The nodules shown in Figure 3 are expressing β-glucuronidase (GUS

The nodules shown in Figure 3 are expressing β-glucuronidase (GUS) from a pJH104 plasmid insertion in Smc00911. The nodules shown were stained for 3.75 hr. There is strong staining throughout the nodule, with slightly weaker staining at the invasion zone near the distal end of the nodule. The nodule expression of the SMc00911::GUS fusion is much stronger than the expression of any of the other fusions tested (see Figure 4 and Table 3). In contrast, SMc00911 is expressed at a very low level by free-living S. meliloti carrying the SMc00911::GUS fusion grown on LBMC plates (Figure 3G

and Table 3). For comparison, Figure 3G also shows that a greA::GUS fusion strain of S. meliloti constructed with the same reporter insertion plasmid, pJH104, is find more strongly expressed under these conditions. Table 3 summarizes

the expression data for all of the GUS fusion strains. Figure 3 Expression of β-glucuronidase (GUS)-encoding reporter gene uidA inserted within SMc00911. S. meliloti within alfalfa root nodules (B–F) express GUS inserted in SMc00911 throughout the nodule. Panel A shows an alfalfa nodule invaded by wild type S. meliloti 1021 that does not express GUS (subjected to the same staining SC79 molecular weight procedure as B–F). (Roots in B, C, and D were inoculated with strain SMc00911. Xsd1. Roots in E and F were inoculated with strain SMc00911.original.) Nodules were stained for 3.75 hr after 5 weeks of growth post-inoculation. Scale bars correspond to 0.1 mm. Panel G shows SMc00911-controlled

GUS expression in S. meliloti grown on solid LBMC medium. Wild type S. meliloti 1021 is shown as a negative control for GUS expression and a strain carrying the same GUS insertion plasmid in the greA gene is shown as a positive control PDK4 for GUS expression in free-living cells. Strain SMc00911.original and a ϕM12 transductant of this strain were tested on plants. Figure 4 Expression of β-glucuronidase (GUS)-encoding gene uidA expressed under the control of the promoter elements of the following ORFs: SMb20360 (B and C); SMc00135 (D and E); SMc01562 (F and G); SMc01266 (H and I); SMc03964 (J and K); SMc01424-22 (L and M); SMa0044 (N and O); SMb20431 (P and Q); SMc01986 (R and S); SMa1334 (T and U). SMb20360 and SMc00135 are strongly expressed in the nodules. (See Table 3 for percentage of nodules with GUS expression and staining times.) SMc01562, SMc01266, SMc03964 and the SMc01424-22 operon are expressed at a moderate level in the nodules. The remaining ORFs are expressed at a very low level in the nodule (or not at all). S. meliloti 1021 wild type is shown in Panel A as a negative control for GUS expression. Scale bars correspond to 0.1 mm.

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