A DELLA protein complex controls the arbuscular mycorrhizal symbiosis in plants

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Plants establish beneficial symbiotic associations with arbuscular mycorrhizal fungi, which colonize the root cortex, building specialized structures called arbuscules that facilitate nutrient exchange. The association occurs following plant recognition of lipochitooligosaccharides (LCOs) from mycorrhizal fungi, which activates the symbiosis signaling pathway prior to mycorrhizal colonization. Here we show that SLR1/DELLA, a repressor of gibberellic acid (GA) signaling, and its interacting partner protein are required for the mycorrhizal symbiosis. GA treatment inhibits mycorrhizal colonization and leads to the degradation of DELLAs. Consistently, rice lines mutated in DELLA are unable to be colonized by mycorrhizal fungi. DELLAs are members of the GRAS family of transcription factors. We further show that rice DELLA interacts with a second GRAS protein, DIP1 (DELLA Interacting Protein 1). DIP1 is also required for mycorrhizal colonization and in turn interacts with a previously characterized mycorrhizal GRAS protein, RAM1, that has been shown to directly regulate mycorrhizal-associated gene expression. We conclude that a complex of GRAS proteins, including DELLAs, is necessary for regulation of mycorrhizal-associated gene expression and thus colonization.

It has previously been shown by whole genome transcriptomic analysis that GA biosynthetic genes are induced during mycorrhizal colonization in rice¹. Furthermore, GA has been shown to inhibit mycorrhizal colonization in Pisum sativum². To understand the role of GA in this regulation, we sought to assess the function of GA during mycorrhizal colonization. Oryzae sativa ssp. japonica cv. Zhonghua11 plants were infected with the arbuscular mycorrhizas (AM) fungus Rhizophagus irregularis and treated with 0.1 μM, 1 μM, 10 μM and 100 μM GA3. Consistent with previous studies, GA treatment greatly promoted the elongation of the shoot and root (Supplementary information, Figure S1). Interestingly, mycorrhizal colonization was severely impaired at 0.1 μM, 1 μM GA3, and was completely blocked at 10 μM and 100 μM GA3, suggesting that GA negatively regulates AM colonization (Supplementary information, Figure S2A). AM1, AM3, AM10, AM14, AM15, AM34 and PT11 were previously shown to be induced during mycorrhizal colonization of rice³. Consistent with the changes that we observed in mycorrhizal colonization, the expression of these AM-specific marker genes was repressed by GA treatment (Supplementary information, Figure S2B and S2C). To explore whether the effect of GA was limited to rice, we also tested the effect of GA treatment on the AM symbiosis in Medicago truncatula. Consistent with the results in rice, colonization levels were greatly reduced by 10 μM GA3 treatment in M. truncatula (Supplementary information, Figure S2E). These results suggest that GA negatively regulates the AM symbiosis in both monocotyledonous and dicotyledonous plants.

GA is perceived by GID1 and promotes the interaction between GID1 and SLR1 (the rice DELLA ortholog). GID1 interaction with SLR1 leads to polyubiquitination of SLR1 and its subsequent degradation⁴. The gid1 mutant of rice is insensitive to GA and as a result, SLR1 accumulates in the nucleus⁵. Both wild-type and gid1 mutant showed comparable levels of AM colonization; however, when treated with 10 μM GA, the AM colonization level in wild-type rice was greatly reduced, while colonization level in the gid1 mutant was similar to that of control plants (Supplementary information, Figure S2D). Thus we conclude that GA signaling negatively regulates mycorrhizal colonization and that this regulation may be associated with the degradation of SLR1, but not associated with the impact on the performance of the mycorrhizal fungi.

