Abstract
KNOTTED1 (KN1)-like homologous box is a homeodomain transcription factor that is transported intercellularly through plasmodesmata and over long distances via phloem transport in plants. In this study, we investigated KN1 long-distance transport and its regulation by the viral movement protein binding protein of ‘Du Li’ (Pyrus betulaefolia, PbMPB2C) in two species of fruit trees, ‘Du Li’ (rootstock) and ‘Ya Li’ (P. bretschneideri, scion), using grafting experiments. The results showed that PbKN1 mRNA was present in the phloem in both transport directions across the graft junctions of tissue-cultured grafted plants and orchard saplings. To investigate the relationship between PbKN1 and PbMPB2C, we conducted interaction assays to demonstrate their physical interaction. An RNA–protein pull-down assay revealed that PbKN1 mRNA interacted with PbMPB2C protein in vitro but not with PbKN1 and that PbKN1 repressed the binding of PbMPB2C to PbKN1 mRNA. Additionally, using p35S:GFP-PbKN1 transgenic tobacco grafted with wild-type plants, we found that the movement of PbKN1 was increased by the silencing of NtMPB2C. Based on the above findings, we first demonstrate evidence of the bidirectional transport of PbKN1 mRNA and propose that PbMPB2C could act as a negative regulator of the phloem-mediated long-distance transport of PbKN1 mRNA.
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Acknowledgments
We thank Dr. Aide Wang (Shenyang Agricultural University, Shenyang, China) and Dr. Alexander Ivakov (Max-Planck Institute of Molecular Plant Physiology-Golm) for giving the helpful comments and examining the English usage in the manuscript. This work was supported by the Doctoral Program Special Fund of the Ministry of Education in China (20130008110006) and National Natural Science Foundation of China (31372035).
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11240_2014_685_MOESM1_ESM.tif
Fig. S1 Alignments of KNOX proteins and the conserved domains. The lines above the sequences mark the KNOX1, KNOX2, GSE box, ELK and homeodomain. (TIFF 1465 kb)
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Fig. S2 Phylogenetic analysis of PbKNOX proteins. Neighbor-joining (NJ) tree of PbKNOX proteins and their relatives in the class 1 KNOX group. Bootstrap values are indicated above the nodes. The species names and accession numbers of the protein sequences used are detailed in Table S2. (TIFF 448 kb)
11240_2014_685_MOESM3_ESM.tif
Fig. S3 Expression patterns of PbKN1 genes. a Semiquantitive RT-PCR analysis of the tissue-specificity of DL-PbKN1 gene expression in ‘Du Li’ (P. betulaefolia). b Separation of the inner tissue (xylem) and outer tissue (bark, including phloem) of the stem. The phloem (SCP) stripped from the samples was used as the material for the RT-PCR experiments. c d e In situ hybridization of transverse sections of the ‘Du Li’ stem. Sections were hybridized with a digoxigenin-labeled 368-bp mRNA copy of a DL-PbKN1 sequence. c Sense probe. d e Antisense probe. The boxed area in d is enlarged in e. The arrows in c indicate DL-PbKN1 mRNA signals. P, phloem; and X, xylem. Scale bars = 50 μm. (TIFF 3354 kb)
11240_2014_685_MOESM4_ESM.tif
Fig. S4 Structural organization of MPB2C genes. a Alignments of MPB2C proteins and the conserved domains. b Schematic representations of PbMPB2C, NtMPB2C and AtMPB2C genes showing the conserved coding regions. (TIFF 1540 kb)
11240_2014_685_MOESM5_ESM.tif
Fig. S5 Confocal images of green fluorescence track the long-distance transport of PbKN1 in transgenic tobacco grafted with wild-type tobacco. a p35S:GFP-PbKN1 scion grafted on p35S:GFP-PbKN1 rootstock. b WT scions grafted on p35S:GFP-PbKN1 rootstock. c p35S:GFP-PbKN1 scion grafted on WT rootstock. d WT scions grafted on WT rootstock. Bar = 100 μm. (TIFF 1248 kb)
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Duan, X., Zhang, W., Huang, J. et al. KNOTTED1 mRNA undergoes long-distance transport and interacts with movement protein binding protein 2C in pear (Pyrus betulaefolia). Plant Cell Tiss Organ Cult 121, 109–119 (2015). https://doi.org/10.1007/s11240-014-0685-z
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DOI: https://doi.org/10.1007/s11240-014-0685-z