A dual regulatory role for the arbuscular mycorrhizal master regulator RAM1 in tomato

Abstract The REQUIRED FOR ARBUSCULAR MYCORRHIZATION1 (RAM1) transcription factor from the GRAS family is well known for its role as a master regulator of the arbuscular mycorrhizal (AM) symbiosis in dicotyledonous and monocotyledonous species, being essential in transcriptional reprogramming for the development and functionality of the arbuscules. In tomato, SlGRAS27 is the putative orthologue of RAM1 (here named SlRAM1), but has not yet been characterized. A reduced colonization of the root and impaired arbuscule formation were observed in SlRAM1-silenced plants, confirming the functional conservation of the RAM1 orthologue in tomato. However, unexpectedly, SlRAM1-overexpressing (UBIL:SlRAM1) plants also showed decreased mycorrhizal colonization. Analysis of non-mycorrhizal UBIL:SlRAM1 roots revealed an overall regulation of AM-related genes and a reduction of strigolactone biosynthesis. Moreover, external application of the strigolactone analogue GR244DO almost completely reversed the negative effects of SlRAM1 overexpression on the frequency of mycorrhization. However, it only partially recovered the pattern of arbuscule distribution observed in control plants. Our results strongly suggest that SlRAM1 has a dual regulatory role during mycorrhization and, in addition to its recognized action as a positive regulator of arbuscule development, it is also involved in different mechanisms for the negative regulation of mycorrhization, including the repression of strigolactone biosynthesis.

The following Supplementary data is available for this article:  S1.Primers used in this study for quantitative reverse transcription polymerase chain reaction (RT-qPCR) experiments.Table S2.MRM conditions for endogenous strigolactones and GR24.Table S3.Number of mapped reads, high quality reads and splices reads for libraries from each sample in the RNA-seq analysis.Table S4.List of DEGs genes.List of DEGs generated by RNA-seq found to be differentially expressed upon UBUL:SlRAM1 expression in non-inoculated roots and by AM inoculation (Displayed as a separate excel file).Table S5.Gene ontology and protein class analyses on UBIL:SlRAM1 induced and repressed genes.

Target sequence for cloning
Primer name Primer sequence (5´à3´) Reference

Table S5 Gene ontology and protein class analyses on UBIL:SlRAM1 induced and repressed genes.
Selection of enriched slim GO terms and protein class annotations obtained by submitting to the PANTHER database the 1232 genes significantly upregulated (Fold change >2; P<0.05) and the 2129 genes significantly repressed (Fold change <-2; P<0.05) by UBIL:SlRAM1 in our RNA-seq analyses.Its overrepresentation in the AM-induced or AM-repressed genes datasets (Fold change >2 or <-2; P<0.05; NCBI Bioproject PRJNA509606) is also indicated.

Fig. S1 .
Fig. S1.Mycorrhizal colonization in UBIL:SlRAM1 roots inoculated with Funneliformis mosseae.Fig. S2.Validation of RNAseq data analysis by RT-qPCR.Fig. S3.Differentially regulated genes by UBIL:SlRAM1.Fig. S4.SlRAM2 gene expression in UBIL:SlRAM1 hairy roots of composite plants.Fig. S5.Expression analysis of SlRAM1 and SlTPSI1 upon UBIL:SlRAM1 and P-starvation conditions.TableS1.Primers used in this study for quantitative reverse transcription polymerase chain reaction (RT-qPCR) experiments.TableS2.MRM conditions for endogenous strigolactones and GR24.TableS3.Number of mapped reads, high quality reads and splices reads for libraries from each sample in the RNA-seq analysis.TableS4.List of DEGs genes.List of DEGs generated by RNA-seq found to be differentially expressed upon UBUL:SlRAM1 expression in non-inoculated roots and by AM inoculation (Displayed as a separate excel file).TableS5.Gene ontology and protein class analyses on UBIL:SlRAM1 induced and repressed genes.

Fig. S1
Fig. S1 Mycorrhizal colonization in UBIL:SlRAM1 roots inoculated with Funneliformis mosseae.The percentage of total root length colonized was measured in the hairy root systems of UBIL:SlRAM1 composite plants and control plants transformed with the empty vector at 68 days after inoculation with the AM fungus F. mosseae.Values correspond to mean ± SE (n=6).Significant difference (Student´s t-test) is indicated with an asterisk (*P<0.05).

Fig. S2
Fig. S2 Validation of RNAseq data analysis by RT-qPCR.(a) Expression level of GRAS38, DLK2, MAX1 and GH3.4 genes in the RNA-seq analysis (n=3).(b) Expression level of the same genes analysed by RT-qPCR using five independent biological replicates from the same experiment.Fold change in gene expression was represented with respect to the control roots transformed with the empty vector (EV).Significant differences (Student´s t test) are indicated with asterisks (*P ≤0.05, **P ≤ 0.01).

Fig. S3
Fig. S3 Differentially regulated genes by RAM1.Diagrams showing the number of genes differentially regulated (DEGs) in response to AM and UBIL:SlRAM1 expression among the present study and the previous published data from Zeng et al.The number of DEGs upregulated or downregulated by UBIL:SlRAM1 expression (RAM1-up or RAM1-down, respectively) or by AM inoculation (AM-up or AMdown, respectively) is indicated.

Fig. S4
Fig. S4 SlRAM2 gene expression in UBIL:SlRAM1 hairy roots of composite plants.Expression level of SlRAM2 gene was analysed by RT-qPCR with five independent biological replicates from the same experiment used for the RNA-seq.Fold change in gene expression was represented with respect to the control roots transformed with the empty vector (EV).Significant differences (Student´s t test) are indicated with asterisks (**P < 0.01).

Fig. S5
Fig. S5 Expression analysis of SlRAM1 and SlTPSI1 upon UBIL:SlRAM1 expression and P-starvation conditions.The effect of SlRAM1 overexpression (UBIL:SlRAM1) and phosphate deficiency (-P) on transcript levels of SlRAM1 (a) and SlTPSI1 (b), the tomato homolog to IPS1 (Liu et al., 1997) was analysed by RT-qPCR with seven independent biological replicates.Fold change in gene expression was represented with respect to the control roots transformed with the empty vector (EV) under normal phosphate conditions (+P).Conditions with similar letters are not significantly different (P>0.05) according to Tukey's multiple comparisons test.

Table S4 List of DEGs genes.
List of DEGs generated by RNA-seq found to be differentially expressed upon UBUL:SlRAM1 expression in non-inoculated roots and by AM inoculation (Displayed as a separate excel file).