A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated ... more A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium. The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy. The clone contained a single open reading frame, and the gene was termed PsNlec1. Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin. Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant. It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule. When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system. The identification...
Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum bv. viciae 3841, as all ot... more Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum bv. viciae 3841, as all other members of the family Rhizobiaceae with the possible exception of Azorhizobium caulinodans, contains a very long chain fatty acid; 27-hydroxyoctacosanoic acid (27OHC28:0) in its lipid A region. The exact function and importance of this residue, however, is not known. In this work, a previously constructed mutant, Rhizobium leguminosarum bv. viciae 22, deficient in the fatty acid residue, was analyzed for its symbiotic phenotype. While the mutant was able to form nitrogen-fixing nodules, a detailed study of the timing and efficiency of nodulation using light and electron microscopy showed that there was a delay in the onset of nodulation and nodule tissue invasion. Further, microscopy showed that the mutant was unable to differentiate normally forming numerous irregularly shaped bacteroids, that the resultant mature bacteroids were unusually large, and that several bacteroids were fre...
In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric din... more In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter–gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein...
The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum i... more The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum in nodule tissue from a symbiotically defective pea mutant, Sprint2Fix¯(sym31), and the parent line, Sprint2. Immunostaining of Western blots (immunoblots) revealed that, whereas wild-type nodules contained three antigenic isoforms of PsNLEC-1, nodule homogenates of mutant sym31 contained only one isoform, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was not associated with symbiosomes in either the mutant or the wild-type parent (unlike the other two isoforms from wild-type nodules). Light microscopy revealed that PsNLEC-1 antigen was more abundant in the infected tissues of wild-type nodules than in nodules of sym31. By contrast, in situ hybridization indicated that the PsNlec1 gene transcript was strongly expressed in infected cells of both Sprint2 and sym31 nodule tissues. At the ultrastructural level, most of the PsNLEC-1 antigen in sym31 nodule tissue was visualized as ...
Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorl... more Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) “phased” intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22...
The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides ... more The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4′ position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain ω-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus , Bartonella henselae , and Legionella pneumophila . In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL , the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity,...
Summary Using a series of antibody probes as markers of symbiosome development, we have investig... more Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix− (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often
Root nodules on peanut (Arachis hypogaea 1.) accumulate a galactose/lactose-binding lectin that i... more Root nodules on peanut (Arachis hypogaea 1.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. lhe function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most
Bacillus subtilis, which belongs to the phylum Firmicutes, is the most widely studied Gram-positi... more Bacillus subtilis, which belongs to the phylum Firmicutes, is the most widely studied Gram-positive model organism. It is found in a wide variety of environments and is particularly abundant in soils and in the gastrointestinal tracts of ruminants and humans. Here, we present the complete genome sequence of the newly described B. subtilis strain UD1022. The UD1022 genome consists of a 4.025-Mbp chromosome, and other major findings from our analysis will provide insights into the genomic basis of it being a plant growth-promoting rhizobacterium (PGPR) with biocontrol potential.
A natural rice rhizospheric isolate abates arsenic uptake in rice by increasing Fe plaque formati... more A natural rice rhizospheric isolate abates arsenic uptake in rice by increasing Fe plaque formation on rice roots. Rice (Oryza sativa L.) is the staple food for over half of the world's population, but its quality and yield are impacted by arsenic (As) in some regions of the world. Bacterial inoculants may be able to mitigate the negative impacts of arsenic assimilation in rice, and we identified a nonpathogenic, naturally occurring rice rhizospheric bacterium that decreases As accumulation in rice shoots in laboratory experiments. We isolated several proteobacterial strains from a rice rhizosphere that promote rice growth and enhance the oxidizing environment surrounding rice root. One Pantoea sp. strain (EA106) also demonstrated increased iron (Fe)-siderophore in culture. We evaluated EA106's ability to impact rice growth in the presence of arsenic, by inoculation of plants with EA106 (or control), subsequently grew the plants in As-supplemented medium, and quantified the ...
Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the ... more Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors ...
Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that i... more Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. The function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most abundant in the nodule parenchyma, where it accumulated in vacuoles, suggesting a possible role as a vegetative storage protein. Lectin was also detected in the extracellular matrix in the nodule parenchyma, a location that corresponds to the tissue layer forming a barrier to oxygen diffusion. The potential for interactions between PNL and other cell wall components, including a previously described high-molecular weight glycoprotein that co-localizes with PNL, is discussed. Within infected cells, lectin was not detectable by immunogold labeling within the cytoplasm, but light labeling was sugges...
We report on a nondestructive clearing technique that enhances transmission of light through spec... more We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process.
