Fig 1.
Gene structure, phylogenetic tree, and expression analysis of CsIVP.
(A) CsIVP gene structure in which exons and the intron are indicated by green boxes and a black line, respectively; red box indicates the RNAi-targeted region, and the orange box represents the bHLH domain. (B) Phylogenetic analysis of CsIVP and its homologs. CsIVP is highlighted in red, and the star indicates the duplication event of HEC3 in the Brassicaceae family. Gymnosperm sequences were used as the outgroup. Bootstrap values over 50% are placed above the branches. (C–N) RNA in situ hybridization analysis of CsIVP in cucumber. (C–F) CsIVP transcripts are highly expressed in the shoot apical meristem and leaf primordia. (C) Longitudinal section of a cucumber shoot apex, (D) transverse section of a shoot apex, (E) enlarged view of the leaf primordia p4 in panel D, (F) transverse section of a shoot apex hybridized with the CsIVP sense probe. p1–p7: leaf primordia 1–7. (G–I) CsIVP is expressed in stem vascular strands (panel G) and floral organ primordium (panels H–I). (J–N) CsIVP transcripts are accumulated in fruit vascular tissues and at the boundary between the developing seed and the fruit placenta (arrow in panel L) (panels K–L). (M, N) In situ hybridization with the CsIVP sense probe. Scale bars represent 200 μm in panels C–D and F–I, 100 μm in panel E, 50 μm in panels J–K and M, and 25 μm in panels L and N. bHLH, basic Helix-Loop-Helix; C, carpel; CsIVP, Cucumis sativus Irregular Vasculature Patterning; HEC3, HECATE3; P, petal; RNAi, RNA interference; S, sepal; St, stamen; v, vascular tissue.
Fig 2.
Phenotypic characterization of CsIVP-RNAi transgenic plants.
(A) Plant morphology of WT and CsIVP-RNAi lines R5, R3, and R2. (B) qRT-PCR analysis indicates reduced expression of CsIVP in the CsIVP-RNAi lines. (C) Immunoblot blot analysis indicates reduced CsIVP protein in the CsIVP-RNAi lines. (D–E) Morphology of young (panel D) and mature leaves (panel E) of WT and CsIVP-RNAi lines. White squares represent the gap between the bilateral leaf margins. The angle between the vertical axis and the primary vein indicates the degree of down-curled leaf. (F) Leaf venation in WT and CsIVP-RNAi leaves. Red arrows represent the primary veins in the leaf. (G) Male flower size at anthesis. (H) Fruit at anthesis in WT and CsIVP-RNAi lines. (I) Reduced mature fruit length and decreased seed viability in the CsIVP-RNAi lines. (J) Seeds after 36 h of germination. (K) Seeds after testa removal. (L–O) Transverse sections of leaf mid-veins of WT (panel L) and CsIVP-RNAi (panels M–O) plants. Red stars in panels M–O indicate extra vascular bundles in CsIVP-RNAi lines. (L’–O’) Amplified vascular bundles in red boxes of panels L–O. (P) Transverse sections of stems from WT and CsIVP-RNAi lines. White numbers indicate the vascular bundles. (Q) IAA content in leaf veins of WT and CsIVP-RNAi transgenic plants. (R–U) IAA distribution in leaf veins as detected by immunolocalization. Anti-IAA monoclonal antibodies and DyLight 488–conjugated goat anti-mouse IgG antibodies were used to detect IAA. (R, T) Differential interference contrast images; (S, U) fluorescent images. Scale bars represent 2 cm in panels A, D–J, and P; 2 mm in panel K; 200 μm in panels L–O; 50 μm in panels L’–O’; and 100 μm in panels R–U. Values are means ± SE (n = 3) in panels B and Q. Double asterisks indicate significant difference at P < 0.01 by t test. The data underlying this figure are included in S1 Data. CsIVP, Cucumis sativus Irregular Vasculature Patterning; ExP, external phloem; IAA, indole-3-acetic acid; IgG, immunoglobulin G; InP, internal phloem; Ph, phloem; qRT-PCR, quantitative real-time PCR; RNAi, RNA interference; WT, wild type; Xy, xylem.
