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Fig. 6. Phenotype of nodules and infection structures induced by A. caulinodans strain ORS571 and ORS571-oac2 on S. rostrata stems. (A) ORS571[pRG960SD-32]-induced stem nodule 30 days postinfection (dpi) lacking β-glucuronidase (GUS) activity on the exterior of the nodule. (B) Hand-cut section through a nodule as in A with a clearly stained fixation zone (asterisk). (C) Semithin section through an ORS571-oac2-infected root primordium 3 dpi showing colonization of the fissure (arrow), the induction of a nodule primordium (NP), and the start of the formation of a primary infection pocket (PI). (D-G) Details of infection structures in ORS571-oac2-induced nodules: PI surrounded by enlarged cells (EC) from where infection threads (arrowheads) depart (21 dpi) (D); secondary infection pocket (SI) surrounded by enlarged cells with departing infection threads (arrowheads) (46 dpi) (E); large infection structure that reaches from the surface deep into the nodule tissues (46 dpi) (F); and mixture of thin (TIT) and enlarged (LIT) ramifying infection threads departing from an infection pocket (IP) (46 dpi) (G). (Bars, 1 mm in A-C, 200 m m D-G.)

Fig. 7. Immunolocalization of two early nodulins in nodules induced by ORS571 and ORS571-oac2. Previous comparison of in situ hybridization and immunolocalization have revealed that the transcripts and the proteins for both nodulins colocalized in wild-type nodules (1-3). During early stages in ORS571-induced nodules, Srchi13 was expressed in cortical cells flanking the infection pockets and the infection center, whereas in a mature nodule, the mRNA was localized in the nodule parenchyma and uninfected cells of the fixation zone. At 10-20 days postinfection (dpi), expression was no longer apparent (1, 3). Srchi24, the second nodulin analyzed, is transiently expressed in wild-type nodules from 24 h post infection until 10 days after inoculation in the outermost cortical layers and those surrounding the infection pockets (2). (A-H) Srchi13. (I-P) Srchi24. (A and B) In ORS571-induced nodules, Srch13 is located in cortical cells flanking infection pockets and in the nodule parenchyma at 4 dpi and 9 dpi, respectively. (C-H) Presence of nodulin Srchi13 in ORS571-oac2-nodules at 8 dpi in cortical cells and around infection pockets (C and D), still surrounding infection structures at 21 (E and F) and 46 (G) dpi. (H) Merged image of immunolocalization and autofluorescence showing that Srchi13 localizes to a layer of small cells that border the enlarged cells flanking the infection structures (46 dpi). Location of Srchi24 at 4 (I) and 9 (J) dpi in ORS571-induced nodules in the outermost cortical cells and surrounding infection pockets. (K-P) ORS571-oac2 nodules at 4 (K) and 8 (L) dpi, whose localization is comparable with that in I and J; at later time points (46 dpi), the nodulin is still present surrounding infection pockets (M and O). (N and P) Merged images of immunolocalization and autofluorescence showing that Srchi24 localizes to a layer of enlarged cells that border the infection structures. C, cortical layer; EC, enlarged cells; F, fissure; FZ, fixation zone; IP, infection pocket; NP, nodule primordium; NPa, nodule parenchyma; PI, primary infection pocket; SI, secondary infection pocket. (Bar, 250 m m.)

1. Goormachtig, S., Lievens, S., Van de Velde, W., Van Montagu, M. & Holsters, M. (1998) Plant Cell 10, 905-915.

2. Van de Velde, W. (2002) Ph.D. thesis (Ghent University, Ghent, Belgium).

3. Goormachtig, S., Van de Velde, W., Lievens, S., Verplancke, C., Herman, S., De Keyser, A. & Holsters, M. (2001) Plant Physiol. 127, 78-89.

Fig. 8. Inability of plant defense responses to block infection thread progression and bacterial release. (A) Autofluorescence in some of the enlarged cells (EC) that surround secondary infection structures; the plant cells that harbor infection structures (asterisk) do not autofluoresce, nor do cells surrounding newly formed infection structures (lozenge) (61 days postinfection). (B and C) Transmission electron microscopy images illustrating the flexibility of thin (TIT) and thick (LIT) walled infection threads; signs of plant cell decay such as protein aggregates (PA) and flattened nuclei (FN) are obvious in cells that flank or are one cell layer away from cells harboring infection structures (46 days postinfection). (Bars, 100 m m in A and 5 m m in B and C.)

For bacterial quantification in infected tissues, 10-20 induced nodules were excised from the stem and ground in yeast extract broth (YEB) medium. Serial dilutions of these bacterial suspensions were plated on YEB medium supplemented with antibiotics. For ORS571, an exponential increase in the number of bacteria was observed during the first 8 days of the interaction, which coincided with the establishment of the fixation zone. From then on, the bacterial population remained constant. For the ORS571-oac2 population, the kinetics were different. At the start of the interaction, very low numbers of bacteria could be recovered, whereas at later time points the bacterial population increased reaching a level 100-fold lower than that of the wild type.

Numbers are the average occupancy of 10 individual nodules. NO, not observed.