Silencing of Nicotiana benthamiana NbRNP1 gene encoding U3 small nucleolar ribonucleoprotein affects leaf development by interference with ASYMMETRIC LEAVES 1 and 2 Abstract Nucleolar ribonucleoprotein (RNP) is involved in the molecular function that contributes to processing of ribosomal RNA precursors. RNP has been implicated in cell-cycle regulation in animals. However, the roles of RNP in plant developmental programs remain to be elusive. In this study, we isolated and characterized NbRNP1genes from Nicotiana benthamiana. Alignment of the NbRNP1 protein sequence with those from animals and plants showed conservation of the MPP10 (M phase phosphoprotein 10) domain. Subcellular localization analysis of NbRNP1 showed that NbRNP1 was predominantly localized in nucleus. The C-terminal of NbRNP1 containing the MPP10 domain was critical to nuclear localization. Functional characterization of NbRNP1 gene by virus- induced gene silencing and overexpression demonstrated its role in leaf and root development. In NbRNP1-silenced plants, newly emerging leaves exhibited needle-like phenotype as compared to vector-control plants. The leaf cells stained with nuclear-specific dye showed lower nuclear fluorescence as compared with the control plants. The knockdown of NbRNP1 gene in leaf significantly increased the percentage of dead cells. In addition, the growth of roots was severely inhibited in NbRNP1-silenced plants. Silencing NbRNP1 interfered with leaf development and the expression of genes involved in leaf polarity ASYMMETRIC LEAVES 1 and 2 (NbAS1 and AS2). In summary, this study suggests a role for NbRNP in leaf and root development. Further study is required to understand the molecular component that involved in NbRNP-mediated development programs. 魏惠美 1 、李勇毅 2 、許再文 3 、楊俊逸 4 、傅士峰 1 1 彰化師範大學生物系 2 自然科學博物館植物園 3 特有生物研究保育中心 4 中興大學生物化學研究所 Results Fig. 1 Alignment of the amino acid sequences of NbRNP1 with representative plant RNP members. 1. Isolation of a full-length cDNA encoding RNP1 from N. benthamiana 2. Subcellular localization and immunoblotting of GFP-NbRNP1 proteins 7. Analysis of the leaf development by slice in VIGS N. benthamiana plants 3.Using BiFC to analysis the interaction between NbRNP1 and Fibrillarin in N. benthamiana plant 4. Analysis of mRNA content in VIGS N. benthamiana plants. 8. Analysis of the root growth in VIGS N. benthamiana plants 9.Analysis of the numbers of nucleus in the leaf of VIGS N. benthamiana plants 10. Analysis of the numbers of the gene expression in the root and leaf of VIGS N. benthamiana plants 6. Aberrant leaf development and senescence of NbRNP1-silenced plants Fig. 6 The typical phenotypes of NbRNP-silenced N. benthamiana plants. (A) VIGS phenotypes of the leaves in TRV empty-vector control- and NbRNP-silenced plants at 3 weeks post inoculation. The photos of upper leaves were collected from the top of the individual plant. (B) Lateral branching shoots phenotypes of the NbRNP-silenced N. benthamiana plants. The morphology of shoot apexes was shown at 6 weeks post inoculation. (C) The shoot length at the similar developmental stage was compared between the control and NbRNP- silenced plants (n=8). * * Fig. 7 Microscopic examination of leaf structures in N. benthamiana plants. Light microscopy of leaf tissues of control (TRV2-Ve) and NbRNP1-silenced (TRV2-NbRNP1) plants. Stars denote cells at the upper endodermis. Typical organization of palisade and spongy mesophyll are marked in red and green, respectively. TRV control leaves had the typical leaf structure of dicotyledonous plants, with normal epidermal, palisade and spongy mesophyll cells. The organization of Fig. 2 Subcellular localization and immunoblotting of NbRNP-GFP proteins in N. benthamiana. 5. Analysis of the gene expression related to rRNA biosynthesis in VIGS N. benthamiana plants Fig. 4 Relative mRNA expression level in the root and apical leaves is stronger, the expression level of mature leaves and flowers is weaker. The results imply NbRNP1 gene may play important role in apical leaves and root development. Fig. 5 Suppression of NbRNP gene expression in N. benthamiana plants by VIGS. The NbRNP, Nb18S, NbITS and NbEF1 cDNA fragments were amplified by RT-PCR with the gene-specific primers (Table S1). NbEF1 was an internal loading control. the epidermal and spongy mesophyll cells was mostly maintained, but differentiation of palisade cells was disturbed. Scanning electron micrography of adaxial epidermis from the same VIGS plant. Fig. 9 Fluorescence microscopy to observe the number of nucleus in leaf tissues. we use the DAPI to stain the nucleus,and place the leaf under UV light.The lower panel show the bright feild. We discovered that the nucleus of NbRNP1- silenced plants is less than the control. Fig. 8 The roots of the control (TRV2- Ve) and NbRNP-silenced (TRV2- NbRNP) plants. The leaves of the N. benthamiana plants were Agro- infiltrated with the TRV-derived constructs. The VIGS-treated plants were transfer to a liquid-culture equipped bottle supplemented with nutrients. The phenotypic analysis of the roots from the TRV2-Ve and TRV2-NbRNP were performed after 3 week post inoculation. The VIGS- treated plants from TRV2-Ve and TRV2-NbRNP were grown in soil for 3 weeks. Fig. 3 Confocal microscopy to observe the interaction between NbRNP1 and Fibrillarin by BiFC analysis in N. benthamiana plant.The results revealed that in the view of merge screen,the interaction occurred between NbRNP1 and Fibrillarin. Fig. 10 We finally verified lots of gene expressins connected with NbRNP1 、 AS1 and AS2 、 Knat1 and 18S 、 28S rRNA biosynthesis in NbRNP1-silenced plants. Conclusions 1.This study suggests a role for NbRNP in leaf and root development. 2. Future study is required to clarify the component that involved in NbRNP-mediated development programs. 1.This study suggests a role for NbRNP in leaf and root development. 2. Future study is required to clarify the component that involved in NbRNP-mediated development programs.