Download Nicotiana benthamiana

Silencing of NbRNP1 gene encoding U3 small nucleolar ribonucleoprotein
affects leaf and root development in Nicotiana benthamiana
1
2
3
4
1
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
Abstract
4. Aberrant leaf development and senescence of NbRNP-silenced plants
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 virusinduced 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.
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 NbRNP-silenced
plants (n=8).
The
experimental
were
repeated three times,
and a representative
result is shown.
Fig. 4
Results
1. Isolation of a full-length cDNA encoding NbRNP from N. benthamiana
5. Silencing of NbRNP damaged mesophyll and vascular cell structures
**
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
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.
Fig. 5 Microscopic examination of
leaf structures in N. benthamiana
plants. Light microscopy of leaf tissues
of control (TRV2-Ve) and NbRNPsilenced (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
(A)
3. Suppression of endogenous transcripts of NbRNP by VIGS
TRV2-Ve
TRV2-NbRNP
(B)
(C)
Fig. 6 The roots of the control (TRV2-Ve) and
NbRNP-silenced (TRV2-NbRNP) plants. The
leaves of the N. benthamiana plants were Agroinfiltrated 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 VIGStreated 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
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.
1.
2.
3.
4.
Subcellular localization studies suggest that MPP10 domain of NbRNP protein is important for nuclear localization.
Suppression of NbRNP gene affected biosynthesis of 18S rRNA.
Silencing of NbRNP affects leaf and root development.
Further study is required to understand the component that involved in NbRNP-mediated development programs.