Article Figures & Data
Figures
- Figure 1.
The B. distachyon ortholog of RAM1 is expressed in roots colonized by AM fungi. A, BdRAM1 gene expression is induced in roots colonized by the AM fungus D. epigaea (gray bars) relative to nonmycorrhizal, mock-inoculated roots (white bars). Plants were harvested 4, 6, and 8 weeks postplanting (wpp). AM-induced BdRAM1 gene expression increases over time. B, Gene expression of the AM marker genes BdPT7 and D. epigaea (D.e.) α-tubulin in D. epigaea-colonized and mock-inoculated control roots over time. A and B, Gene expression was measured by reverse transcription quantitative PCR (RT-qPCR) and normalized to the B. distachyon elongation factor BdEF1α. Bar graphs show the mean, with error bars representing sd. Single points represent individual measurements. Pairwise comparisons of gene expression in AM and control roots were analyzed separately for each time point using Student’s t test (***P < 0.001, **P < 0.01, and *P < 0.05).
- Figure 2.
Arbuscule formation is impaired in B. distachyon ram1 mutants. A, A root piece with ram1-like D. epigaea arbuscules in CRISPR ram1 mutants. ram1-like infections contain solely arbuscules that are not fully developed and show only sparse branching, reminiscent of the ram1 mutant phenotype described previously in dicots (indicated by asterisks). B, A root piece with wild type-like D. epigaea arbuscules in CRISPR ram1 mutants. Wild type-like infections contain fully developed arbuscules (indicated by arrows). D. epigaea fungal structures visualized using WGA-Alexa Fluor 488 (green), with plant cell walls counterstained using propidium iodide (pink). C, Quantification of ram1-like infections relative to the total number of infections in ram1 CRISPR plants and B. distachyon plants transformed with the empty vector (E.V.). The proportion of aberrant infections is increased in two ram1 alleles (ANOVA, P = 2.38 × 10−5). D, Quantification of total D. epigaea root-length colonization in CRISPR ram1 plants relative to E.V. controls. Root length colonization is significantly decreased in two ram1 mutant alleles (ANOVA, P = 0.0014). Pairwise comparisons in C and D were performed using Tukey’s HSD post-hoc test. Different lowercase letters denote significant differences. Box and whisker plots show lower and upper quartiles and minimum and maximum values. The horizontal bar represents the median and the points individual measurements. All results presented in this figure were obtained from the T3 generation.
- Figure 3.
Ectopic overexpression of BdRAM1 promotes arbuscule formation and expression of AM marker genes. A, Gene expression levels of B. distachyon orthologs of MtRAM1 target genes in noncolonized 35S:NLS-GFP (denoted as GFP) and 35S:BdRAM1ox line 1 (denoted as ox1) roots. 35S:BdRAM1ox roots display induced expression of BdRAM1, as well as BdRAM2, BdPT7, BdFatM1, and BdFatM2 in the absence of symbiosis relative to 35S:NLS-GFP control roots. BdSTR gene expression is not affected in these roots. B, Gene expression of B. distachyon RAD1 and WRI5 orthologs. Only expression of BdWRI5.1 is induced in noncolonized 35S:BdRAM1ox roots relative to 35S:NLS-GFP control roots. A and B, Bar graphs show the mean, with error bars representing sd. Single points represent individual measurements. Pairwise comparisons were estimated using the Student’s t test (***P < 0.001, **P < 0.01, and *P < 0.05).; n.s., Not significant. C, Quantification of total root colonization in independent lines transformed with 35S:BdRAM1. There is no difference in overall root colonization between three lines ectopically overexpressing 35S:BdRAM1 (35S:BdRAM1ox, denoted as ox1, ox2, and ox3) and control plants (35S:BdRAM1WT, which does not overexpress BdRAM1 and was therefore denoted as WT [wild type]; and 35S:NLS-GFP, labeled as GFP). ANOVA (P = 0.71). Root-length colonization was quantified using the grid-line method (McGonigle et al., 1990). D, Quantification of D. epigaea arbuscules in a defined area at the fungal hyphopodium. Roots of three independent transgenic 35S:BdRAM1ox lines (denoted as ox1, ox2, and ox3) contain more arbuscules than roots transformed with the control construct 35S:NLS-GFP (GFP) or roots that contain 35S:BdRAM1 but do not overexpress the gene (WT). Arbuscule number was normalized to the volume of the confocal stack. Kruskal-Wallis test (P = 1.32 × 10−4), pairwise comparisons were conducted using the Dunn’s post-hoc test. Different lowercase letters denote significant differences. Box and whisker plots show lower and upper quartiles and minimum and maximum values. The horizontal bar represents the median and the points individual measurements. E, Representative image of a 35S:NLS-GFP root colonized by D. epigaea. F, Representative image of a RAM1-overexpressing 35S:BdRAM1ox (line 1) root colonized by D. epigaea. In E and F, arbuscules are highlighted with arrows.
- Figure 4.
RAM1 overexpressors show altered shoot development and constitutively express root AM marker genes in their shoots. A, Photograph of 4.5-week-old B. distachyon plants transformed with 35S:NLS-GFP or 35S:BdRAM1. The three independent transformant lines overexpressing BdRAM1 (ox1, ox2, and ox3) display a bushy stature, whereas the 35S:BdRAM1-transformant line not overexpressing BdRAM1 (wild type [WT]) resembles the 35S:NLS-GFP control plant. B, Gene expression of BdRAM1 and of several root AM marker genes in shoots. All tested genes are strongly induced in 4.5-week-old shoots of three 35S:BdRAM1ox lines (ox1, ox2, and ox3) relative to control plants transformed with 35S:NLS-GFP (GFP) or the 35S:BdRAM1-transformant line not overexpressing BdRAM1 (wild type). Gene expression was measured by RT-qPCR. Bar graphs show the mean, with error bars representing sd. Single points represent individual measurements. Significance values (ANOVA) for each gene are indicated in the figure. Pairwise comparisons were conducted using Tukey’s HSD post-hoc test. Different lowercase letters denote significant differences.
- Figure 5.
Expression of hormone biosynthesis genes is altered in roots of RAM1 overexpressors. A, B. distachyon orthologs of three genes involved in the SL biosynthesis pathway (BdD27, BdCCD7, and BdCCD8) are down-regulated in noncolonized roots ectopically overexpressing BdRAM1 (35S:BdRAM1ox line 1, denoted as ox1) relative to 35S:NLS-GFP (“GFP”) control roots. B, Two genes with a putative function in GA biosynthesis (BdGA3ox1 and BdGA20ox1) are down-regulated in 35S:BdRAM1ox roots. C, Two B. distachyon genes orthologous to known brassinosteroid biosynthesis genes (BdCPD and BdD2/BdCYP91D) are induced in 35S:BdRAM1ox roots. A third gene, BdDWF4, is not affected. Gene expression was measured by RT-qPCR. Bar graphs show the mean, with error bars representing sd. Single points represent individual measurements. Pairwise comparisons were estimated using the Student’s t test (***P < 0.001, **P < 0.01, and *P < 0.05). n.s., Not significant.
Additional Files
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-7 and Supplemental Tables 1 and 2
