Journal: Plant Physiology

Article Title: A Select and Resequence Approach Reveals Strain-Specific Effects of Medicago Nodule-Specific PLAT-Domain Genes ^{1 [OPEN]}

doi: 10.1104/pp.19.00831

Figure Lengend Snippet: Phenotypes of plants with all five NPD genes knocked out (npd1–5) differed from wild-type (WT) M. truncatula plants more than plants with the NPD2 gene knocked out differed from wild type. Measured phenotypes are as follows: mean nodule size (A), nodule number per plant (B) colony-forming rhizobia per nodule (C), percent pink nodules (D), and plant biomass (E). Letters indicate between-group differences in the multi-strain inoculation experiment based on ANOVA (P < 0.05; P adjusted for three contrasts via Tukey method). Solid points indicate average values in multi-strain inoculations for each pot replicate (median of 5.5 plants per pot). Hollow triangles indicate single-strain (Sm1021-only) inoculations.

Article Snippet: Recently, the phenotypic effects of NPD1 , a member of the nodule-specific polycystin-1, lipoxygenase, alpha toxin (PLAT) domain ( NPD ) gene family studied here, were shown to be strain specific in Medicago truncatula ( Pislariu et al., 2019 ).

Techniques:

Journal: Plant Physiology

Article Title: A Select and Resequence Approach Reveals Strain-Specific Effects of Medicago Nodule-Specific PLAT-Domain Genes ^{1 [OPEN]}

doi: 10.1104/pp.19.00831

Figure Lengend Snippet: Characterization of shifts in E. meliloti strain communities in nodules of M. truncatula wild-type (WT) plants, plants with NPD2 knocked out (npd2), and plants with all five NPD genes knocked out (npd1–5). A, Nodule strain diversity was greater in wild-type hosts and npd2 mutants than npd1–5 mutants. Higher diversity communities have more strains that are at similar frequencies. Different lowercase letters indicate genotypes that are significantly different from each other (P < 0.05). B, Similarly, the number of strains with fitness greater than zero (i.e. those that increased in frequency in nodules as compared to the initial inoculum) were greater in wild-type hosts and npd2 mutants than npd1–5 mutants. C, Redundancy analysis of relative strain fitness shows distinct effects of npd2 and npd1–5 mutants (host genotype explains 17.2% of strain fitness variation [adj. r2], more than expected by chance [P = 0.005]). ANOVA test results from A, wild type versus npd1–5 (P = 0.011), npd2 versus npd1–5 (P = 0.03), and wild type versus npd2 (P = 0.20).

Article Snippet: Recently, the phenotypic effects of NPD1 , a member of the nodule-specific polycystin-1, lipoxygenase, alpha toxin (PLAT) domain ( NPD ) gene family studied here, were shown to be strain specific in Medicago truncatula ( Pislariu et al., 2019 ).

Techniques:

Journal: Plant Physiology

Article Title: A Select and Resequence Approach Reveals Strain-Specific Effects of Medicago Nodule-Specific PLAT-Domain Genes ^{1 [OPEN]}

doi: 10.1104/pp.19.00831

Figure Lengend Snippet: Strain frequency estimates for 68 E. meliloti strains in nodules of wild-type M. truncatula (WT) plants, plants with the NPD2 gene knocked out (npd2), and plants with all five NPD genes knocked out (npd1–5) . Points indicate median frequency across replicates, and lines span from minimum to maximum values. Scales have been split for readability, and strains are ordered based on frequency in wild-type nodules. Dashed line indicates median overall strain frequency in the initial inoculum.

Article Snippet: Recently, the phenotypic effects of NPD1 , a member of the nodule-specific polycystin-1, lipoxygenase, alpha toxin (PLAT) domain ( NPD ) gene family studied here, were shown to be strain specific in Medicago truncatula ( Pislariu et al., 2019 ).

Techniques:

Journal: Plant Physiology

Article Title: A Select and Resequence Approach Reveals Strain-Specific Effects of Medicago Nodule-Specific PLAT-Domain Genes ^{1 [OPEN]}

doi: 10.1104/pp.19.00831

Figure Lengend Snippet: Heatmaps (left) show that NPD mutations in M. truncatula have differential effects on relative fitness of E. meliloti strains in nodules. Strain fitness in wild-type M. truncatula (WT) plants are compared to plants with the NPD2 gene knocked out (npd2) and plants with all five NPD genes knocked out (npd1–5). The effects of NPD disruption on strain fitness are not clearly related to the effects of strains on plant biomass (right). Strains are ordered from high relative fitness in wild type (WT; red) to low (blue), and values are the median of three replicates). Right shows plant benefit in wild-type R108 background in terms of plant dry weight (green is high, gray is median, yellow is low, and white is N/A). Plant benefit estimates are from single-strain inoculation experiments (Burghardt et al., 2018).

