Fig 1.
SL-RNAi plants have more branches.
(A) Representative branching phenotypes of EV, two independent transgenic lines in which the target genes were individually silenced: max2#1 and max2#2, d14#1 and d14#2, ccd7#1 and ccd7#2, and kai2#1 and kai2#2 plants. Scale bar, 5 cm. (B) Plant height and primary branch numbers per plant of indicated plants (±SE, n = 12). Branches longer than 5 cm were counted. (C) Silencing efficiency of target genes. Relative transcript abundance of MAX2a and MAX2b in axillary buds of EV, max2#1, and max2#2 plants; D14 in axillary buds of EV, d14#1, and d14#2 plants; CCD7 in seedlings of EV, ccd7#1, and ccd7#2 plants; KAI2a and KAI2b in leaves of EV, kai2#1, and kai2#2 plants (±SE, n = 3–4) (***P < 0.001; two-tailed Student t test). Values for graphs (B) and (C) are listed in S1 Data. ccd, carotenoid cleavage dioxygenase; d14, dwarf 14; EV, empty vector; kai2, karrikin insensitive 2; max2, more axillary growth 2; n.s., not significant; RNAi, RNA interference; SL, strigolactone.
Fig 2.
Anthocyanin accumulations in stems of max2, d14, ccd7, and kai2 plants.
(A) Representative stem phenotypes of 75-day-old EV, max2, d14, ccd7, and kai2 plants. (B) Relative anthocyanin levels in epidermis of stems of indicated plants (±SE, n = 8). (C) Levels of JA, JA-Ile, and IAA in stems of indicated plants (±SE, n = 6) (*P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student t test). Values for graphs (B) and (C) are listed in S1 Data. ccd, carotenoid cleavage dioxygenase; d14, dwarf 14; EV, empty vector; IAA, indole-3-acetic acid; JA, jasmonate; JA-Ile, jasmonic acid-isoleucine; kai2, karrikin insensitive 2; max2, more axillary growth 2; n.s. not significant.
Fig 3.
max2, d14, and ccd7 plants are more susceptible to T. m. attack.
(A) Schematic diagram of a T. m.–attacked plant. Stem pith and RSJ part used in this study are marked. (B) Representative pictures of EV, max2, and aoc plants after 3 weeks of T. m. attack; red pigmentation occurred along stems after attack or in max2 plants regardless of attack as indicated by red arrows. (C) Biomass of T. m. larvae after attacking EV and aoc plants for 2 weeks (n = 8–15). (D) Representative pictures of T. m. larvae after attacking EV, max2#1, and max2#2 plants for 2 weeks and biomass of T. m. larvae after attacking EV, max2#1, and max2#2 plants for 2 weeks (n = 15). Kinetic analysis of T. m. larvae biomass accumulations after 1, 2, and 3 wpi (n = 10–18). Scale bar, 2 mm. (E) Biomass of T. m. larvae after attacking EV, d14#1, and d14#2 plants for 2 weeks (n = 16). (F) Representative pictures and biomass of T. m. larvae after attacking EV, ccd7#1, and ccd7#2 plants for 2 weeks (n = 20). (G) Biomass of T. m. larvae after attacking EV, kai2#1, and kai2#2 plants for 3 weeks (n = 20). (H) Relative transcript abundance of BRC1a and CCD8 in stem pith of EV plants without (Control) or with T. m. attack (T. m.) for 2 weeks (±SE, n = 4). Levels of (±)-2′-epi-orobanchol in roots with indicated treatments (±SE, n = 8). (I) Levels of JA and JA-Ile in piths of EV and max2 plants without or with T. m. attack for 3 weeks (±SE, n = 6–10). (J) Levels of JA, JA-Ile, and IAA in piths of EV, max2, and ccd7 plants with T. m. attack for 2 weeks (±SE, n = 6–10) (two-tailed Student t test). Values for graphs (C–J) are listed in S1 Data. aoc, allene oxide cyclase; BRC1, branched 1, ccd, carotenoid cleavage dioxygenase; d14, dwarf14; EV, empty vector; IAA, indole-3-acetic acid; JA, jasmonate; JA-Ile, jasmonic acid-isoleucine; kai2, karrikin insensitive 2; max2, more axillary growth 2; n.s. not significant; RSJ, root–shoot junction; T. m., T. mucorea; wpi, weeks postinoculation.
