Additional file 2 of Soil conditions and the plant microbiome boost the accumulation of monoterpenes in the fruit of Citrus reticulata ‘Chachi’

Additional file 2: Figure S1. Differentially expressed genes in leaf, peel and root samples between the two regions. (a) The numbers in the figure represent the number of differentially expressed genes. (b) FPKM of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) between regions. Statistical differences in peel, leaves, and roots between the two regions were evaluated by the Wilcoxon rank sum test. (c) Relative expression of DXS between regions was measured using qRT–PCR. Statistical differences in peel, leaves, and roots between the two regions were evaluated by the Wilcoxon rank sum test. Figure S2. Correlation network of transcript KOs and monoterpenes. The correlation-based network between highly expressed genes in the leaves (a) and peels (b) (nodes) and monoterpenes (triangles). Node size corresponds to the degree of each monoterpene. The thickness and colour of the edges denote the strength and significance, respectively. Solid and dashed edges indicate positive and negative correlations, respectively. Figure S3. The taxonomic composition of the rhizosphere soil microbiome at the phylum level. Only the microbial phyla with the top 10 relative abundances among bacteria (a) and archaeal phyla (b) are shown. Figure S4. Microbial composition of the root-associated microbiome and its relationship to soil chemical properties. (a) PCoA based on the genus abundance profile was performed to assess the influences of geographical location and microhabitat on microbial communities. (b) Pairwise comparisons of environmental factors are shown, with a colour gradient denoting Spearman’s correlation coefficient. Taxonomic (endophyte and metagenomes) and functional composition relationships with each environmental factor were detected by partial Mantel tests. Edge width corresponds to Mantel’s R statistic for the corresponding distance correlations, and edge colour denotes the statistical significance based on 9,999 permutations. Solid and dashed edges indicate positive and negative correlations, respectively. Figure S5. The correlation-based network between highly expressed genes (nodes) in the root and MAGs positively correlated with monoterpenes (triangles). Node size corresponds to the degree of each genus. The thickness and colour of the edges denote the strength and significance, respectively. Solid and dashed edges indicate positive and negative correlations, respectively. Figure S6. Information on secondary metabolite BGCs in newly reconstructed MAGs. Distribution of different biosynthetic gene cluster (BGC) compositions in phyla. Figure S7. The abundance of terpene BGCs in the microbial community shifted between the core region and non-core region. Figure S8. PCoA based on the endophyte OTU profile was performed to assess the influences of geographical location. Figure S9. The endophyte OTUs annotated to the genera enriched in the rhizosphere soil of the core region. The left boxplot represents the relative abundances of genera enriched in rhizosphere soil samples from the core region, and the right boxplot represents the relative abundances of endophyte OTUs in the core region. Figure S10. The genome of the endophyte strain Serra-11. (a) Circular representation of the genome of Serratia marcescens strain rb-red1. The inner circle shows the 103 contigs sorted by size. The middle circle shows GC skew. The outer circle shows the functional genes annotated to the terpene backbone synthesis pathway. (b) Synthesis process of monoterpenoid precursors. The blue circle represents the substrate or product, and the orange box represents enzymes. Figure S11. The expression of microbial biosynthetic gene clusters in citrus peel, leaf and root samples. (a) The number of microbial biosynthetic gene clusters found in citrus peel, leaf and root samples. (b) The expression of biosynthetic gene clusters in citrus peel, leaf and root samples. Figure S12. Microscopic observation of root endophytes. (a-f) Microstructure of root endophytic bacteria in C. reticulata ‘Chachi’; a, c and e show the overall view of the root in C. reticulata ‘Chachi’; b, d and f show the local enlargement of the root in C. reticulata ‘Chachi’. b is the local magnification in the black box in a. Gram-positive bacteria were distributed in the root cortical cells (black arrow). d shows local magnification of the black box area in c. Gram-positive bacteria were distributed in the intercellular space of root cortex cells (black arrows). f shows local magnification of the black box area in e. Gram-negative bacteria were distributed in the root cortical cells (black arrows). Bar = 50 μm for a, c and e. Bar = 25 μm for b, d and f. (g-i) Ultrastructure of root endophytic bacteria in C. reticulata ‘Chachi’ detected by TEM; (g) Endophytic bacteria in the intercellular space of the root cortex in C. reticulata ‘Chachi’. The white arrows indicate the endophytic bacteria. Bar = 1 μm. (h) Endophytic bacteria in the cells of the root cortex in C. reticulata ‘Chachi’. The white arrows indicate endophytic bacteria. Bar = 1 μm. (i-l) Endophytic bacteria in developing cells of the root vascular cylinder in C. reticulata ‘Chachi’. The white arrow shows the black osmiophilic droplets in the bacterial membrane and the intimal structure of the bacterium, and the black arrow shows the grey osmiophilic droplets in the cytoplasm of the bacterium. Bar = 500 nm. IS: intercellular space. (m, n). Ultrastructure of the root of aseptic seed culture in C. reticulata ‘Chachi’. No endophytic bacteria occurred in the intercellular space and cytoplasm of the root cortical cells (o) and root vascular cylinder cells (p) in C. reticulata ‘Chachi’. Bar = 2 μm. M: mitochondrion. G: Golgi apparatus; S: starch; N: nucleus; ER: endoplasmic reticulum. Figure S13. The Sudan black dye lipid droplet method shows the distribution of lipid droplets in the root and peel. (a-c) Few lipid droplets in the vascular cylinder and cortical cells of aseptic culture plants produced by seeds. (d-i) Abundant lipid droplets in the vascular cylinder and cortical cells of young roots (d-f) and more mature roots (g-i) collected from the core region. (j-l) Vascular bundles of the peels collected from the core region. j provides an overall view of the peel with two secretary cavities and a vascular bundle. k and l are longitudinal sections and cross-sections of the vascular bundle with lipid droplets, respectively. Black arrows indicate lipid droplets.