Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr

Abstract

Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 µM), nevertheless SSE-supplemented medium contained 4.7 µM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.

Genomic comparison of Bradyrhizobium japonicum strains with different symbiotic nitrogen-fixing capabilities and other Bradyrhizobiaceae members

“…Twenty-five nanograms of fragmented DNA was labeled with [a-33 P] dCTP (2500 Ci mmol À1 ; Amersham Biosciences, Pittsburgh, PA, USA) by using a Rediprime II random primer labeling system (Amersham Biosciences). DNA macroarray of B. japonicum USDA110 (Ito et al, 2006;Wei et al, 2008) was used for CGH analyses. The macroarray contains 3960 spots on a nylon membrane, on which 2.7-kb DNA segments (on average) of USDA110 brb libraries (Kaneko et al, 2002) were mainly spotted.…”

“…The macroarray contains 3960 spots on a nylon membrane, on which 2.7-kb DNA segments (on average) of USDA110 brb libraries (Kaneko et al, 2002) were mainly spotted. Hybridization was carried out as described earlier (Uchiumi et al, 2004;Ito et al, 2006;Wei et al, 2008). The membrane was washed twice for 15 min at 55 1C in 2 Â saline sodium citrate (SSC) containing 0.1% sodium dodecyl sulfate and twice for 15 min at 55 1C in 0.1 Â SSC (0.2 Â SSC was used for bacteria other than B. japonicum) containing 0.1% sodium dodecyl sulfate (1 Â SSC is 0.15 M NaCl plus 0.015 M sodium citrate).…”

“…The membrane was washed twice for 15 min at 55 1C in 2 Â saline sodium citrate (SSC) containing 0.1% sodium dodecyl sulfate and twice for 15 min at 55 1C in 0.1 Â SSC (0.2 Â SSC was used for bacteria other than B. japonicum) containing 0.1% sodium dodecyl sulfate (1 Â SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Image acquisition and data analysis were performed as described earlier (Uchiumi et al, 2004;Ito et al, 2006;Wei et al, 2008). At least three sets of biologically independent array analyses were performed for each strain.…”

“…Total RNA of bacteroids (isolated from 2 g of 'Enrei' nodules) and free-living cells (cultured in 100 ml of Yeast Mold medium) was prepared by the hot phenol method as described earlier (Ditta et al, 1987;Uchiumi et al, 2004). Bacteroid preparation, cDNA labeling, hybridization, image acquisition and data analysis were performed as described earlier (Uchiumi et al, 2004;Ito et al, 2006;Wei et al, 2008).…”

“…The CGH approach is suitable for efficient determination of the global genome variations among target bacterial populations, although the comparison has been limited to the original gene repertoires in template genomes. In rhizobia, DNA micro-and macroarrays have been used mainly for global gene expression analysis of B. japonicum (Chang et al, 2007;Pessi et al, 2007;Brechenmacher et al, 2008;Wei et al, 2008), Mesorhizobium loti (Uchiumi et al, 2004) and Sinorhizobium meliloti (Becker et al, 2004). CGH analyses of four natural isolates of S. meliloti by microarray revealed a significant fraction of variable genes including transposease and unknown genes on pSymA megaplasmid (Giuntini et al, 2005).…”