tpj12966-sup-0009-Legends

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SUPPORTING FIGURE LENGENDS
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Figure S1. Schematic of the predicted domains of CMPG proteins and phylogenetic analysis of
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CMPG amino acid sequences from plants. Nucleotide sequence alignment of CMPG1-V and its
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orthologs in common wheat.
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(a) Schematic representation of CMPG1-V and other U-Box/ARM proteins from plants.
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CMPG1-V (H.villosa), AK375870 (Hordeum vulgare), Os02g0738200 (Oryza sativa),
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NM_001154388 (Zea mays), XM_003573078 (B. distachyon), PcCMPG1 (Petroselinum crispum),
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SlCMPG1 (Solanum lycopersicum), NtCMPG1 (Nicotiana tabacum), AtCMPG2, AtPUB17, AtPU22
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and AtPUB12 (Arabidopsis thaliana), StPHOR (Solanum tuberosum) were used for analysis. The
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orange columns at N-terminus represent the U-Box domain, and the percentages in orange columns
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represent the similarities of U-Box domain. Purple columns represent the ARM repeats, and the
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number of ARM repeats is represented by the numbers in the purple columns, the percentages in the
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second purple column represent the similarities of ARM repeat domain. The numbers show the start
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and stop amino acids of each domain, and the most right percentages represent the similarities of the
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full length of different U-Box/ARM proteins with CMPG1-V.
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(b) Phylogenetic analysis of CMPG amino acids sequences from plants. The scale bar of 0.1 indicates
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10% sequence divergence. 1000 bootstrap repetitions were made.
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(c) Nucleotide sequence alignment of CMPG1-V and its orthologs from common wheat Chancellor,
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Funo, Baimian 3 (BM), Kavkaz and Ulka/8cc (UL). The red lines indicate positions of primers
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CMPG-NP-F/CMPG-NP-R.
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Figure S2. Sequence alignment of the specific amplicons amplified using primers
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CMPG-NP-F/CMPG-NP-R.
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From up to down: CMPG sequences amplified from plasmid pBI-220-CMPG1-V, H.villosa and
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T6AS·6VL.
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Figure S3. Schematic of the pedigree of the CMPG1-V transgenic plants from T0 to T4
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generation.
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T0-26, T0-46 and their derived lines were identified as positive transgenic plants while T0-33 and its
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derived plants were negative control.
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Figure S4. PCR identification of CMPG1-V transgenic lines T2-26 and T2-46.
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NAU02Y393, Yangmai158 and T2-N (negative transgenic plant) represent negative control. Plasmid
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(pBI-220-CMPG1-V), T1-26-3 and T1-46-25 represent positive control. The arrow indicates that the
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target band of amplification (206bp).
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Representative PCR results of plants derived from (a) T1-26-3 (T2-26-1~ T2-26-6) and plants from (b)
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T1-46-25 (T2-46-1~ T2-46-6) were shown.
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Figure S5. Determination of H2O2 content and activities of antioxidase enzymes in leaves of
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Yangmai158, T4-N, T4-26, T4-46, T6AS·6VL, NAU9918 and 91C43 (H.villosa) after inoculation of
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Bgt.
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(a) The content of H2O2, (b) enzyme activity of guaiacol peroxidase (POD) and (c) ascorbate
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peroxidase (APX) in leaves of Yangmai158, T4-N, T4-26, T4-46, T6AS·6VL, NAU9918 and 91C43
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(H.villosa) were assayed. Values are means ±SE of three independent experiments with at least three
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replicates for each. Different letters represent statistically significant differences (P<0.05, one-way
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ANOVA).
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Figure S6. Expression patterns of CMPGs in different materials in response to Bgt infection.
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RT-qPCR showed the relative expression of CMPG genes in 91C43 (H.villosa), NAU9918, T6AS·6VL
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and Yangmai158 leaves. Tubulin gene was used as a reference gene. The RT-qPCR values were
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normalized to tubulin and then presented as fold-changes relative to 91C43, NAU9918, T6AS·6VL and
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Yangmai158 without treatment respectively. Similar results were obtained from three independent
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experiments, and standard deviation (n = 3) is represented by error bars. The letters represent
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statistically significant differences (P<0.01, one-way ANOVA) of 91C43, NAU9918, T6AS·6VL and
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Yangmai158 respectively.
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Figure S7. Expression patterns of CMPGs in T4 transgenic plants in response to Bgt treatment.
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RT-qPCR showed the relative expression of CMPG genes in Yangmai158, negative transgenic plants
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(T4-N) and positive transgenic plants T 4-46 and T4-26 leaves. The RT-qPCR values were normalized to
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tubulin and then presented as fold-changes relative to Yangmai158 without treatment. Values represent
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the mean and standard deviations obtained from three replicates of each sample. Different letters
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represent statistically significant differences (P<0.01, one-way ANOVA).
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