Article Figures & Data
Figures
- Figure 1.
Cytokinin Signaling Genes Are Transcriptionally Induced in Response to Short-Term Cytokinin Treatments.
Real-time RT-PCR analysis of Mt HK (A) and A- and B-type Mt RR (B) induction in response to the cytokinin BAP at different concentrations (10−8, 10−7, or 10−6 M) and various incubation times (15 min, 30 min, 1 h, 3 h, or 6 h). Histograms represent the quantification of specific PCR amplification products for each gene normalized with the constitutive control Mt ACTIN11. The value of untreated roots (t = 0) is set to 1 as reference. A representative example out of three biological experiments is shown, and error bars indicate standard deviation for three technical replicates. Arrows indicate significant induction of specific genes mentioned in the text.
- Figure 2.
Mt CRE1 RNAi Roots Are Cytokinin Insensitive.
(A) to (D) Representative examples of root growth observed in A. rhizogenes–transformed M. truncatula roots expressing GUS RNAi, Mt CRE1 RNAi, Mt HK2 RNAi, or Mt HK3 RNAi constructs 6 d after transfer to 10−7 M BAP. Black lines indicate the position of root tips at the moment of transfer. Cytokinin insensitivity is visualized by the root's ability to grow on this inhibitory concentration of cytokinin. The transgenic roots were selected on kanamycin as described by Boisson-Dernier et al. (2001).
(E) Mean length of independent transgenic roots transformed with these different constructs and grown 6 d on media with or without 10−7 M BAP. A representative example out of seven biological experiments is shown (n > 30 per construct and condition), and error bars represent standard deviation (ANOVA, P < 0.001)
(F) Expression analysis of Mt CRE1 and a cytokinin-inducible A-type response regulator, Mt RR4. Real-time RT-PCR analysis was performed on A. rhizogenes–transformed M. truncatula roots carrying either a control (GUS RNAi) or Mt HKs RNAi constructs (n > 15) after a short-term cytokinin treatment (10−7 M BAP for 1 h). Histogram represents the quantification of specific PCR amplification products normalized to the constitutive control Mt ACTIN11. The value of control transgenic roots (i.e., non-BAP-treated GUS RNAi root) is set at 1. A representative example out of three biological experiments is shown, and error bars represent standard deviation for three technical replicates.
(G) Expression analysis of Mt CRE1, Mt HK2, and Mt HK3. Real-time RT-PCR analysis was performed on independent A. rhizogenes–transformed M. truncatula roots carrying either a control (GUS RNAi) or Mt HK RNAi constructs and grown 7 d on 10−7 M BAP. Histograms represent the quantification of each specific PCR amplification product normalized to the constitutive control Mt ACTIN11. The value of control transgenic roots (i.e., GUS RNAi) is set up at 1 as reference. A representative example out of three biological experiments is shown, and error bars represent standard deviation for three technical replicates.
- Figure 3.
Mt CRE1 RNAi Roots Are Affected in Lateral Root and Nodule Development.
(A) Representative examples of root growth observed in A. rhizogenes–transformed M. truncatula roots expressing GUS RNAi (left panel and close-up) and Mt CRE1 RNAi (right panel and close-up) constructs 6 d after transfer to LRIM. Black lines indicate the position of root tips at the moment of transfer. Lateral root density is calculated in A. rhizogenes–transformed M. truncatula roots in the newly grown root region 6 d after transfer to LRIM. Histograms represent lateral root densities in one (n > 25) out of three biological experiments for GUS RNAi, Mt CRE1 RNAi, Mt HK2 RNAi, and Mt HK3 RNAi roots. Error bars indicate standard deviations (ANOVA, P < 0.001).
(B) One-month-old transgenic roots expressing GUS RNAi or Mt CRE1 RNAi constructs were S. meliloti–inoculated in a greenhouse. General view of GUS RNAi and Mt CRE1 RNAi composite plants (left panel) and close-up of S. meliloti–inoculated GUS RNAi (close-up, left panel) and Mt CRE1 RNAi (close-up, right panel) roots. Arrowheads indicate nodule locations. Quantification of nodules was done on GUS RNAi, Mt CRE1 RNAi, Mt HK2 RNAi, and Mt HK3 RNAi roots 15 DAI with S. meliloti 2011 strain. Histogram represents nodule numbers per transgenic root in one out of five biological experiments (n > 25). Error bars represent standard deviations (ANOVA, P < 0.001).
- Figure 4.
Early Nodulation Is Perturbed in Mt CRE1 RNAi Roots.