To assess whether DELLA proteins play a role during AM colonization, we analyzed the slr1 mutant of rice and SLR1-YFP overexpression line. Unlike Arabidopsis thaliana, rice has only a single DELLA protein, SLR1, making analysis of the role of DELLAs easier in this species. Mycorrhizal colonization was severely impaired in the slr1 mutant and was improved in SLR-YFP overexpression line, implying a direct role for this DELLA protein during AM colonization (Figure 1A, 1B and Supplementary information, Figure S3). This is surprising as DELLA proteins are generally considered to be negative regulators, yet during AM colonization SLR1 is essential. The fact that GA promotes SLR1 degradation (Supplementary information, Figure S2F) and that SLR1 is essential for AM colonization, explains why GA treatment negatively regulates AM colonization.

Figure 1.

DELLA protein complex is required for the mycorrhizal symbiosis. (A) Confocal images of R. irregularis colonization stained with wheat germ agglutinin (WGA)-conjugated Alexa Fluor 488 at 6 weeks post inoculation. Left, middle and right panels show normal arbuscule development in wild-type, no colonization in slr1 and occasional colonization events in slr1 mutant. Scale bars, 150 μm. (B) Internal root length colonization of slr1 mutants at 6 weeks post inoculation with R. irregularis. The experiment was repeated three times and representative data is shown. Error bars show standard errors. (C) Interactions between SLR1 and DIP1 in a Y2H assay. (D) Protein-protein interactions in N. benthamiana leaves revealed by BiFC. YFP fluorescence of leaves co-transformed with SLR1-YFPc (C-terminal half of YFP) or OsRAM1-YFPc and DIP1-YFPn (N-terminal half of YFP). No YFP fluorescence in OsRAM1-YFPn/YFPc, OsRAM1-YFPn/SLR1-YFPc, DIP-YFPn/YFPc and YFPn/SLR1-YFPc. (E) The MBP-DIP1 fusion protein interacts with His-SLR1 in pull-down assays using heterologously expressed proteins in E. coli. (F) A time course (10-40 DPI) of relative expression of DIP1 following R. irregularis inoculation. These experiments were replicated two times. Bars represent standard error. (G) Ink stained roots of rice reveal a greatly reduced level of infection in the DIP1 RNAi lines at 6 weeks post inoculation. Scale bars, 150 μm. (H) Quantification of R. irregularis colonization levels in 8 independent DIP1 rice RNAi lines at 6 weeks post inoculation (CK, empty vector control). (I) Interactions between DIP1, OsRAM1 and MtRAM1 are shown in a Y2H assay. (J) The MBP-DIP1 fusion protein can interact with His-OsRAM1 in pull-down assays with heterologously expressed proteins in E. coli. In (C, I): BD, binding domain; AD, activation domain. -LT, SD/-Leu-Trp; -LTHA, SD/-Leu-Trp-His-Ade. 30 mM 3-AT (3-aminotriazole) was used to suppress protein autoactivation.

The slr1 mutant showed greatly reduced AM colonization, with reductions in internal hyphae, arbuscules and vesicles (Figure 1A, 1B). However, hyphopodia (the infection structures on the root surface) formation appeared to be unaffected in slr1 (Figure 1B). Consistent with the defect in AM colonization, the induction of the AM-specific genes AM1, AM3, AM11, AM14, AM15, AM34 and PT11 was severely suppressed in slr1 (Supplementary information, Figure S4A and S4B). Interestingly, SLR1 was slightly induced at 30 and 40 days post inoculation (DPI) with R. irregularis (Supplementary information, Figure S4C), consistent with its role in the AM colonization.

To further investigate the function of SLR1 in the mycorrhizal signaling pathway, we searched for its interacting proteins using a yeast two-hybrid (Y2H) screen. We identified an interacting protein (LOC_Os12g06540) from this screen (Figure 1C), which interestingly encoded a GRAS-domain protein (Supplementary information, Figure S5). SLR1 itself is a member of the GRAS family of transcriptional regulators. We named this new GRAS protein, DELLA Interacting Protein 1 (DIP1). We further confirmed the SLR1-DIP1 interaction using bimolecular fluorescence complementation (BiFC) analysis in Nicotiana benthamina, which revealed an interaction between these two proteins in the nucleus (Figure 1D). We further validated this interaction using pull-down asssy with heterologously expressed proteins and detected a clear interaction (Figure 1E).

