Fig 1.
MiR156 suppressed age-related resistance to Pto DC3000 in Arabidopsis.
A, developmental phenotype of a 4-week (left) and 7-week (right) Col-0 plant grown under short-day conditions. Arrows point to leaves 1,2,3,4 and leaf 8 in the left and right plants, respectively. B, Pto DC3000 bacterial growth in juvenile and adult leaves of Col-0. C, developmental phenotype of a 7-week Col-0 and 35S::MIR156A plant. Arrows indicate an adult leaf of Col-0 or a leaf from a similar position in 35S::MIR156A. D, bacterial growth in adult leaves of Col-0 and 35S::MIR156A. E, developmental phenotype of a 4-week Col-0 and 35S::MIM156 plant. Arrows indicate leaves 1 and 2 on each plant. F, Pto DC3000 bacterial growth in juvenile Col-0 and MIM156 leaves 1–2 on Day 0 and Day 2. Scale bar = 1 cm. Day 0, the day of Pto infection. Day 2, two days post-infection. CFU/cm2, bacterial colony forming unit per square centimeter of a leaf. Juv, juvenile leaves, Adu, adult leaves. The student t-test was used for statistical analysis. Each genotype was compared with Col-0, ns, not significant, *, p < 0.05, **, p < 0.01. The same annotation is used for bacterial growth dot-box plots shown in Figs 2 and 7. Repeats of bacterial growth are presented in S2 Table.
Fig 2.
MiR156-targeted SPL10 promoted resistance to Pto DC3000.
A, disease phenotype of Pto DC3000 in SPL gain and loss-of function mutants. NA, not available. B, bacterial growth in leaves 1–2 and leaves 11 from Col-0 and rSPL10. C, developmental phenotype of Col-0, and early phase change phenotypes of transgenic plants expressing stable MIM156, rSPL10 and rSPL3 on ½ MS plate (top panel); developmental phenotype of Col-0, estradiol-inducible(in)MIM156, inrSPL10 and inrSPL3 at 3 days after estradiol treatment (bottom panel). D, bacterial growth in leaves 1–2 from 4 -week-old Col-0, inMIM156, inrSPL10 and inrSPL3 on Day 0 and Day 3. Emmeans package in R was used for statistical analysis in 2 A and 2 B. The student t test was used for statistical analysis in 2 A and 2 C, and each genotype was compared with Col-0 wild type, ns, not significant, *, p < 0.05, **, p < 0.01. Repeats for the experiments here are shown in S2 Table.
Fig 3.
Basal transcript level of defense genes were heightened over vegetative phase change.
A, experimental settings of RNA-seq. Non-infected state and challenged (3 hours after Pto inoculation) transcriptomes from leaf 1–2 (Col-0 and rSPL10) and leaf 11 (Col-0) were compared. * indicates examples of leaf samples that were collected for RNA-seq. Green: juvenile leaves; brown: adult leaves. B, an expression profiling of DEGs identified in mock-treated adults against mock-treated juvenile leaves. DEGs, differently expressed genes with LFC ≥ ±0.58 and padj ≤ 0.05. LFC, log2 fold change of gene expression. Padj, adjusted p value. Color-codes in the heatmap, blue is for down-regulated DEGs and red is for up-regulated DEGs. Euclidean distance was used for calculating distance. Complete linkage was applied to structure the hierarchical clustering. Expressing profiles of DEGs derived from Adu-M/Juv-M are mapped in the first column of the map. The expressing profiles of those DEGs in Adu-P/Juv-P are shown in the second column of the map. Genes that were DEGs in both Adu-M/Juv-M and Adu-P/Juv-P are marked by the black bar on the right. 38% indicates the percentage of those overlapping DEGs in Adupto. C, a profile of Pto-triggered DEGs came from Juv-P/Juv-M and Adu-P/Adu-M. Adult-specifically Pto-triggered DEGs (dark red), Juvenile-specifically triggered (light brown), adult preferentially triggered (deep blue), and commonly triggered in both adults and juveniles, i.e., shared (light grey) are marked by the first column of bars on the right. The percentages (20.6%, 3.4%, 5.3%, 12.6%) and corresponding black bars indicate the proportion of each DEG category that overlaps with Adupto. D, representative GO terms enriched in DEGs of Adunof, DEGs triggered by Pto DC3000 in both adult and juvenile leaves (the Shared) and DEGs specifically triggered by Pto in adult phase within the total Adupto (20.6%). Red and blue color blocks refer to GOs enriched in up- and down-regulated DEGs, respectively. Only GO terms with FDR < 0.05 were deemed as enriched here. Fold enrichment was based on hypergeometric tests within the range of the DEG set used for each GO analysis relative to Arabidopsis genome. The analysis was done using the TAIR Gene ontology website (geneontology.org). The same GO analysis and color-coding are used for Fig 4C.