(A) to (C) Histochemical staining of an S. meliloti strain expressing a ProHemA:LACZ reporter construct to follow early events of nodulation. Representative infection threads observed in an S. meliloti–inoculated GUS RNAi root (A) and nodule-deficient Mt CRE1 RNAi roots ([B] and [C]) are shown. Arrowheads indicate cortical cell divisions. Note the lack of cortical cell divisions in Mt CRE1 RNAi roots. Bars = 25 μm.
(D) Real-time RT-PCR analysis of Mt CRE1 and Mt HK2 expression in representative independent transgenic roots expressing GUS RNAi, Mt CRE1 RNAi, or Mt HK2 RNAi constructs 15 DAI by S. meliloti. Histograms represent the quantification of each specific PCR amplification product normalized to the constitutive control Mt ACTIN11. The value of control transgenic roots (i.e., GUS RNAi) is set to 1 as reference. Error bars represent standard deviation for three technical replicates.
(E) Expression analysis of the early nodulation markers Mt ENOD11, Mt ENOD12, and Mt NIN by semiquantitative RT-PCR in independent transgenic roots expressing GUS RNAi, Mt CRE1 RNAi, or Mt HK2 RNAi constructs 15 DAI by S. meliloti. Histogram represents the quantification of each specific PCR amplification product normalized to the constitutive control Mt ACTIN11.
- Figure 5.
Differential Expression of Cytokinin-Related Response Regulator Genes during Nodule Formation.
Real-time RT-PCR analysis of roots or nodules at different days after S. meliloti infection (dpi). Expression analysis of A-type (A) and B-type (B) response regulator genes. Histograms represent the quantification of specific PCR amplification products normalized to the constitutive control Mt EF1α. The value for uninfected control roots is set to 1 as reference. A representative example out of three biological experiments is shown, and error bars represent standard deviation for three technical replicates. Arrows indicate significant induction of specific genes mentioned in the text.
- Figure 6.
Crosstalk between Cytokinin and Nod Factor Signaling Pathways.
(A) Real-time RT-PCR analysis of several nodulation and cytokinin early response genes in wild-type and Nod factor signaling pathway mutant (dmi1-Y6, dmi2-TR25, dmi3-TRV25, and nsp2-1) roots 1 DAI with S. meliloti nod+ or nod− strain.
(B) Real-time RT-PCR analysis of Mt NIN and Mt NSP2 in response to short-term cytokinin treatments: BAP at different concentrations (10−8 or 10−7 M) and various incubations times (15 min, 30 min, 1 h, 3 h, or 6 h).
(C) Real-time RT-PCR analysis of Mt NIN expression in response to short-term cytokinin treatments (BAP 10−7 M for 1 h) in GUS RNAi, Mt CRE1 RNAi, Mt HK2 RNAi, or Mt HK3 RNAi A. rhizogenes–transformed M. truncatula roots (n > 15).
In all cases, the histogram represents the quantification of specific PCR amplification products for each gene normalized with the constitutive control Mt ACTIN11. The value of wild-type, mutant, or transgenic nontreated roots (S. meliloti nod− strain or no BAP [e.g., t = 0]) is set to 1 as reference. A representative example out of three biological experiments is shown, and error bars represent standard deviation for three technical replicates. Arrows indicate significant induction of specific genes mentioned in the text.
Tables
- Table 1.
Cellular Localization of Infection Thread Ends in GUS RNAi and Mt CRE1 RNAi Roots to Monitor Infection Progression
GUS RNAi Mt CRE1 RNAi Root hairs 2 13 Root epidermis 5 7 Outer cortex 6 0 Inner cortex 7 0 Nodule primordia formed 10 0 Total number of infections scored 30 20
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure 1 - Unrooted relationship trees of A. thaliana and M. truncatula cytokinin signaling genes.
- Supplemental Figure 2 - Expression analysis of cytokinin signaling genes in wild-type versus A. rhizogenes-transformed GUS RNAi roots.
- Supplemental Figure 3 - Effect of ethylene on Mt CRE1 RNAi root growth.
- Supplemental Figure 4 - Induction of lateral roots in M. truncatula seedlings.
- Supplemental Figure 5 - Structure of nodules formed on A. rhizogenes-transformed GUS RNAi or Mt CRE1 RNAi roots.
- Supplemental Figure 6 - Real-time RT-PCR analysis of nodulation and/or cytokinin early response genes in response to short-term cytokinin treatment.
- Supplemental Figure 7 - Nodule number in Mt RR1 RNAi and control roots.
- Supplemental Table 1 - List of Mt HKs, Mt HPs and Mt RRs accession numbers.
- Supplemental Table 2 - List of primers used in real-time and semi-quantitative RT-PCR experiments.
