Silencing of NbRNP1 gene encoding U3 small nucleolar ribonucleoprotein affects leaf and root development in Nicotiana benthamiana Abstract Results 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 NbRNP protein sequence with those from animals and plants showed conservation of the MPP10 (M phase phosphoprotein 10) domain. Subcellular localization analysis of NbRNP showed that NbRNP was predominantly localized in nucleus. The C-terminal of NbRNP containing the MPP10 domain was critical to nuclear localization. Functional characterization of NbRNP gene by virus- induced gene silencing demonstrated its role in leaf and root development. In NbRNP-silenced plants, newly emerging leaves exhibited needle-like phenotype as compared to vector-control plants. In addition, the growth of roots was severely inhibited in NbRNP-silenced plants. 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. 1.Isolation of a full-length cDNA encoding NbRNP from N. benthamiana 4. Aberrant leaf development and senescence of NbRNP-silenced plants Fig. 1 Alignment of the amino acid sequences of NbRNP with RNPs from plants and animals. 2. Subcellular localization and immunoblotting of NbRNP-GFP proteins 5. Silencing of NbRNP damaged mesophyll and vascular cell structures 3. Suppression of endogenous transcripts of NbRNP by VIGS Fig. 3 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. Fig. 4 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). The experimental were repeated three times, and a representative result is shown. Fig. 5 Microscopic examination of leaf structures in N. benthamiana plants. Light microscopy of leaf tissues of control (TRV2-Ve) and NbRNP- silenced (TRV2-NbRNP) 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 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. 7. NbRNP-silenced plants showed stunt root growth Fig. 6 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. (A and B) 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. (C) The VIGS- treated plants from TRV2-Ve and TRV2-NbRNP were grown in soil for 3 weeks. The dry weight of the roots from one individual plant corresponding to TRV2-Ve and TRV2-NbRNP was shown. Conclusions and future work 1.Subcellular localization studies suggest that MPP10 domain of NbRNP protein is important for nuclear localization. 2.Suppression of NbRNP gene affected biosynthesis of 18S rRNA. 3.Silencing of NbRNP affects leaf and root development. 4.Further study is required to understand the 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 Fig. 2 Subcellular localization and immunoblotting of NbRNP-GFP proteins in N. benthamiana. A schematic diagram of the constructs used for transient transformation of N. benthamiana leaf epidermal cells. TRV2-Ve TRV2-NbRNP * * (A) (B) (C)