Article Snippet: Recently, the phenotypic effects of NPD1 , a member of the nodule-specific polycystin-1, lipoxygenase, alpha toxin (PLAT) domain ( NPD ) gene family studied here, were shown to be strain specific in Medicago truncatula ( Pislariu et al., 2019 ).

Techniques: Disruption

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: Symbiotic phenotype of the NF4608 mutant. A and B, Wild-type R108 (A) and NF4608 mutant (B) plants at 21 DPI with S. meliloti strain Sm1021. C and D, Average nodule number and size in wild-type R108 (C) versus NF4608 mutants (D). Vertical bars represent the sd from measurements in 20 R108 and 35 NF4608 mutants. Asterisks indicate a significant difference compared to the control, as determined using Student’s t test (P ≤ 0.01). E to J, Histochemical staining of S. meliloti Sm1021 carrying the hemA:LacZ reporter in wild-type (E–G) and NF4608 mutants (H–J). The rhizobia stain blue. Shown are 50-µm-thick slices from nodules at 6 DPI (E and H), 8 DPI (F and I), and 10 DPI (G and J). K, Schematic representation of the MtNPD1 gene structure displaying exons in black, introns as a line, and the 5′ and 3′ untranslated regions in gray. Tnt1 insertion sites in three independent alleles are shown. Symbiotic phenotypes of wild-type (L) and three npd1 insertion alleles: npd1-1 (M; NF4608); npd1-2 (N; NF6576); npd1-3 (O; NF9985) are shown. Scale bars = 1 cm (A and B), 50 µm (E–J), 100 bp (K), and 1 mm (L–O).

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Mutagenesis, Control, Staining

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: Genetic complementation of the npd1-1 mutant by p35S:MtNPD1. R. rhizogenes-mediated transformation was used to rescue the wild-type phenotype in the npd1-1 mutant by cloning the coding sequence of wild-type MtNPD1 driven by the 35S promoter into npd1-1 plants. A, C, and D, Images of npd1-1 plants transformed with an empty vector control. B, E, and F, Images of npd1-1 plants transformed with p35S:MtNPD1. Green fluorescence in D and F is given by the GFP marker in the complementation plasmid backbone and indicates transgenic roots. The white arrows point towards npd1-1 roots that failed to transform with the vector control (D) and with the p35S:MtNPD1 construct (F). G, Acetylene reduction activity in whole nodulated roots from 10 composite plants transformed with the vector control (VC) and 11 npd1-1 plants transformed with p35S:MtNPD1. Vertical bars represent the se. Photomicrographs in C to F were acquired with an Olympus SZX12 fluorescence stereomicroscope. CDS = coding sequence. Scale bars = 1 mm.

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Mutagenesis, Transformation Assay, Cloning, Sequencing, Plasmid Preparation, Control, Fluorescence, Marker, Transgenic Assay, Construct, Activity Assay

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: MtNPD1 is required for the accommodation of rhizobia inside host cells. Longitudinal, 1 µm-thick sections of 12 DPI R108 (A to D) and npd1-1 (E to H) nodules were stained with toluidine blue O. A and E, images show the different zones in wild-type (A) and mutant (E) nodules. Other images correspond to cells in the proximal infection zone (B and F; INF) and the distal nitrogen fixation zone (C; dFIX) in the wild type, infected cells in the junction area between the infection zone (INF) and the senescence zone (SEN) in the npd1-1 mutant (G), infected cells in the nitrogen fixation zone (FIX) in the wild type (D), and infected cells in the senescence-like zone (SEN) in the npd1-1 mutant (H). N, nuclei; IC, infected cell; UC, uninfected cell; MER, meristem.. Asterisks, vacuoles; arrowheads, nucleoli; arrows, ITs; ☆, newly released rhizobia; ★, differentiated rhizobia. Scale bars = 100 µm.

