SUPPLEMENTARY DATA. Supplementary Table 2; Supplementary Figure 6; Supplementary Figure 7
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1 SUPPLEMENTARY DATA Supplementary Table 1; Supplementary Table 2; Supplementary Figure 1; Supplementary Figure 2; Supplementary Figure 3; Supplementary Figure 4; Supplementary Figure 5; Supplementary Figure 6; Supplementary Figure 7
2 Supplementary Tables Supplemental Table 1. List of proteins/peptides identified from pull-down screening. Ratio in candidate Protein/peptide name proteins(%) GenBank Acc. No. Peptides matched nucleotide binding site-leucine rich repeat protein (NBS-LRR) AAU Ethylene responsive factor (ERF) AY Phospho-specific binding protein AEH Calcium-dependent protein kinase (CDPK) 9.1 ADZ Auxin response factor 3 (ARF3) 7.76 ABO ACC oxidase 3 (ACO3) 6.5 AAZ Nonexpresser of pathogenesis-related 1 (NPR1) 5.83 ABC Xyloglucan endotransglycosylase (XTH) 5.43 ABU Lipin domain-containing protein (LIPN) 4.54 AEE Endo-alpha-1,4-glucanase (EG) 3.38 ADZ
3 Supplemental Table 2. Primers used in this study. Primer sequence 5'-5 -AAAGAGCTCTCA TTAACTTGCTTGGGTGAGGT-3 5'-CCGAATTCTTAACTTGCTTGGGTGAGGTGGCCGTCAA-3 5 -CCGGATCCAGGATGCGCAATCCCACCGTGCAGCTTC-3 5 -CCGAATTCAAATCACTCCCACATCAAACAAACAAACCA-3 5'-AAAGGATCCATGACGTCTTCAGCTCTGACAGG-3 5'-CCGAATTCTTAACTTGCTTGGGTGAGGTGGCCGTCAA-3 5'-AAGAATTCTCATTAGCGCCGTTGCTTATACTGAGTTA -3' 5'-AAGGATCCATGTGTGGAGGTGCAATTATTTCCGA-3' 5'-AAGGATCCATGTGTGGAGGTGCAATTATTTCCGA-3' 5'-AAGAATTCTTAGCGCCGTTGCTTATACTGAGTTA -3' 5'-CCGAATTCCGGCTGTGTTATAGGTACCGAGCCAAACTC-3' 5'-CCGGATCCTATTGGGTCGGTCTATAAATCCTAGACC-3' 5'-GGGAATTC TTCGGTGACCCTTATAACTCGGACCCGGAG-3' 5'-GGGGATCC ATCAAACCGTGCTCTTTCCTCCACAATCTT-3' Primer names and Purpose GhMLP28-F1, pet28a-ghmlp28 GhMLP28-R1, pet28a-ghmlp28 GhMLP28-F2, ptrv-ghmlp28 GhMLP28-R2, ptrv-ghmlp28 GhMLP28-F3, 35S-GhMLP28 and 35S-GFP-GhMLP28 GhMLP28-R3, 35S-GhMLP28 and 35S-GFP-GhMLP28 GhERF6-F1, pgex6p-1-gherf6 GhERF6-R1, pgex6p-1-gherf6 GhERF6-F2, 35S-GhERF6 and 35S- GhERF6-mcherry GhERF6-R2, 35S-GhERF6 and 35S-mcherry- GhERF6 GhERF6-F3, ptrv-gherf6 GhERF6-R3, ptrv-gherf6 GhCLA1-F, ptrv-ghcla1 GhCLA1-R, ptrv-ghcla1
4 5'-ATCAAATATAATCCTTATTAATAATTCAAT-3' 5'-GGTTGGTTTTTACTTGGTGATTTGTTCATA-3' 5'-GCTGAAGAAGAGGATGGAAGAGATCTGGGA-3' 5'-ATTCCTCGAAAAATAATAATCTAGAAAGA-3' 5'-TAAATATTATTATAACCGAGCGAATGATTGAAA-3' 5'- CACATCAAACAAACCACTCTCACAT-3' 5'-TACGCTGCCACTACCCTCGCTCTTG-3' 5 -TAACAAAGAGAGAAGCGAGACGAT-3 5 -CCATACCCAAGACCCATAGGATAAG-3 5 -CGCCCTTCCTCATGCAAT-3 5'- TTAACATCGCGGACAATTTCC-3' 5'-TCCTGGGCAATTTCACGAACAAGC-3' 5'-TGCCCGTAAATCTACTGGAGGCAA-3' 5'-TCCTGGGCAATTTCACGAACAAGC-3' 5'-TGCCCGTAAATCTACTGGAGGCAA-3' 5'-ACTGAAACGCCTCTCGGAAACA-3' GhPR5Pro-F1, pgwb435-ghpr5pro GhPR5Pro-F2, pgwb435-ghpr5pro GhPR5 Pro-R, pgwb435-ghpr5 Pro GhPDF1.2Pro-F, pgwb435-ghpdf1.2pro GhPDF1.2Pro-R, pgwb435-ghpdf1.2pro GhMLP28-QF, qrt-pcr GhMLP28-QR, qrt-pcr GhERF6-QF, qrt-pcr GhERF6-QR, qrt-pcr NtActin-QF, qrt-pcr NtActin-QR, qrt-pcr NtHistone 3-F, RT-PCR NtHistone 3-R, RT-PCR GhHistone 3-QF, qrt-pcr GhHistone 3-QR, qrt-pcr NtNPR1-QF, qrt-pcr
5 5'-CCAACACACTCACCACAGCATC-3' 5'-ACTGCAACCTCGTACATTCT-3' 5'-CACTTAACCCTAGCACATCC-3' 5'-CACAACAAGAAGCAAATCCT-3' 5'-TATCAAAGTGAAACCAGAGT-3' 5'-AAACCACAAATAATTTGTCACTATA-3' 5'-CATGTACCCCTACCAGCAGCATTAAA-3' 5'-GCCTTGCCTCAACCCATT-3' 5'-GCCGCTTCGGAATGGAAG-3' 5'-AGCCGCCTCA GCGTTTATTT TTAAA-3' 5'-TACGAGCCATGGCTGTGCCAGCAG-3' 5'-AATTGGTCCTTCCCTCCCATGCCCAAGA-3' 5'-AACCTGCTGCGCACCTGTTAGAGTTG-3' NtNPR1-QR, qrt-pcr NtPR1b-QF, qrt-pcr NtPR1b-QR, qrt-pcr NtPR2-QF, qrt-pcr NtPR2-QR,qRT-PCR NtPR5-QF, qrt-pcr NtPR5-QR, qrt-pcr GhNPR1-QF, qrt-pcr GhNPR1-QR, qrt-pcr GhPR5-QF, qrt-pcr GhPR5-QR, qrt-pcr GhPR1b-QF, qrt-pcr GhPR1b-QR, qrt-pcr
