Identification of new potential regulators of the Medicago truncatula-Sinorhizobium meliloti symbiosis using a large-scale suppression subtractive hybridization approach.
We set up a large-scale suppression subtractive hybridization (SSH) approach to identify Medicago truncatula genes differentially expressed at different stages of the symbiotic interaction with Sinorhizobium meliloti, with a particular interest for regulatory genes. We constructed 7 SSH libraries covering successive stages from Nod factor signal transduction to S. meliloti infection, nodule organogenesis, and functioning. Over 26,000 clones were differentially screened by two rounds of macroarray hybridizations. In all, 3,340 clones, corresponding to genes whose expression was potentially affected, were selected, sequenced, and ordered into 2,107 tentative gene clusters, including 767 MtS clusters corresponding to new M. truncatula genes. In total, 52 genes encoding potential regulatory proteins, including transcription factors (TFs) and other elements of signal transduction cascades, were identified. The expression pattern of some of them was analyzed by quantitative reverse-transcription polymerase chain reaction in wild-type and in Nod- M. truncatula mutants blocked before or after S. meliloti infection. Three genes, coding for TFs of the bHLH and WRKY families and a C2H2 zinc-finger protein, respectively, were found to be upregulated, following S. meliloti inoculation, in the infection-defective mutant lin, whereas the bHLH gene also was expressed in the root-hair-curling mutant hcl. The potential role of these genes in early symbiotic steps is discussed.
Godiard
Laurence
L
Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR CNRS-INRA 2594/441, BP 52627, F- 31 326 Castanet Tolosan, France. lgodiard@toulouse.intra.fr
Niebel
Andreas
A
Micheli
Fabienne
F
Gouzy
Jérôme
J
Ott
Thomas
T
Gamas
Pascal
P
D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant; D046913:Medicago truncatula / Q000235:genetics* / Q000382:microbiology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D010940:Plant Proteins / Q000235:genetics; D018517:Plant Roots / Q000235:genetics / Q000382:microbiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D016962:Sinorhizobium meliloti / Q000254:growth & development*; D013559:Symbiosis / Q000235:genetics*
D018076:DNA, Complementary; D010940:Plant Proteins