Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice

Abstract

Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

Figure 1.

Myc-LCO-Induced Calcium Responses in M. truncatula. (A) Images showing a single calcium transient measured using the Yellow Cameleon calcium sensor in a trichoblast of M. truncatula in close proximity to R. irregularis (upper panels) and cells treated with S-LCO at 10⁻⁸ M (lower panels). Note that the calcium changes are restricted to the nuclear region. Bars = 50 μm. (B) Representative traces of M. truncatula trichoblasts responding to Nod factor (NF), R. irregularis, and Myc-LCOs: the LCO mix (M-LCOs), fungal purified (Pur) S-LCOs and NS-LCOs, and synthetic (Syn) S-LCOs and NS-LCOs. The y axis is the ratio of YFP to CFP in arbitrary units.

Figure 2.

Dose–Response Curves for Myc-LCOs and CO4 in Seedling Roots and Lateral Roots of M. truncatula. Quantification of M. truncatula calcium responses in seedling roots (A) and lateral roots (B) to S. meliloti Nod factor (blue), S-LCO (red), NS-LCO (green), and CO4 (purple). The y axis denotes the percentage of cells measured that show calcium oscillations relative to the concentration of the molecule denoted on the x axis.

Figure 3.

The Structure of LCO-Induced Calcium Oscillations in M. truncatula. (A) Bayesian spectrum analysis was used to generate binned probability distributions (PDF) of period length for 10 calcium traces from 10 M. truncatula plants, with each trace consisting of at least 2 h of imaging from each treatment: 10⁻¹⁰ M Nod factor (NF), 10⁻⁹ M S-LCO, 10⁻⁶ M NS-LCO, M-LCO (1:100 dilution), or 10⁻⁵ M CO4. Each sample size represents a group of periods, such that <50 = periods ranging from 1 to 50 s; <100 = periods ranging from 51 to 100 s, etc. (B) The bar plots show the mean duration for upward (red) and downward (orange) phases of spikes, with their associated standard deviations. A minimum of three traces was used for each treatment, representing ∼80 spikes. No significant difference was observed in the structure of the calcium oscillations induced by the different signals. (C) The box plots show the interspike intervals during the first 40 min of response. The whiskers mark the minimum and maximum values, and the box marks the lower and upper quartiles, so 50% of the values are inside the box. A thick black line is drawn at the median value. Each group consists of 10 traces that were detrended using the moving average method. The Mann-Whitney U-test detected a statistically significant difference (P < 0.05) in NS-LCO and CO4, compared with the other treatments; however, the NS-LCO responses were not significantly different when it was applied at 10⁻⁵ M NS-LCO.

Figure 4.

Specific Gene Induction in M. truncatula by the Different LCOs. Induction of NIN ([A] and [B]), ERN1 ([C] and [D]), MSBP1 ([E] and [F]), MNR ([G] and [H]), and Annexin1 ([I] and [J]) following 1-, 6-, and 24-h treatments with 10⁻¹⁰ M Nod factor (NF), 10⁻⁹ M S-LCO, 10⁻⁶ M NS-LCO, and 10⁻⁵ M CO4 in seedling roots. For (B), (D), (F), (H), and (J), 6 h of Nod factor treatment (black bars) and 24 h of NS-LCO treatment (gray bars) were used to assess gene induction in the mutants. These time points were selected based on the intensity of gene induction observed for NIN and ERN with NF and MSBP1, MNR, and Annexin1 with NS-LCO. Values represent averages from three biological replicates and error bars are se. Samples are grouped according to their significance as denoted by letters, calculated with a two-tailed t test (P < 0.05).

Figure 5.

Differential Induction of pENOD11-GUS in Seedling Roots versus Lateral Roots. Induction of pENOD11-GUS in M. truncatula by 10⁻⁷ M Nod factor, S-LCO, NS-LCO, and CO4 in seedling roots (2-d-old plants) and lateral roots. These images are representative roots. The experiment was repeated three times with similar outcomes. Bars = 200 μm.

Figure 6.

Calcium Responses in L. japonicus to the Myc-LCOs and CO4. Representative calcium traces from L. japonicus atrichoblasts on lateral roots treated with either 10⁻⁵ M or 10⁻⁸ M S-LCO, NS-LCO, and CO4. The number of cells showing calcium responses, relative to the total number of cells analyzed, is indicated.

Figure 7.

Calcium Responses in Rice to the Myc-LCOs, Nod Factors, and CO4. Representative calcium traces from rice atrichoblasts on lateral roots treated with 10⁻⁵ M Myc-LCOs and LCO isolations from NGR234 and R. tropici, as well as 10⁻⁵ M and 10⁻⁸ M treatments of CO4. The number of cells showing calcium responses, relative to the total number of cells analyzed, is indicated.

Figure 8.

Calcium Responses in Trichoblasts and Atrichoblasts. The percentage of calcium-responsive cells among trichoblasts (gray) and atrichoblasts (black) of M. truncatula, L. japonicus, and rice. For M. truncatula treatments of 10⁻¹¹ M and 10⁻⁹ M S. meliloti Nod factor and 10⁻⁸ M and 10⁻⁵ M NS-LCO were analyzed, for L. japonicus 10⁻⁹ M and 10⁻¹¹ M M. loti Nod factor, and 10⁻⁸ M and 10⁻⁵ M NS-LCO were analyzed, while for rice 10⁻⁸ M and 10⁻⁵ M CO4 was analyzed. For all plants, the response of trichoblasts in close proximity to R. irregularis hyphae was analyzed. Where indicated in the figure, the R. irregularis treatments were performed alone, without any additional signaling molecules added. Unfortunately, due to technical difficulties, it is not possible to assess atrichoblasts in close proximity to R. irregularis hyphae; therefore, responses in these cells were not determined (ND). The asterisks show significant differences (P < 0.05), measured using a χ² test, between the data points indicated.

Figure 9.

Rice Trichoblasts Respond to Mixes of Myc-LCOs and CO4. Representative calcium traces of rice trichoblasts treated with mixes of 10⁻⁵ M CO4, S-LCO, and NS-LCO. Note that mixes of Myc-LCOs with CO4 induced calcium oscillations, but the Myc-LCOs alone do not. The number of cells showing calcium responses, relative to the total number of cells analyzed, is indicated.

Figure 10.

Myc-LCOs and CO4 Promote Lateral Root Emergence in Rice. The affect of 10⁻⁸ M CO4, S-LCO, and NS-LCO treatments on the mean number of lateral roots per rice plant (A) and the mean root system length (B). These results are based on two independent experiments. Values are means ± se. The P value was calculated using a t test, assuming a normal distribution of the data, or a Wilcoxon signed-rank test when a normal distribution was not observed. The asterisk indicates significant difference relative to the control (P < 0.05).