Using RNAi silencing to explore gene function during soybean nodulation

1. . 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. Finding the Promoter GUS Staining were used to analyze the expression levels of different promoters. The Process of Nodulation Cloning and Plant Transformation Reference: Stacey G, Clough S, Taylor C, Functional Genomics of Root Hair Infection. Small cDNA fragments of the genes to be studied were isolated and cloned into an entry vector. GUS Expressing Soybean Hairy Roots- 1 week 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 RNAi Fad2-Intron tNOS ccdB CmR CmR ccdB Promoters cloned at EcoRV. EcoRV pCMV::GUS p35S::GUS AscI AscI attRI attR2 attR2 attRI Reference: Stacey G, Clough S, Taylor C, Functional Genomics of Root Hair Infection. Reffrence: Perret X, Stahelin C, Broughton W. Molecular Basis of Symbiotic Promiscuity. Microbiology and Molecular Biology Reviews. Mar. 2000 p.180-201 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) Fad2-Intron sequence pFMV::GUS pSBV::GUS 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 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. GUS Expressing Soybean Nodules on Soybean Hairy Roots- 1 week Genes to be Studied We are focusing on the genes listed below that were found by DNA microarray anslysis to be significantly regulated during nodulation. Phenotypes of Transgenic Roots pCvMV::GUS p35S::GUS Later: Previously studied: Early: pFMV::GUS pSBV::GUS pFMV was chosen for its high expression in both the root and the nodules Research funded by the National Science Foundation.