Fig 4.
Untargeted metabolic profiling reveals the JA regulation of specific sectors of specialized metabolism in the RSJ of max2 plants with JA-deficient aoc plants as reference.
(A) PLS-DA of metabolomes of EV, aoc, and max2 plants in two independent experiments (batch 1 and 2). (B) Number of up-regulated and down-regulated features in total after statistical filtering. (C) TOM plot for visualizing coexpression outcome by WGCNA. (D and E) Extracted subnetworks visualized by Cytoscape (left panels) and correlations with JA contents with each subnetwork were calculated as PCC and their P values. Bar charts display the representative compounds from each subnetwork across all samples from two batches (dark gray: first batch; purple: second batch). Values for graphs (D) and (E) are listed in S1 Table. aoc, allene oxide cyclase; CP, N′-caffeoylputrescine; DCS, N′, N″-decaffeospermidine; EV, empty vector; JA, jasmonate; JDC, JA-dependent cluster; JIC, JA-independent cluster; max2, more axillary growth 2; PCC, Pearson’s correlation coefficient; PLS-DA, partial least squares discriminant analysis; RSJ, root–shoot junction; TOM, topological overlap matrix; WGCNA, weighted correlation network analysis.
Fig 5.
Increased phenolamide accumulations in SL-RNAi RSJ are positively related to JA signaling via MYB8.
(A) Relative contents of phenolamides such as CP and DCS in the RSJs of indicated plants (±SE, n = 6). (B) Relative contents of CP and DCS in RSJs of EV and myb8 plants after attack. (C) Biomass of T. m. larvae after attacking EV and myb8 plants for 2 weeks (n = 15). (D) Relative transcript abundance of MYB8 and DH29 in RSJ of EV and aoc plants (±SE, n = 4). (E) Relative contents of CP and DCS in the pith of EV and aoc plants without (Control) or with T. m. attack for 2 weeks. (F) Relative transcript abundance of MYB8 and DH29 in the RSJs of indicated plants (±SE, n = 4). (G) Relative content of CP and DCS in the pith of EV and max2#2 plants without (Control) or with T. m. attack for 2 weeks (±SE, n = 6–10) (*P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student t test). Values for graphs (A–G) are listed in S1 Data. aoc, allene oxide cyclase; ccd, carotenoid cleavage dioxygenase; CP, N′-caffeoylputrescine; d14, dwarf14; DCS, N′, N″-decaffeospermidine; DH29, ACYLTRANSFERASE DH29; EV, empty vector; JA, jasmonate; kai2, karrikin insensitive 2; max2, more axillary growth 2; n.s., not significant; RNAi, RNA interference; RSJ, root–shoot junction; SL, strigolactone; T. m., T. mucorea.
Fig 6.
Phenolamide and anthocyanin accumulations are regulated by SL signaling via JA signaling.
(A) Representative pictures of stems of EV, EV×d14#1, aoc×d14#1, EV×max2#2, and aoc×max2#2 plants. (B) Relative anthocyanin levels in epidermis of stems of indicated plants (n = 8). (C) Relative contents of CP and DCS in stem pith of indicated plants after 2 weeks of T. mucorea attack (n = 8) (two-tailed Student t test). Values for graphs (B) and (C) are listed in S1 Data. aoc, allene oxide cyclase; CP, N′-caffeoylputrescine; d14, dwarf 14; DCS, N′, N″-decaffeospermidine; EV, empty vector; max2, more axillary growth 2; JA, jasmonate; SL, strigolactone.
