AP2 transcription factor CBX1 with a specific function in symbiotic exchange of nutrients in mycorrhizal Lotus japonicus

Abstract

The arbuscular mycorrhizal (AM) symbiosis, a widespread mutualistic association between land plants and fungi, depends on reciprocal exchange of phosphorus driven by proton-coupled phosphate uptake into host plants and carbon supplied to AM fungi by host-dependent sugar and lipid biosynthesis. The molecular mechanisms and cis-regulatory modules underlying the control of phosphate uptake and de novo fatty acid synthesis in AM symbiosis are poorly understood. Here, we show that the AP2 family transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), a WRINKLED1 (WRI1) homolog, directly binds the evolutionary conserved CTTC motif that is enriched in mycorrhiza-regulated genes and activates Lotus japonicus phosphate transporter 4 (LjPT4) in vivo and in vitro. Moreover, the mycorrhiza-inducible gene encoding H⁺-ATPase (LjHA1), implicated in energizing nutrient uptake at the symbiotic interface across the periarbuscular membrane, is coregulated with LjPT4 by CBX1. Accordingly, CBX1-defective mutants show reduced mycorrhizal colonization. Furthermore, genome-wide-binding profiles, DNA-binding studies, and heterologous expression reveal additional binding of CBX1 to AW box, the consensus DNA-binding motif for WRI1, that is enriched in promoters of glycolysis and fatty acid biosynthesis genes. We show that CBX1 activates expression of lipid metabolic genes including glycerol-3-phosphate acyltransferase RAM2 implicated in acylglycerol biosynthesis. Our finding defines the role of CBX1 as a regulator of host genes involved in phosphate uptake and lipid synthesis through binding to the CTTC/AW molecular module, and supports a model underlying bidirectional exchange of phosphorus and carbon, a fundamental trait in the mutualistic AM symbiosis.

Keywords: AP2 transcription factor; CTTC cis-regulatory element; fatty acid biosynthesis; mycorrhizal symbiosis; phosphate transport.

Fig. 1.

Sequence-specific DNA-binding properties of CBX1. (A) CTTC is required for LjPT4 gene regulation in mycorrhizal roots. The schematic diagram shows pLjPT4:GUS and pLjPT4-mCTTC:GUS with mutations (Upper). Lower shows GUS activity in transgenic hairy roots harboring different reporters in the presence of R. irregularis. EV, pRedRoot-GUS vector; 4*CTTC, a quadruple tandem repeat of CTTCTTGTTC fused to minimal 35S cauliflower mosaic virus promoter; 4-MU, 4-methylumbelliferone. Mean ± SD (n = 3). Kruskal–Wallis test followed by Fisher’s least significant difference test was used [Kruskal–Wallis χ² = 9.7, degree free (df) = 3, P < 0.05]. Three independent experiments were performed with similar results. (B) EMSA of CBX1 binding to CTTC motif. Unlabeled CTTC CRE or mCTTC CRE were used as competitor. Increasing amounts of competitor DNA is indicated on top. Red arrow indicates the protein–DNA complex. (C) The sequence logo of CTTC CRE was created from 21 putative CTTC motifs in promoters of 19 mycorrhiza-inducible Pi transporter genes shown in SI Appendix, Table S2 using WebLogo (weblogo.berkeley.edu/logo.cgi). Stack height represents the degree of conservation and the letter size represents relative frequency. (D) CBX1 DNA-binding preference for the CTTC motif in EMSA. Nine Cy5-labeled oligonucleotides carrying single base-pair substitutions were synthesized. WT, wild type CTTC motif; red letters, base changes within CTTC; black letters, wild-type bases. (E) Schematic diagram of truncated CBX1 proteins. AP2, APETALA2 domain; NLS, nuclear localization signal. Protein regions are labeled at left. (F) Relative binding affinities of truncated CBX1on CTTC motif (w) or mutated CTTC (m) in EMSA.

Fig. 2.

CBX1 is required for mycorrhizal colonization. (A) Mycorrhization rate in Gifu-129, cbx1-2, and cbx1-3 mutant lines grown under low Pi (100 µM) in the presence of R. irregularis. A, arbuscules; H, hyphae; V, vesicles; A + H (%), percentage of root sectors with arbuscules and hyphae; A + V + H (%), percentage of root sectors with arbuscules, vesicles, and hyphae; H (%), percentage of root sectors with only hyphae; V + H (%), percentage of root sectors with vesicles and hyphae. Mean ± SD (n = 3) is shown. One-way ANOVA followed by Tukey’s honestly significant difference (HSD) test was used [F (A + H)_2.6 = 9.261; F (V + H)_2.6 = 9.874; F (A + V + H)_2.6 = 70.54; F (Total)_2.6 = 73.2; P < 0.05]. Different letters indicate different statistical groups. n.s., not significant. (B) Mycorrhizal gene expression in cbx1 allelic mutants in the absence (−) or presence (+) of R. irregularis (n = 3). One-way ANOVA followed by Tukey’s HSD test [F (CBX1)_5.12 = 30.73; F (LjHA1)_5.12 = 37.54; P < 0.05] and the nonparametric equivalent Kruskal–Wallis test [χ² (RAM2) = 15.082; χ² (LjPT4) = 14.342; df = 5, P < 0.05] were used to determine the significance. This experiment was independently repeated three times with similar results. (C and E) pCBX1:GUS activation in L. japonicus roots in the presence of R. irregularis. (D and F) WGA488 staining of AM fungal structures in the same sectors. (Scale bars: C and D, 500 μm; E and F, 200 μm.)

