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Over-expression of the red plant gene R1 enhances anthocyanin production and resistance to bollworm and spider mite in cotton

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Abstract

Anthocyanins are a class of pigments ubiquitously distributed in plants and play roles in adoption to several stresses. The red plant gene (R1) promotes light-induced anthocyanin accumulation and red/purple pigmentation in cotton. Using 11 markers developed via genome resequencing, the R1 gene was located in an interval of approximately 136 kb containing three annotated genes. Among them, a PAP1 homolog, GhPAP1D (Gohir.D07G082100) displayed differential transcript level in the red- and green-plant leaves. GhPAP1D encoded a R2R3-MYB transcription factor and its over-expression resulted in increased anthocyanin accumulation in transgenic tobaccos and cottons. Dual luciferase assay indicated that GhPAP1D activated the promoters of several cotton anthocyanin structural genes in tobacco leaves. Importantly, we found that the GhPAP1D-overexpressing cotton leaves had increased resistance to both bollworm and spite mite. Our data demonstrated that GhPAP1D was the controlling gene of the red plant phenotype in cotton, and as the major anthocyanin regulator, this gene was potential to create transgenic cottons with resistance to a broad spectrum of herbivores.

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Funding

This study was partially funded by the Genetically Modified Organisms Breeding Major Project of China (2016ZX08005005-001 to Y.H.X.), the National Natural Science Foundation of China (31571582 and 31871539 to Y.H.X.) and the Chongqing Project of Basic and Frontier Research (cstc2015jcyjA80001 to Y.H.X.).

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Authors and Affiliations

  1. Biotechnology Research Center, Southwest University, Chongqing, China

    Xin Li, Xufen Ouyang, Yi Wang, Yaohua Li, Jia Zhao, Zhong Chen, Chuannan Wang, Lingli Ding, Yan Pei & Yuehua Xiao

  2. College of Agronomy and Biological Science and Technology, Southwest University, Chongqing, China

    Zhengsheng Zhang

  3. College of Plant Protection, Southwest University, Chongqing, China

    Lin He

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  1. Xin Li
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Correspondence to Yuehua Xiao.

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438_2018_1525_MOESM1_ESM.doc

S1 Figure 1. Phenotypic differences between red- (R1, left) and green- (right) plant cottons. Two recombinant inbred lines (RIL103 and RIL130) derived from T586×Yumian 1 were photographed to show the phenotypic variations between red- and green-plant cotton, respectively. The R1 cotton (RIL103) displayed significant red–purple coloration in various organs, but not in fibers. A, 1-week seedlings; B, 2-month plants; C, stems; D, flowers; E, Bolls of 25 days post anthesis (DPA); F and G, Peeled 25-DPA bolls to show fibers, respectively. S2 Figure 2. Sequence analyses of cotton PAP1 homologs. A, A phylogenetic tree of cotton PAP1 homologs and homologous proteins. The phylogenetic tree was constructed using Neighbor Joining method with 1000 replicates of bootstrap test in MEGA6.0. Numbers indicate the branches supported by > 50% bootstrap. The subgroups were named according to Arabidopsis MYB proteins. Solid dots indicate the sequences from cotton species (G. hirsutum, G. arboreum and G. raimondii). AtMYB75, 90, 123, 12, 11, and 111 are from Arabidopsis thaliana loci AT1G56650, AT1G66390, AT5G35550, AT2G47460, AT3G62610 and AT5G49330, respectively. AN2, Ruby and ANT1 are from Petunia×hydrida (GenBank accession No. AAV98200), Citrus sinensis (JN402334) and Solanum lycopersicum (AY348870), respectively. B, Detection of genome specificity of cotton PAP1 homologs. T, Y, A and D represent the template genomic DNA from G. hirsutum (AD1) T586 and Yumian 1, G. arboreum (A2) and G. raimondii (D5). M indicates DNA markers showing three bands of 500, 250 and 100 bp. C, Multiple sequence alignment of cotton PAP1 homologs and other Sg6 MYBs. The conserved and consensus sequences are shadowed in black and grey, respectively. The R2 and R3 MYB domains and the signature motifs of Sg6 MYB proteins (S1 and S2) are indicated by solid bars. S3 Figure 3. Overexpression of cotton PAP1 homologs (GhPAP1A/1D and GhPAP2A/2D) significantly enhances anthocyanin accumulation in flowers (upper panel) and leaves (lower panel) of transgenic tobaccos. S4 Figure 4. GhPAP1D expression (A) and anthocyanin contents (B) in T0 transgenic cotton leaves. S5 Figure 5. Anthocyanin contents and expression levels of GhPAP1D and representative anthocyanin structural genes in T2 homogenous transgenic cotton leaves. OP1 and OP2 represent two transgenic lines, and NU is the null segregant of OP1. * and ** indicate significant difference (t test, p < 0.05 and 0.01, respectively) compared with the non-transgenic segregant. Gene names are as in the S10 Table. S6 Figure 6. Silencing of GhPAP1D in the red-plant cotton reduces anthocyanin accumulation in leaves. The cotyledons of R1 line (RIL103) were infiltrated with Agrobacterium harboring empty (TRV00) or GhPAP1D (TRV-PAP1) VIGS vector. The control was grown in parallel condition, but not inoculated with Agrobacterium. ** indicated significant difference compared with the control (p < 0.01, t test). A and B, GhPAP1D expression levels and anthocyanin contents in the second true leaves. Error bar indicated the SD of three biological replicates. C, Seedlings of two weeks after infiltration. S7 Figure 7. Dual luciferase assay of the activation effects of GhPAP1D on its own promoters from red- and green-plant cottons, T586 (pPAP1-T) and Yumian 1, (pPAP1-Y) respectively. A, Schematic of the expression cassettes of effector and reporter vectors. Arrows indicate promoters and boxes show coding sequences. B, the activation effects on the promoters pPAP1-Y and -T. The activation effects on different promoters are calculated as the activity ratio of firefly to Renilla luciferases, and normalized to that of GFP. Error bars represent standard deviation of three biological replicates. ** indicate significant activation compared to the effector control GFP (t test, p < 0.01). S8 Table 1. Molecular markers used in mapping of R1 S9 Table 2. Primers used for cloning. S10 Table 3. Primers used for qRT-PCR analyses. S11 Table 4. Annotations of the coding genes in R1 region. S12 Table 5. PAP1 homologs in R1 region and their cotton homeologs. S13 Table 6. Transcript levels (FPKM) of the coding genes in the R1 region in the leaves of red- (L-R1) and green-plant (L-r1) cottons. S14 Table 7. Differentially expressed flavonoid-related genes identified in transgenic (GhPAP1D over-expresser, TGPA vs null segregant, NUPA) and natural (R1 leaf, L-R1 vs r1 leaf, L-r1) red cotton leaves. S15 Table 8. Life table of the spider mites feeding on GhPAP1D-over-expressing cotton (OP1) and its null segregant. S16 Table 9. Reproductive parameters of the spider mites feeding on GhPAP1D-over- expressing cotton (OP1) and its null segregant (DOC 18655 KB)

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Li, X., Ouyang, X., Zhang, Z. et al. Over-expression of the red plant gene R1 enhances anthocyanin production and resistance to bollworm and spider mite in cotton. Mol Genet Genomics 294, 469–478 (2019). https://doi.org/10.1007/s00438-018-1525-3

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