Fig 4.
rSPL10 transcriptomes resembled that of adult leaves.
A, an expression profiling of co-regulated DEGs by adult and rSPL10 leaves at non-infected state compared with juvenile leaves. EDS1 and PAD4 were identified as Adu/r10 co-upregulated DEGs under non-infected state, which are indicated in the heatmap with arrows. B, expressing profiles of co-regulated DEGs by adult and rSPL10 from Adu-P or r10-P against Juv-P. For the clustered heatmaps in 4 A and B, blue represents down-regulated DEGs and red is for up-regulated DEGs. Euclidean distance was used for calculating distance in the partition around medoids (PAM) clustering (k = 4). C, GO enrichment of co-regulated adult and r10 vs juvenile DEGs in non-infected and Pto-infection states.
Fig 5.
Salicylic acid (SA) biosynthesis and signaling were enhanced by SPL10 in adult phase.
A, 672 up-regulated core SA markers were defined via overlapping DEGs pools from Ding Y et al., 2018 (dark circle) [44] and Yang L et al., 2017 (light circle) [43]. B, expression patterns of 527 detected (out of 672) up-regulated core SA markers in adult and r10 leaves, under non-infected and Pto infection states. Four random pools of detected genes (450 genes per set) derived from each pair-wise comparison exhibits here were chosen as negative controls. Up, upregulated. No, no change. Down, downregulated. Y axis shows the negative logarithm transformed adjusted p value, -log10(padj). X axis displays the value of Log2 fold change (LFC) of gene expressions. M, mock. P, Pto DC3000. P < 0.0001 (Hypergeometric test, done by GeneOverlap R package) was reproducibly output from each overlap between core upregulated SA markers and Adunof, Adupto, r10nof and r10pto (S1 Information). The P values for 3 out of 4 random controls were not significant (S1 Information). C, endogenous SAG and free SA accumulation in juvenile, r10 and adult leaves at non-infected state. DW, dry weight. Repeats for the experiment are shown in S2 Table. D, age-associated expression of four SA markers in adult leaves from Col-0 and in comparable leaves of spl2/10/11. Similar results were seen two times. E, the qPCR of the four SA marker genes in juvenile, r10 and adult leaves on sterile 1/2 MS plates. Similar results were seen three times. F, overrepresentation of EDS1-PAD4 core regulon (EP core) in Adu/r10nof and Adu/r10pto. The EP core markers were defined in Cui et al., 2017 [45]. 127/155 of the EP core markers were detected in this work. Randomly selected 127 genes from our RNA-seq dataset were used as controls.
Fig 6.
PAD4 was a direct target of SPL10.
A, overlap between Adu/r10nof co-regulated genes and potential SPL10 targets defined according to ChIP-seq data from Ye et al. (upper panel) [24]. A motif discovery and enrichment analysis of the 203 Adu/r10nof co-upregulated DEGs (lower panel). B, SPL10 bound to a GTAC-containing motif upstream of PAD4. qPCR following chromatin immunoprecipitation of rSPL10-YFP for motifs (M1 and M2) and negative control sites (nc1 and nc2), the latter of which are at least 600 bp away from M1 and M2 at the PAD4 genomic region. Three primer sets (p1-p3) were used to amplify the M1 site. Relative locations of ChIP peaks (dark grey) derived from Ye et al, primers (arrow pairs) and the tested sites (color blocks) were indicated in the schematic diagram. The student t test was performed to compare and indicate the significance of difference between the sites. C, qPCR of PAD4 transcripts level in juvenile (Juv) and adult (Adu) leaves of Col-0 and spl2/10/11. The student t test, ns, not significant, *, p < 0.05, **, p < 0.01.
Fig 7.
SA biosynthesis and signaling components were required for the SPL10 regulated ARRVPC.
A, a comparison of Pto DC3000 growth between the juvenile and adult leaves (ARR phenotype) of Col-0, sid2-1 and npr1-1. B, developmental phenotype (top) and the bacterial growth (bottom) in 4-week-old leaves 1 and 2 from Col-0, sid2-1, MIM156 and MIM156/sid2-1. C, developmental phenotype of 4-week-old Col-0, eds1.2, r10 and r10 /eds1.2. Note the similar leaf shape between r10 and r10/eds1.2; (bottom panel) bacterial multiplication in leaves 1 and 2 derived from Col-0, eds1.2, r10 and rSPL10/eds1.2. Arrows indicate leaves 1–2. D, the ARR phenotyping of eds1.2 and pad4-1 infected with Pto DC3000. Emmeans package in R was used for statistical analysis in 7 A and D. The student t test was used for statistical analysis in 7 B-C, and each genotype was compared with Col-0 wild type, ns, not significant, *, p < 0.05, **, p < 0.01.