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Staining, Mutagenesis, Infection

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: MtNPD1 belongs to a nodulation-specific clade of PLAT single-domain proteins. Full-length amino acid sequences of 40 plant PLAT domain proteins were aligned with ClustalW, and an unrooted maximum-likelihood phylogenetic tree was generated using PhyML and the LG substitution model (Geneious 10.2.5; Le and Gascuel, 2008). Numbers at branch nodes represent confidence values from 1,000 bootstrap replicates and indicate tree robustness. The scale bar represents the number of substitutions per site. The tree includes all members of the Medicago PLAT domain proteins found in the updated genome (Mt4.0v1; http://jcvi.org/medicago/). Proteins that have not previously been characterized are labeled with the two-letter species designation followed by the UniProt identifier. The nodule-specific clade is highlighted in red; the clade of proteins involved in biotic and abiotic stress response is highlighted in green, and the monocot clade is highlighted in blue. Bold fonts highlight MtNPD1. Asterisks mark proteins that have previously been described.

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Generated, Labeling

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: Spatiotemporal expression of MtNPD1. A, MtNPD1 expression during a nodule development time course was measured by RT-qPCR. Nodulation-susceptible zones were used at 0 DPI and at 2 and 4 DPI (after inoculation with Sm1021). Samples at 6 to 21 DPI represent nodules dissected out of the roots. B, MtNPD1 expression was also measured by RT-qPCR in the following hand-dissected nodule zones: the meristem (MER), the infection zone (INF), the distal nitrogen fixation zone (dFIX), the nitrogen fixation zone (FIX), and the senescence zone (SEN). RT-qPCR measurements were carried out on three biological replicates. C to F, pMtNPD1:GUS expression was observed in composite M. truncatula roots and nodules at 4 DPI (C and D), 7 DPI (E), and 10 DPI (F). Arrowheads point toward ITs. GUS coloration for promoter activity is blue. S. meliloti Sm1021 carrying the hemA:LacZ reporter was used for inoculation. Bacteria are seen as magenta precipitate in C to E, following histochemical staining for β-galactosidase activity. In F, only GUS coloration is shown. GUS and LacZ staining were carried out on whole roots. Images in C to E represent whole root mounts and that in F is a 50-μm-thick section. Scale bars = 100 µm. Error bars in A and B represent the mean ± se. ****P < 0.0001, one-way ANOVA test followed by the Tukey posttest for multiple comparisons. Comparison is with 0 DPI.

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Expressing, Quantitative RT-PCR, Infection, Activity Assay, Bacteria, Staining, Comparison

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: Relative expression of MtNPD1 in Fix− nodules. Measurement of the relative MtNPD1 transcript level was done in 10 DPI Fix− nodules from mutant plants with defects in plant genes (A) and bacterial genes (B). Column heights correspond to mean values from three independent biological replicates. Asterisks indicate comparison with wild-type (R108) nodules. Error bars show the se. **P < 0.005; ****P < 0.0001; one-way ANOVA test with correction for multiple comparisons.

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Expressing, Mutagenesis, Comparison

Journal: Plant Physiology

Article Title: The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis ^{1 [OPEN]}

doi: 10.1104/pp.18.01613

Figure Lengend Snippet: MtNPD1:GFP fusion driven by the native promoter localizes to the space surrounding symbiosomes in nodule infected cells. Transgenic nodules were harvested at 15 DPI, hand-sliced longitudinally, and immediately observed using a confocal laser scanning microscope. Images were acquired by sequential scanning using green and red fluorescence channels. A and B, GFP fluorescence driven by the MtNPD1 promoter in R108 transgenic roots, used as a control. Accumulation of MtNPD1 transcripts can be seen in the infection zone and in infected cells. The close-up image (B) shows a high level of MtNPD1 promoter activity in the infection zone. C to F, Fluorescence of GFP fused to the C terminus of MtNPD1, driven by the native promoter, in complemented nodules of the npd1-1 mutant. A and C represent overlays of GFP and transmitted light images. Red fluorescence in B, E, and F is from Sm1021 rhizobia carrying the mCherry reporter plasmid. (C and D) MtNPD1p:MtNPD1:GFP; (E) mCherry rhizobia; (F) Merge of D and E. Scale bars = 100 µm (A and C), 20 µm (C), and 10 µm (D to F).

Article Snippet: MtNPD1 Belongs to a Cluster of Five Nodule-Specific PLAT Domain-Encoding Genes A BLAST search of the M. truncatula A17 genome, using the MtNPD1 genomic sequence ( http://medicago.jcvi.org ) yielded 13 homologous genes ( Supplemental Table S3 ).

Techniques: Infection, Transgenic Assay, Laser-Scanning Microscopy, Fluorescence, Control, Activity Assay, Mutagenesis, Plasmid Preparation