Detailed expression analysis showed that DIP1 was induced at 30 DPI with R. irregularis and was highly expressed at 40 DPI (Figure 1F). This expression pattern is suggestive of a role for DIP1 in the mycorrhizal association. Unfortunately, rice T-DNA insertion lines were not available in DIP1. Therefore, to assess whether DIP1 functioned during AM colonization, we generated transgenic rice lines expressing a DIP1 hairpin allowing RNA-mediated interference (RNAi) of DIP1. We observed that DIP1 expression levels were reduced in several RNAi lines and those lines with significant DIP1 reductions showed defects in AM colonization (Figure 1G, 1H). The expression level of DIP1 in the RNAi lines correlated well with the AM colonization levels (Figure 1H and Supplementary information, Figure S6), implying a role for DIP1 during AM colonization.

GRAS-domain proteins have already been shown to function during AM associations, with RAM1 playing a major role⁶, and NSP1/NSP2 playing minor roles^7,8. We found that OsRAM1 can complement Medicago ram1, indicating that OsRAM1 served the same function as MtRAM1 during mycorrhizal colonization (Supplementary information, Figure S7). Considering that we have identified two additional GRAS proteins functioning in the mycorrhizal association, we wished to ask whether these two proteins interacted with those previously defined mycorrhizal GRAS proteins. We found that DIP1, but not SLR1, interacted with OsRAM1 and MtRAM1 (Figure 1C, 1I). The interaction between DIP1 and OsRAM1 was validated by BiFC and the interaction occurred in the nucleus (Figure 1D). We further confirmed this interaction using pull-down assays (Figure 1J). Taken together, our results imply that a large complex of GRAS-domain proteins functions in the AM symbiosis with at least SLR1, DIP1 and RAM1 interacting together. As MtRAM1 has been shown to bind to the promoter of an AM-induced gene^6,9, we propose that this GRAS-domain protein complex is directly associated with mycorrhizal gene expression.

The mycorrhizal symbiosis is extremely ancient and was found in the earliest land plants, suggesting that the role of DELLAs in the AM symbiosis is probably very ancient. A role for SLR1 during the AM symbiosis provides a direct link between GA levels and AM colonization. The fact that SLR1 is necessary for AM colonization explains why GA treatment inhibits AM infection: GA-induced degradation of SLR1 will limit AM signaling. Linking AM signaling with a component of GA signaling should allow direct hormonal regulation of AM colonization. Thus, GA, acting as a general signal during plant development, can regulate the AM association, presumably in accordance with the developmental status of the plant. DELLAs act as a node for crosstalk during nutrient responses, abiotic stress, light perception and sigaling of several interacting hormones including auxin, ethylene, abscisic acid and brassinosteroids. The colonization by AM is tightly regulated by the host in response to its local environment and our results suggest that this is at least partly achieved by integrating GA signaling and AM signaling through DELLAs, which function directly in AM colonization.

Detailed methods are described in the Supplementary information, Data S1.

Supplementary Information

Supplementary information, Figure S1

GA3 promotes rice plant growth.

Supplementary information, Figure S2

Gibberellic acid inhibits mycorrhizal colonization.

Supplementary information, Figure S3

SLR1-YFP fusion protein functions in mycorrhizal colonization and plant development.

Supplementary information, Figure S4

Expression of AM specific marker genes in slr1 mutant and expression pattern of SLR1 during mycorrhizal colonisation.

Supplementary information, Figure S5

Phylogenetic tree revealed DIP1 is conserved in mycorrhizal colonised plants.

Supplementary information, Figure S6

Expression of DIP1 in DIP1 RNAi lines.

Supplementary information, Figure S7

OsRAM1 serves the same function as MtRAM1 in Medicago.

Supplementary information, Data S1

Materials and Methods