Fig 3.
Transcriptome and interaction analysis between CsIVP and putative downstream vascular-related targets.
(A) Venn diagrams of the overlapping DEGs that were up-regulated or down regulated in the vein and fruit of CsIVP-RNAi plants, as compared to WT plants. (B) Gene category enrichment of up- and down-regulated genes in the vein of CsIVP transgenic compared to WT plants. (C) Yeast one-hybrid assays identify interactions between CsIVP and the E-box from the CsYAB5, CsBP, and CsAUX4 promoters. Activation of AbAr occurred when CsIVP bound to the E-box sequence. The SD/-Leu medium with 100 ng/ml or 500 ng/ml inhibitory AbA was used to screen for interactions. (D) Visualization of direct binding of CsIVP to promoters of CsYAB5, CsBP, CsAUX4, and CsCCR1 via EMSAs. Three concentrations of labeled probe were used (80, 120, and 160 fmol). Cold (unlabeled) probes were used as competitors. (E) ChIP-PCR showing the in vivo binding of CsIVP to the pCsYAB-1626, pCsBP-333, and pCsAUX4-1492 promoters. The cucumber alpha-tubulin gene (CsTUBULIN, GenBank: AJ715498) was used as the internal gene control, the pCsCCR1-1939 was used as a negative amplification. Values are means ± SE (n = 3). Double asterisks indicate significant difference at P < 0.01 by t test. (F) Luciferase activity measured in tobacco leaves after co-expression of 35S:CsIVP with proCsYAB5:LUC, or proCsBP:LUC, or proCsAUX4:LUC. Values are means ± SE (n = 6). Double asterisks indicate significant difference at P < 0.01 by t test. The data underlying this figure are included in S2 Data. AbA, aureobasidin A; AbAr, AbA resistance gene; AUX4, AUXIN/INDOLEACETIC ACIDS4; BP, BREVIPEDICELLUS; CCR1, CINNAMOYL COA REDUCTASE1; ChIP, chromatin immunoprecipitation; Cs, Cucumis sativus; CsIVP, Cucumis sativus Irregular Vasculature Patterning; DR, down-regulated; EMSA, electrophoretic mobility-shift assay; RNAi, RNA interference; SD/-Leu, synthetical dropout/-leucine; UR, up-regulated; WT, wild type; YAB5, YABBY5.
Fig 4.
Functional characterization of CsYAB5 in cucumber.
(A–D) RNA in situ hybridization analysis of CsYAB5 in the cucumber shoot apex and flower buds. Longitudinal (panel A) and transverse sections (panel B) of cucumber shoot apex. p1–p6: leaf primordia 1–6. (C) Longitudinal image of a flower bud. (D) Transverse section of leaf primordia in the shoot apex, hybridized with the CsYAB5 sense probe. (E) Plant morphology of WT and CsYAB5-RNAi line R1, R2, and R3. (F) Leaf morphology of WT and CsYAB5-RNAi lines. White squares show the gap between the bilateral leaf margins. (G) qRT-PCR analysis indicates reduced expression of CsYAB5 in the CsYAB5-RNAi lines. (H–I) Fruit at anthesis in WT and CsYAB5-RNAi lines (panel H); quantification of ovary length at anthesis (panel I). (J–K) Reduced mature fruit length (panel J) and decreased seed viability (panel K) in the CsYAB5-RNAi line. (L) Morphology of mature seeds of WT and CsYAB5-RNAi line. (M–P) Transverse sections of leaf mid-veins of WT (panel M) and CsYAB5-RNAi (panels N–P) plants. (M’–P’) Amplified vascular bundles in red boxes of panels M–P. Blue stars in panels N’–P’ indicate extra vascular bundles in CsYAB5-RNAi lines. Scale bars represent 100 μm in panels A–D; 2 cm in panels E–F, H, and J–K; 1 mm in panel L; 500 μm in panels M–P; and 200 μm in panels M’–P’. Values are means ± SE (n = 3) in panels G and I. Double asterisks indicate significant difference at P < 0.01 by t test. The data underlying this figure are included in S3 Data. C, carpel; Cs, Cucumis sativus; ExP, external phloem; InP, internal phloem; P, petal; Ph, phloem; qRT-PCR, quantitative real-time PCR; RNAi, RNA interference; S, sepal; St, stamen; v, vascular tissue; WT, wild type; Xy, xylem; YAB5, YABBY5.
