Using RNAi silencing to explore gene function during soybean nodulation

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.

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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

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)

Later: Previously studied:

Finding the PromoterGUS Staining were used to analyze the expression levels of different promoters.

GUS Expressing Soybean Hairy Roots- 1 week

pCMV::GUS p35S::GUS

pSBV::GUS pFMV::GUS

GUS Expressing Soybean Nodules on Soybean Hairy Roots- 1 week

pCvMV::GUS p35S::GUS

pSBV::GUS pFMV::GUS

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Noduleprimordia

The Process of Nodulation

Gene Name

Calcium kinase

Calcium exchanger

Trans kinase

Zinc finger Protein

Transcription Factior

Genes to be StudiedWe are focusing on the genes listed below that were found by DNA microarray anslysis to be significantly regulated during nodulation.

Early:

Gene name

Map Kinase

Chitinase

Sucrose Cleavage Protein

Potassium Transporter

Phospholipase D

Cellulose synthase

Phenotypes of Transgenic Roots

Cloning and Plant Transformation

RNAi

Research funded by the National Science Foundation.

Gene name

bax inhibitor

Calmodulin-like protein

Cytokinin

oxidase

Fbox protein

Hypersensitive induced protein

hydrolase

Lectin

Transfactor myb

Subtilisin protease

Small cDNA fragments of the genes to be studied were isolated and cloned into an entry vector.

Fad2-Intron

AscI

EcoRV

AscI

ccdB

attR2attRI

CmR

tNOSCmR

attRIattR2

ccdB

Promoters cloned at EcoRV.

Invitrogen’s Gateway™ is the system used, wherein a binary vector receives the same gene fragment from the entry vector in inverted orientation between the two attR1/ Attr2 sites. This binary vector is introduced into Agrobacterium rhizogenes (K599), which is used to generate the composite plants

Reffrence: Perret X, Stahelin C, Broughton W. Molecular Basis of Symbiotic Promiscuity. Microbiology and Molecular Biology Reviews. Mar. 2000 p.180-201

Reference: Stacey G, Clough S, Taylor C, Functional Genomics of Root Hair Infection.

Reference: Stacey G, Clough S, Taylor C,

Functional Genomics of

Root Hair Infection.

Fad2-Intron

sequence

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.

pFMV was chosen for its high expression in both the root and the

nodules

Reference: Collier R, Fuchs B, Walter N, Lutke W, Taylor C. Ex vitro composite plants: an inexpensive, rapid method for root biology. Plant Science. 2005 43, 449–457