  
Type Iii Secretion ◽  
Root Nodules ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Dependent Manner ◽  
Nitrogen Fixing ◽  
Sinorhizobium Fredii ◽  
Type Iii

ABSTRACT Sinorhizobium fredii is a nitrogen-fixing legume symbiont that stimulates the formation of root nodules. S. fredii nodulation of roots is influenced by Nop proteins, which are secreted through a type III secretion system (T3SS). We demonstrate that S. fredii injects NopP into Vigna unguiculata nodules in a T3SS-dependent manner.

Rhizobium infection threads in root hairs of Glycine max (L.) Merr., Glycine soja Sieb. & Zucc, and Vigna unguiculata (L.) Walp.

1983 ◽  
Vol 29 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Steven G. Pueppke
Keyword(s):  
Glycine Max ◽  
Vigna Unguiculata ◽  
Glycine Soja ◽  
Primary Root ◽  
Wild Soybean ◽  
Root Hairs ◽  
Infected Root ◽  
Infection Threads ◽  
Basal Portion ◽  
Rhizobium Sp

Eight lines of soybean (Glycine max), four of wild soybean (Glycine soja), and one cowpea (Vigna unguiculata) cultivar were inoculated with 18 Rhizobium strains. After 4 days, root hairs were examined for infection threads. Threads were produced by all hosts but exclusively in nodulating combinations. Only Rhizobium sp. strains 3G4b9a and 3G4b19 were inconsistent; they nodulated soybean and G. soja in some experiments, but rarely formed infection threads. Soybean and G. soja were indistinguishable in their interactions with rhizobia, as were lele soybean lines (genetically lack soybean lectin), Hardee soybean (contains the noduation-influencing genes Rj2 and Rj3), and several other soybean cultivars. Threads formed in cowpea with all of the R. japonicum strains and most Rhizobium sp. but not with R. lupini. Infection of all three host species occurred in portions of the primary root containing immature or no root hairs at the time of inoculation; proximal tissues having elongated root hairs lacked infection threads. Infected root hairs were short and commonly shaped like question marks. Threads usually branched and sometimes intertwined prior to elongation into the basal portion of root hairs.

scholarly journals Sinorhizobium fredii HH103 cgs Mutants Are Unable to Nodulate Determinate- and Indeterminate Nodule–Forming Legumes and Overproduce an Altered EPS

2009 ◽  
Vol 22 (5) ◽  
pp. 575-588 ◽  
Author(s):  
Juan C. Crespo-Rivas ◽  
Isabel Margaret ◽  
Ángeles Hidalgo ◽  
Ana M. Buendía-Clavería ◽  
Francisco J. Ollero ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Glycine Max ◽  
Vigna Unguiculata ◽  
Distilled Water ◽  
Sinorhizobium Fredii ◽  
Indeterminate Nodule ◽  
Nodulation Factors ◽  
Indeterminate Nodules ◽  
Gentamicin Resistance ◽  
K Antigen

Sinorhizobium fredii HH103 produces cyclic β glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgs∷lacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs∷Ω) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

scholarly journals Disruption of the Glycine Cleavage System Enables Sinorhizobium fredii USDA257 To Form Nitrogen-Fixing Nodules on Agronomically Improved North American Soybean Cultivars

2010 ◽  
Vol 76 (13) ◽  
pp. 4185-4193 ◽  
Author(s):  
Julio C. Lorio ◽  
Won-Seok Kim ◽  
Ammulu H. Krishnan ◽  
Hari B. Krishnan
Keyword(s):  
North American ◽  
Genetic Locus ◽  
Open Reading Frames ◽  
Soybean Cultivar ◽  
Sinorhizobium Fredii ◽  
Amino Acid Homology ◽  
Soybean Cultivars ◽  
Glycine Cleavage ◽  
P Proteins ◽  
Cultivar Specificity

ABSTRACT The symbiosis between Sinorhizobium fredii USDA257 and soybean [Glycine max (L.) Merr.] exhibits a high degree of cultivar specificity. USDA257 nodulates primitive soybean cultivars but fails to nodulate agronomically improved cultivars such as McCall. In this study we provide evidence for the involvement of a new genetic locus that controls soybean cultivar specificity. This locus was identified in USDA257 by Tn5 transposon mutagenesis followed by nodulation screening on McCall soybean. We have cloned the region corresponding to the site of Tn5 insertion and found that it lies within a 1.5-kb EcoRI fragment. DNA sequence analysis of this fragment and an adjacent 4.4-kb region identified an operon made up of three open reading frames encoding proteins of deduced molecular masses of 41, 13, and 104 kDa, respectively. These proteins revealed significant amino acid homology to glycine cleavage (gcv) system T, H, and P proteins of Escherichia coli and other organisms. Southern blot analysis revealed the presence of similar sequences in diverse rhizobia. Measurement of β-galactosidase activity of a USDA257 strain containing a transcriptional fusion of gcvT promoter sequences to the lacZ gene revealed that the USDA257 gcvTHP operon was inducible by glycine. Inactivation of either gcvT or gcvP of USDA257 enabled the mutant to nodulate several agronomically improved North American soybean cultivars. These nodules revealed anatomical features typical of determinate nodules, with numerous bacteroids within the infected cells. Unlike for the previously characterized soybean cultivar specificity locus nolBTUVW, inactivation of the gcv locus had no discernible effect on the secretion of nodulation outer proteins of USDA257.

scholarly journals NolR Regulates Diverse Symbiotic Signals of Sinorhizobium fredii HH103

2004 ◽  
Vol 17 (6) ◽  
pp. 676-685 ◽  
Author(s):  
JoséMaría Vinardell ◽  
Francisco Javier Ollero ◽  
Ángeles Hidalgo ◽  
Francisco Javier López-Baena ◽  
Carlos Medina ◽  
...  
Keyword(s):  
Glycine Max ◽  
Vigna Unguiculata ◽  
Sr Proteins ◽  
Nod Factors ◽  
Sinorhizobium Fredii ◽  
Nodulation Capacity ◽  
Multiple Copies ◽  
Inducible Genes

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Strr) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysac-charide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

scholarly journals The Sinorhizobium fredii HH103 type III secretion system effector NopC blocks nodulation with Lotus japonicus Gifu

2020 ◽  
Vol 71 (19) ◽  
pp. 6043-6056 ◽  
Author(s):  
Irene Jiménez-Guerrero ◽  
Sebastián Acosta-Jurado ◽  
Carlos Medina ◽  
Francisco Javier Ollero ◽  
Cynthia Alias-Villegas ◽  
...  
Keyword(s):  
Host Range ◽  
Type Iii Secretion ◽  
Lotus Japonicus ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Infection Thread ◽  
Sinorhizobium Fredii ◽  
Model Legume ◽  
Type Iii ◽  
Infection Threads

Abstract The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.

scholarly journals New methods for confocal imaging of infection threads in crop and model legumes

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Angus E. Rae ◽  
Vivien Rolland ◽  
Rosemary G. White ◽  
Ulrike Mathesius
Keyword(s):  
Cell Wall ◽  
Root Hair ◽  
Periodic Acid ◽  
Confocal Imaging ◽  
Wall Material ◽  
Future Research ◽  
Thin Sections ◽  
New Methods ◽  
Infection Threads ◽  
Imaging Of Infection

Abstract Background The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. Results We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. Conclusions Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

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