6 Supplemental Figure Supplemental Figure 1. Phylogenetic relationship between GhMLP28 and MLPs in other plants. Phylogenetic analysis was performed using the neighbor-joining method. The scale indicates branch lengths. The accession numbers of the proteins are indicated.
7 Supplemental Figure 2. EMSA analysis on the binding of GhERF6 to the GhPDF1.2 promoter. GhERF6 proteins were incubated with a biotin-labeled probe (GhPDF1.2 promoter sequence) in the absence or presences of 2-6 fold unlabeled probe for 30min.
8 Supplemental Figure 3. EMSA analysis on the binding of GhERF6 to the GhPR5 promoter. GhERF6 proteins were incubated with a biotin-labeled probe (GhPR5 promoter sequence) in the absence or presences of 2-6 fold unlabeled probes for 30min.
9 Supplemental Figure 4. Transient expression analysis of GhMLP28-enhanced transcriptional activation activity of GhERF6 on GhPR5 promoter. (A) Expression levels of GhERF6 and GhMLP28 in tobacco leaves transformed with GhPR5Pro:Luc. (B) Luminescence signal on N. benthamiana leaves. Luminescence imaging was performed 48h after co-infiltration with the same amount of Agrobacterium cells harboring constructs indicated on the left panel. (C) Luminescence intensity in N. benthamiana leaves measured by IndiGo software. Error bars in (C) represent the SD (n = 60) of three biological repeats. Asterisks indicated statistically significant differences, as determined by Student s t-test (*P<0.05, **P<0.01).
10 Supplemental Figure 5. Effect of GhMLP28/GhERF6 silencing on GhPR5 promoter-driven Luc expression. (A) Transient expression assay of Luc in GhMLP28- or GhERF6-silenced cotton leaves. Top leaves (similar size) of GhERF6- or GhMLP28-silenced plants were injected with equal amounts of Agrobacterium cells harboring GhPR5Pro:Luc or 35S:Luc. Luminescence imaging of cotton leaves was performed 48h after infiltration with the constructs indicated on the left panel. (B) Luminescence intensity in cotton leaves measured by IndiGo software. Error bars indicate the SD, n = 60. Asterisks indicate statistically significant differences, as determined by Student s t-test (*P<0.05, **P<0.01).
11 Supplemental Figure 6. Increased susceptibility of GhERF6-silenced cotton plants to V. dahliae infection. (A) Disease symptom of GhERF6-silenced plants infected by V. dahliae. Photo was taken at 35d post inoculation. (B) Analysis of GhERF6 expression levels. Total RNAs were extracted from leaves at 14 days post agroinfiltration, and the expression level of GhERF6 in VIGS plant was compared with that of control plant (transfected with TRV:00).
12 Supplemental Figure 7. Albino phenotype of GhCLA1-silenced cotton plant. The cdna of GhCLA1 was inserted into ptrv2 vector. A mixture of Agrobacterium cultures containing ptrv1 or ptrv2-ghcla1 were co-infiltrated into cytoledons of 14-day-old cotton plants. The albefaction phenomenon was visualized on true leaves at 10 days post agroinfiltration.
Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae
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- 1 - Supplemental Data
- 1-1 Supplemental Data 2 3 4 5 6 7 8 9 Supplemental Figure S1. Differential expression of AtPIP Genes in DC3000-inoculated plants. Gene expression in leaves was analyzed by real-time RT-PCR and expression
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Supplementary Material
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Supplementary Information
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