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Silencing of Nicotiana benthamiana NbRNP1 gene encoding U3 small nucleolar ribonucleoprotein affects leaf development by
interference with ASYMMETRIC LEAVES 1 and 2
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魏惠美 、李勇毅 、許再文 、楊俊逸 、傅士峰
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
2. Subcellular localization and immunoblotting
of GFP-NbRNP1 proteins
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 virusinduced 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
d e v e l o p m e n t
p r o g r a m s .
Fig. 2 Subcellular localization and immunoblotting of NbRNP-GFP
proteins in N. benthamiana.
3.Using BiFC to analysis the interaction between
NbRNP1 and Fibrillarin in N. benthamiana plant
8. Analysis of the root growth in VIGS N.
benthamiana plants
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.
Fig. 8 The roots of the control (TRV2Ve) and NbRNP-silenced
(TRV2NbRNP) plants. The leaves of the N.
benthamiana plants were Agroinfiltrated 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 VIGStreated plants from TRV2-Ve and
TRV2-NbRNP were grown in soil for
3 weeks.
6.Aberrant leaf development and senescence of
NbRNP1-silenced plants
The typical phenotypes of
NbRNP-silenced N. benthamiana
plants. (A) VIGS phenotypes of the
leaves in TRV empty-vector controland 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 NbRNPsilenced plants (n=8).
9.Analysis of the numbers of nucleus in the leaf of
VIGS N. benthamiana plants
Fig. 6
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.
Results
1. Isolation of a full-length cDNA encoding RNP1 from N.
benthamiana
5. Analysis of the gene expression related to rRNA
biosynthesis in VIGS N. benthamiana plants
4. Analysis of mRNA content in VIGS N.
benthamiana plants.
7. Analysis of the leaf development by slice 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. 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
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. 1 Alignment of the
amino acid sequences of
NbRNP1 with
representative plant
RNP members.
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 NbRNP1silenced plants is less than the
control.
10. Analysis of the numbers of the gene expression in
the root and leaf of VIGS N. benthamiana plants
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.