Download Use of RNAi silencing to explore gene function during soybean

Using RNAi silencing to explore gene
function during soybean nodulation
Britney A. Koepf¹, Manju Govindarajulu ², Marc Libault¹, Laurent Brechenmacher¹, Chris Taylor ² and
Gary Stacey¹ 1National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri,
Columbia, MO 65211; 2 Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, Missouri 63132
Introduction
Nodulation is the result of a symbiotic association between bacteria within the family Rhizobiaceae and a specific legume host. The interaction between the plant host and the bacterium
leads to the formation of a novel, highly efficient, nitrogen-fixing organ, the nodule. The symbiotic partners recognize one another through the exchange of chemical signals; such as,
isoflavonoids secreted by the plant and the lipo-chitin Nod factors excreted by the bacterium. These chemical signals affect root hair morphology; a critical step in the infection process.
Genes involved in both the early (3 hours post inoculation until 18 hours) and later stages (4 days post inoculation until 16 days) of nodule development were identified by DNA
microarray analysis and then confirmed by quantitative RT-PCR. A large number of genes were found to respond to inoculation and, of these, 22 were chosen for more in-depth analysis.
One means to determine gene function is to silence expression using RNAi and then examine the resulting phenotypic changes. We are now applying this approach to all 22 genes under
investigation.
Zinc finger
Protein
Transcription
Factior
Subtilisin
protease
Chitinase
Sucrose
Cleavage Protein
Potassium
Transporter
Phospholipase D
Cellulose
synthase
Half sized
nodules
4
3
Small
sized
nodules
2
1
Nodule
primordia
0
Ce
l2
R
Trans kinase
pSBV::GUS
pFMV::GUS
5
(c
o
Calcium
exchanger
Map Kinase
6
to
r
bax inhibitor
Calmodulin-like
protein
Cytokinin
oxidase
Fbox protein
Hypersensitive
induced protein
hydrolase
Lectin
Transfactor myb
Gene name
Full formed
nodules
ve
c
Calcium
kinase
Previously studied:
7
pFMV was chosen for its high
expression in both the root and the
nodules
pt
y
Gene Name
Gene name
p35S::GUS
m
Later:
Phenotypes of Transgenic Roots
64
19
A*
E
Early:
pCvMV::GUS
DR
We are focusing on the genes listed below that were
found by DNA microarray anslysis to be significantly
regulated during nodulation.
PL
Genes to be Studied
GUS Expressing Soybean
Nodules on Soybean Hairy
Roots- 1 week
Plant shoots are grown first in rock-wool cubes soaked with the A.
rhizogenes. Some of the roots that emerge will be transgenic and will be
inoculated with B. japonicum. In these roots the inserted vector will be
transcribed forming the hairpin (diagram above) structure, which will
utilize the plants own defense mechanisms to effectively silence the
gene being studied (see above). Different RNAi constructs were used
and the resulting phenotypes toward nodulation were analyzed.
HK
TR
pFMV::GUS
cR
Once the rhizobia ® recognize the root hair (rh) signal (A),
the bacteria attach to the root hair (B). The rhizobial Nod
factors induce root hair curling entrapping the bacteria,
which penetrate the cell creating a center of infection (ci)
where the infection thread (it) begins its journey (C)
following the nucleus (n). (D) The thread elongates crossing
from the epidermal cells (ep) into the nodule primodium
where cortical (c) cells divide in preparation. (Ed) The
infection thread releases bacteroids (b) creating
symbiosomes (s) in the newly formed infected nodule cells.
Other abbreviations ramified infection thread (rit) and
endodermis (ed)
Su
pSBV::GUS
Reffrence: Perret X, Stahelin C, Broughton W. Molecular Basis of
Symbiotic Promiscuity. Microbiology and Molecular Biology
Reviews. Mar. 2000 p.180-201
R
p35S::GUS
R
Ch
i
pCMV::GUS
R
2R
GUS Expressing Soybean
Hairy Roots- 1 week
Small cDNA fragments of the genes to be studied were isolated
and cloned into an entry vector.
Invitrogen’s Gateway™
Fad2-Intron
tNOS
RNAi
is the system used,
ccdB Cm
Cm
ccdB
Promoters cloned at EcoRV.
wherein a binary vector
EcoRV
receives the same gene
fragment from the
AscI
AscI
attRI
attR2
attR2
attRI
entry vector in inverted
orientation between the
Reference: Stacey G, Clough S, Taylor C, Functional Genomics of Root Hair Infection.
two attR1/ Attr2 sites.
This binary vector is
Fad2-Intron
introduced into
s
Agrobacterium
e
rhizogenes (K599),
q
u
which
is
used
to
e
generate the composite
n
Reference: Collier R, Fuchs B, Walter N, Lutke W, Taylor C.
c
Ex vitro composite plants: an inexpensive, rapid method for
plants
e
root biology. Plant Science. 2005 43, 449–457
M
ap
GUS Staining were used to analyze the
expression levels of different promoters.
Cloning and Plant Transformation
nt
ro
l)
Reference:
Stacey G,
Clough S,
Taylor C,
Functional
Genomics of
Root Hair
Infection.
Finding the Promoter
Total # of nodules/transgenic root
The Process of Nodulation
Research funded by the National Science Foundation.