Fig 7.
Interplay of SL and JA signaling through SMXL6/7-JAZ interactions.
(A) Schematic diagram of amplified JA signaling resulting from silenced SL signaling that leads to accumulations of anthocyanins and phenolamides. (B) Phylogenetic analysis of NaSMXL gene family from N. attenuata. (C) Interactions between NaSMXL6/7 and NaD14 proteins in the presence of rac-GR24 by Y2H assays. GAL4 DNA-BD-D14 and AD-SMXL6/7 were cotransformed into yeast. The transformants were grown on QDO (SD−Ade/−His/−Leu/−Trp). (D) The interactions of NaSMXL6 /7 and NaD14 in the presence of 20 μM rac-GR24 by pull-down assays. NaSMXL6/7-YFP was transiently expressed in N. benthamiana leaves. Purified GST-D14 was used. Input of GST-D14 is shown at the second panel. (E) Interaction between NaSMXL6/7 and NaJAZs proteins by Y2H assays. GAL4 DNA-BD-SMXL6/7 and AD-JAZs were cotransformed into yeast. The transformants were grown on QDO (SD −Ade/−His/−Leu/−Trp with 40 μg/mL X-α-gal). (F) Relative transcript abundance of SMXL6, SMXL7, and those JAZs that encode JAZ proteins that interact with SMXL in different tissues. Expression levels were analyzed from microarray data. (G) Interactions of SMXL6/7-YFP and MYC-JAZb by in vivo Co-IP. (H) JAZb degradation in EV and max2 (#1, #2) crude proteins. Purified His-JAZb was incubated in EV or max2 crude proteins extracted from stem pith for the indicated times. His-JAZb was detected by anti-His. The CBB staining represented protein loading levels. (I-J) JAZb degradation in EV, d14#1, or kai2#1 crude proteins, with the same condition mentioned in (H). (K) Interference of SMXL6/7 on interaction of JAZb and MYC2a/b by Y3H assay. The expression of SMXL6/7 was gradually induced by the addition of decreasing amounts of Met. The transformants were grown on QDO (SD−Leu/−His/−Trp/−Met with 40 μg/mL X-α-gal). Raw images for blots are listed in S1 Raw Images. AD, activation domain; ANT, anther; BD, binding domain; CBB, Coomassie Brilliant Blue; COI1, coronatine insensitive 1; Co-IP, co-immunoprecipitation; COL, corolla late; d14, dwarf 14; EV, empty vector; FLB, flower bud; GST, glutathione S-transferase; JA, jasmonate; JAR4/6, JASMONIC ACID RESISTANT 4/6; JAZ, JASMONATE ZIM-DOMAIN; kai2, karrikin insensitive 2; LDOX, LEUCOANTHOCYANIDIN DIOXYGENASE; LEA, leaf; LT, Leu and Trp; LTHA, Leu, Trp, His, and Ade; max2, more axillary growth 2; Met, methionine; MYB8, MYB DOMAIN PROTEIN 8; OFL, opening flower; OVA, ovary; PED, pedicel; QDO, quadruple dropout medium; ROT, root; SD, synthetic defined medium; SED, seed; SL, strigolactone; SMXL, suppressor of max2-like; STE, stem; STI, stigma; Y2H, yeast two-hybrid; Y3H, yeast three-hybrid; YFP, yellow fluorescent protein.
Fig 8.
Decreased nicotine mediated by increased auxin accounts for the susceptibility of SL-RNAi plants to T. mucorea attack.