A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated ... more A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium. The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy. The clone contained a single open reading frame, and the gene was termed PsNlec1. Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin. Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant. It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule. When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system. The identification...
Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum bv. viciae 3841, as all ot... more Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum bv. viciae 3841, as all other members of the family Rhizobiaceae with the possible exception of Azorhizobium caulinodans, contains a very long chain fatty acid; 27-hydroxyoctacosanoic acid (27OHC28:0) in its lipid A region. The exact function and importance of this residue, however, is not known. In this work, a previously constructed mutant, Rhizobium leguminosarum bv. viciae 22, deficient in the fatty acid residue, was analyzed for its symbiotic phenotype. While the mutant was able to form nitrogen-fixing nodules, a detailed study of the timing and efficiency of nodulation using light and electron microscopy showed that there was a delay in the onset of nodulation and nodule tissue invasion. Further, microscopy showed that the mutant was unable to differentiate normally forming numerous irregularly shaped bacteroids, that the resultant mature bacteroids were unusually large, and that several bacteroids were fre...
In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric din... more In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter–gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein...
The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum i... more The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum in nodule tissue from a symbiotically defective pea mutant, Sprint2Fix¯(sym31), and the parent line, Sprint2. Immunostaining of Western blots (immunoblots) revealed that, whereas wild-type nodules contained three antigenic isoforms of PsNLEC-1, nodule homogenates of mutant sym31 contained only one isoform, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was not associated with symbiosomes in either the mutant or the wild-type parent (unlike the other two isoforms from wild-type nodules). Light microscopy revealed that PsNLEC-1 antigen was more abundant in the infected tissues of wild-type nodules than in nodules of sym31. By contrast, in situ hybridization indicated that the PsNlec1 gene transcript was strongly expressed in infected cells of both Sprint2 and sym31 nodule tissues. At the ultrastructural level, most of the PsNLEC-1 antigen in sym31 nodule tissue was visualized as ...
Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorl... more Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) “phased” intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22...
The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides ... more The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4′ position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain ω-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus , Bartonella henselae , and Legionella pneumophila . In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL , the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity,...
Summary Using a series of antibody probes as markers of symbiosome development, we have investig... more Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix− (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often
Root nodules on peanut (Arachis hypogaea 1.) accumulate a galactose/lactose-binding lectin that i... more Root nodules on peanut (Arachis hypogaea 1.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. lhe function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most
Bacillus subtilis, which belongs to the phylum Firmicutes, is the most widely studied Gram-positi... more Bacillus subtilis, which belongs to the phylum Firmicutes, is the most widely studied Gram-positive model organism. It is found in a wide variety of environments and is particularly abundant in soils and in the gastrointestinal tracts of ruminants and humans. Here, we present the complete genome sequence of the newly described B. subtilis strain UD1022. The UD1022 genome consists of a 4.025-Mbp chromosome, and other major findings from our analysis will provide insights into the genomic basis of it being a plant growth-promoting rhizobacterium (PGPR) with biocontrol potential.
A natural rice rhizospheric isolate abates arsenic uptake in rice by increasing Fe plaque formati... more A natural rice rhizospheric isolate abates arsenic uptake in rice by increasing Fe plaque formation on rice roots. Rice (Oryza sativa L.) is the staple food for over half of the world's population, but its quality and yield are impacted by arsenic (As) in some regions of the world. Bacterial inoculants may be able to mitigate the negative impacts of arsenic assimilation in rice, and we identified a nonpathogenic, naturally occurring rice rhizospheric bacterium that decreases As accumulation in rice shoots in laboratory experiments. We isolated several proteobacterial strains from a rice rhizosphere that promote rice growth and enhance the oxidizing environment surrounding rice root. One Pantoea sp. strain (EA106) also demonstrated increased iron (Fe)-siderophore in culture. We evaluated EA106's ability to impact rice growth in the presence of arsenic, by inoculation of plants with EA106 (or control), subsequently grew the plants in As-supplemented medium, and quantified the ...
Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the ... more Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors ...
Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that i... more Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. The function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most abundant in the nodule parenchyma, where it accumulated in vacuoles, suggesting a possible role as a vegetative storage protein. Lectin was also detected in the extracellular matrix in the nodule parenchyma, a location that corresponds to the tissue layer forming a barrier to oxygen diffusion. The potential for interactions between PNL and other cell wall components, including a previously described high-molecular weight glycoprotein that co-localizes with PNL, is discussed. Within infected cells, lectin was not detectable by immunogold labeling within the cytoplasm, but light labeling was sugges...
We report on a nondestructive clearing technique that enhances transmission of light through spec... more We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process.
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Papers by D. Sherrier