Fig. 3.

CBX1 regulates mycorrhizal marker genes across different dicot species. (A) Diagram of reporter and effector utilized in the transactivation assay. DsRed was used to test transformation efficiency. p35S:GFP, negative control. (B) Transactivation assay with AP2 transcription factors on four chimeric reporter genes. GUS staining of suspension cultured cells is shown at the top of the graph. Mean ± SD (n = 3). One-way ANOVA followed by Tukey’s HSD was performed (F_27.56 = 44.38, P < 0.001). (C) Overexpression of CBX1 increased expression of LjPT4, LjHA1, and RAM2 genes in L. japonicus in the absence of AM fungi. Box limits indicate the 25th and 75th percentiles. Bar-plot whiskers extend to the value that is no more than 1.5× interquartile range from the upper or lower quartile. Outliers were plotted by dots. Student’s t test was used (n = 7). (D) Ectopic expression of CBX1 in hairy roots of potato led to transcript accumulation of mycorrhiza-induced Pi transporters and H⁺-ATPase genes. Student’s t test was used (n = 3). (E) Induction of MUP-related genes in ectopic expression of CBX1 in tobacco leaves. Student’s t test was used (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001. Three independent experiments were performed with similar results. (F) Transactivation by CBX1 of Pi transporter genes from different plant species, and of L. japonicus LjHA1 and RAM2 in A. thaliana suspension cultured cells. Mean values ± SD of GUS activity from three biological replicates are shown (n = 3; Student’s t test; *P < 0.05; ***P < 0.001). This experiment was repeated three times independently with similar results.

Fig. 4.

Genome-wide identification of CBX1 target genes by ChIP-seq. (A) ChIP-seq analysis shows CBX1-binding peaks enriched near the transcription start site (TSS). The peaks shared in two replicate experiments were used. Immunoprecipitated DNA fragments from 1-mo-old hairy roots harboring pUB:CBX1-YFP or pUB:CFP negative control were subjected to DNA sequencing. (B) Distribution of 136 CBX1-binding sites in the L. japonicus genome. (C) Venn diagram depicting the overlap between CBX1 targets from ChIP-seq and mycorrhiza-regulated genes. Unique genes (392 and 226) were significantly enriched by CBX1 in two experiments (two times higher in surrounding 10-kb region; fold change relative to CFP control >2; P < 0.0001). rep, replicate. (D) IGV browser view of CBX1 binding on LjPT4 gene. Tracks display data from Input, ChIPed CFP, and ChIPed CBX1 (two replicates) samples. Number on the upper left of each track indicates track height (300 reads per bin). Peak identified in Homer is indicated in blue bar. Thick lines represent exons and thin lines introns in gene structure. Black arrow indicates TSS. Blue and red ticks under the gene structure indicate CTTC core sequence (TCTTGT) or AW-box (CnTnG(n)₇CG) on the positive and negative DNA strand, respectively. (E) Schematic representation of genomic regions of LjPT4 and LjPT3 at scale. Black bars represent coding region. Lines represent noncoding DNA. CTTC CRE and AW box are indicated in promoter regions as black or red bars, respectively. P1 to P4 are DNA fragments designed for ChIP-qPCR. (F) Validation of ChIP-seq by ChIP-qPCR that CBX1 bound to the promoter of LjPT4. After normalization with input, fold enrichment was calculated, compared with anti-GFP ChIPed CFP. Mean values ± SD of three independent biological replicates were shown. Student’s t test was used. *P < 0.05. (G) Mycorrhiza-inducible lipid-related genes were targeted by CBX1 in ChIP-seq. Heatmap of CBX1 target gene expression profiles based on log₁₀ transformed counts per million (cpm) depicted from RNA-seq data analysis (50). The number of AW box and CTTC core (TCTTGT) were counted in the homer peaks from ChIP-seq. (H) Transcript accumulation of CBX1 targets in L. japonicus hairy root overexpressing CBX1 in the absence of AM fungi. Student’s t test was used (n = 7). *P < 0.05; **P < 0.01; ***P < 0.001. Three experiments were performed independently with similar results. (I) Transactivation assay with CBX1 on pLjPT4:GUS reporter with mutations on CTTC or/and AW box. Mean ± SD (n = 3). One-way ANOVA followed by Tukey’s HSD was performed (F_15.32 = 14.17, P < 0.05).