(A) Relative contents of nicotine in the RSJs of indicated plants (±SE, n = 6). (B) Relative levels of nicotine in the pith of EV and pmt plants (n = 6). (C) Representative pictures and biomass of T. mucorea larvae after attacking EV and pmt plants for 2 weeks (n = 15). Scale bar, 2 mm. (D) Relationship of JA and nicotine levels in RSJ of aoc, EV, and SL-RNAi (max2, d14, and ccd7) plants. Data collected from same samples are indicated by gray lines. (E) Relationship of IAA and nicotine levels in RSJ of EV and SL-RNAi (max2, d14, and ccd7) plants. Data collected from the same sample are connected with a gray line. (F) Schematic diagram of nicotine biosynthesis regulated by auxin and JA-Ile. (G) Levels of JA-Ile, JA, and IAA in roots of EV plants without (Control) or with decapitation treatment for 3 days (n = 6–8). Nicotine content in the RSJs of EV plants with or without decapitation treatments for 3 days (n = 5–6). Biomass of larvae that had attacked intact or decapitated EV plants for 2 weeks (n = 12). (H) Levels of JA-Ile, JA, and IAA in roots of EV plants without (Control) or with treatments with the IAA transport inhibitor CFM for 3 days (n = 6). Nicotine levels in RSJ of EV without or with CFM treatment for 3 days (n = 6). Pith-preference bioassay with T. mucorea larvae between EV stem halves EV (Control)-EV (Control), EV (Control)-EV (CFM treatment) (±SE, 3 replicates, each replicate includes 8–10 plants). (I) Relative transcript abundance of nicotine biosynthetic genes BBL and A622 in EV and aoc seedlings without (Mock) or with 10 μM IAA treatment for 8 hours (±SE, n = 4). (J) Nicotine contents in RSJ of aoc plants without (Control) or with decapitation treatment (n = 6). Biomass of larvae that had attacked aoc control plants or decapitated plants for 2 weeks (n = 12) (two-tailed Student t test). Values for graphs (A–E and G–J) are listed in S1 Data. A622, ISOFLAVONE REDUCTASE-LIKE PROTEIN; aoc, allene oxide cyclase; BBL, BERBERINE BRIDGE ENZYME-LIKE; ccd, carotenoid cleavage dioxygenase; CFM, methyl-2-chloro-9-hydroxyfluorene-9-carboxylate; COI1, CORONATINE INSENSITIVE 1; d14, dwarf14; EV, empty vector; IAA, indole-3-acetic acid; JA, jasmonate; JA-Ile, jasmonic acid-isoleucine; JAR, JASMONIC ACID RESISTANT; kai2, karrikin insensitive 2; max2, more axillary growth 2; n.s., not significant; OPR, OPDA reductase; pmt, putrescine n-methyltransferase; RNAi, RNA interference; RSJ, root–shoot junction; SL, strigolactone.
Fig 9.
SL signaling regulates anthocyanin accumulations and plant defense against T. mucorea larval attack via its interaction with JA and auxin signaling.
(A) A ternary analysis illustrating metabolite distributions across three genotypes: aoc, d14, and aoc×d14 plants. For JIC#1 and JDC#1–3, see Fig 4 and S6 Fig. (B) A model of the role of SLs as regulators that fine-tune resistance to endophytic stem-feeding weevils by disrupting the SL-mediated homeostasis between auxin and JA signaling. In N. attenuata, SL-deficient (d14, max2, and ccd7) plants produce more branches (right panel) relative to EV (left panel), have lower pith nicotine levels and higher anthocyanin and phenolamine levels, and are more susceptible to T. mucorea attack. In EV plants, SL levels maintain auxin and JA homeostasis, but once SL signaling is impaired, auxin and JA levels increase and become unbalanced, decreasing pith nicotine levels and increasing susceptibility to larval attack. Values for the graph (A) are listed in S1 Data. aoc, allene oxide cyclase; CP, N′-caffeoylputrescine; d14, dwarf14; DCS, N′, N″-decaffeospermidine; EV, empty vector; JA, jasmonate; IAA, indole-3-acetic acid; JDC, JA-dependent cluster; JIC, JA-independent cluster; max2, more axillary growth 2; SL, strigolactone.