Fig 5.
Response of CsIVP-RNAi plants to pathogen infection.
(A) Increased tolerance of CsIVP-RNAi plants to pathogen attack during growth under greenhouse conditions. (B) Disease level of WT and CsIVP-RNAi plants under greenhouse and growth chamber conditions. (C–E) Phenotypic characterization of WT and CsIVP-RNAi plants 11 dpi with Pseudoperonospora cubensis in a growth chamber. The second, third, and fourth leaves (from bottom to top) are displayed in panel E. (F–G) Sporulation of P. cubensis on WT and CsIVP-RNAi plants imaged by scanning electron microscopy. (H) PsITS expression after inoculation with P. cubensis at 0, 3, 7, and 11 dpi. (I) CsIVP expression after inoculation with P. cubensis at 0, 3, 7, and 11 dpi. Scale bars represented 2 cm in panels A and C–E and 50 μm in panels F–G. Values are means ± SE (n = 3) in panels H–I. Asterisk indicate significant difference at P < 0.05 by t test. The data underlying this figure are included in S4 Data. CsIVP, Cucumis sativus Irregular Vasculature Patterning; dpi, days post inoculation; PsITS, P. cubensis internal transcribed spacer; RNAi, RNA interference; WT, wild type.
Fig 6.
Interaction of CsIVP with CsNIMIN1, a repressor in the SA-responsive pathway.
(A) SA content in leaf veins of WT and CsIVP-RNAi transgenic plants. (B) Schematic diagram of the major components in the SA-mediated plant defense pathway. (C) Expression analyses of selected genes involved in the induced systemic responses in WT and CsIVP-RNAi plants, at the times indicated after P. cubensis inoculation. (D–E) Physical interactions between CsIVP and CsNIMIN1, as revealed by Y2H (panel D) and BiFC assays (panel E). Protein interactions are indicated by green YFP fluorescent signals in nuclei (left panels); differential interference contrast images of tobacco cells are shown in the middle panels; and merged channels are shown in right panels. Values are means ± SE (n = 3) in panels A and C. Double asterisks indicate significant difference at P < 0.01 by t test. The data underlying this figure are included in S5 Data. BiFC, bimolecular fluorescence complementation; Cs, Cucumis sativus; CsIVP, Cucumis sativus Irregular Vasculature Patterning; NIMIN1, NIM1-INTERACTING 1; RNAi, RNA interference; SA, salicylic acid; WT, wild type; Y2H, yeast two-hybrid; YFP, yellow fluorescent protein.
Fig 7.
A working model for vasculature regulator CsIVP functioning in organ morphogenesis and downy mildew resistance in cucumber.
(A) CsIVP regulates organ morphogenesis via 2 pathways, one by directly promoting the expression of vascular-related genes—including CsYAB5 and CsBP—to regulate vascular development, and the other by directly binding to the E-box of CsAUX4 to mediate auxin signaling. Grey arrow indicates the putative positive role of auxin in vascular development. (B) CsIVP may act as a repressor for SA production and physically interacts with CsNIMIN1 to compromise downy mildew resistance in cucumber. Black arrows, positive regulation; black crosses, inhibition regulation; red arrows, transcription start; black dotted crosses, putative inhibition regulation. AUX4, AUXIN/INDOLEACETIC ACIDS4; BP, BREVIPEDICELLUS; Cs, Cucumis sativus; CsIVP, Cucumis sativus Irregular Vasculature Patterning; NIMIN1, NIM1-INTERACTING1; SA, salicylic acid; YAB5, YABBY5.