Abstract
Callose is a β-l,3-glucan with diverse roles in the viral pathogenesis of plants. It is widely believed that the deposition of callose and hypersensitive reaction (HR) are critical defence responses of host plants against viral infection. However, the sequence of these two events and their resistance mechanisms are unclear. By exploiting a point inoculation approach combined with aniline blue staining, immuno-electron microscopy and external sphincters staining with tannic acid, we systematically investigated the possible roles of callose deposition during viral infection in soybean. In the incompatible combination, callose deposition at the plasmodesmata (PD) was clearly visible at the sites of inoculation but viral RNA of coat protein (CP-RNA) was not detected by RT-PCR in the leaf above the inoculated one (the upper leaf). In the compatible combination, however, callose deposition at PD was not detected at the site of infection but the viral CP-RNA was detected by RT-PCR in the upper leaf. We also found that in the incompatible combination the fluorescence due to callose formation at the inoculation point disappeared following the injection of 2-deoxy-d-glucose (DDG, an inhibitor of callose synthesis). At same time, in the incompatible combination, necrosis was observed and the viral CP-RNA was detected by RT-PCR in the upper leaf and HR characteristics were evident at the inoculation sites. These results show that, during the defensive response of soybean to viral infection, callose deposition at PD is mainly responsible for restricting the movement of the virus between cells and it occurs prior to the HR response.
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Abbreviations
- HR:
-
Hypersensitive reaction
- PD:
-
Plasmodesmata
- CP:
-
Coat protein
- DDG:
-
2-Deoxy-d-glucose
- TMV:
-
Tobacco mosaic virus
- PVX:
-
Potato virus X
- CMV:
-
Cucumber mosaic virus
- SMV:
-
Soybean mosaic virus
- PVY:
-
Potato virus Y
- PIPES:
-
1,4-Piperazinediethanesulfonic acid
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Acknowledgments
We thank Dr. Haijian Zhi (University of Nan Jing Agricultural University, China) for providing the virus strains used and advice on their propagation. We thank Shanjin Huang (Institute of Botany, the Chinese Academy of Sciences, China) for reading the manuscript and providing helpful comments. This work was supported by the National Natural Science Foundation of China (no.30971706) and by the Natural Science Foundation of Hebei Province (no.C2008000321).
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Communicated by A.-C. Schmit.
W. Li and Y. Zhao contributed equally to this work.
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Fig. S1 Distributions of necrotic lesions in large and small areas of infection caused by the two different inoculation methods (with a brush or with a tooth-pick) used in the incompatible combination between the soybean cv. Jidou 7 and the SMV strain N3. a showing the symptom of large infection areas at 2 h post-inoculation with a brush. b showing the random distribution of necrotic lesions (arrows) appeared on the large infection area at 96 h post-inoculation with a brush. c showing the symptom of small infection area (arrow) at 2 h post-inoculation with a tooth-pick. d showing the specific distribution of necrotic lesions (arrows) appeared on the small area of infection at 96 h post-inoculation with a tooth-pick. Bar = 1 cm (a and c) and 2 cm (b and d).
Fig. S2 Fluorescence due to mechanical damage caused by either the mimic-inoculation or cutting during sampling on soybean cv. Jidou 7 leaves. a–d showing fluorescence at inoculation sites at 2, 8, 12 and 168 h. Irregular fluorescence of callose appeared at 2 and 8 h post mimic-inoculation (indicated by arrows), likely caused by mechanical damage of inoculation (a, b). The fluorescence disappeared at 12 h after mimic-inoculation (c, d). e showing fluorescence of callose at the edge of cutting with a blade (indicated by arrows). a’–e’ were light micrographs of a–e. Bar = 0.2 mm (a-d and a’-d’) and 0.1 mm (e and e’).
Fig. S3 Fluorescent labeling of callose with aniline blue at the sites of inoculation in the compatible combination between the soybean cv. Jidou 7 and SMV strain SC-8. a–j showing photographs of fluorescence at the sites of inoculation at 2, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h post-inoculation, respectively. The presence of irregular fluorescence caused by inoculation was clear visible during the early stages (2 and 8 h) of inoculation (a and b, arrows). Fluorescence at the sites of inoculation was not detected at 12 to168 h post-inoculation. a’–j’ were the light micrographs of a–j. Bar = 0.1 mm.
Fig. S4 Immunogold-labeling of callose at sites of inoculation in the compatible combination between the soybean cv. Jidou 7 and SMV strain SC-8 and mimic-inoculated soybean cv. Jidou 7 leaves. a–d showing the absence of immunogold particles at PD at 2, 12, 96 and 168 h post-inoculation from the compatible combination. e–h showing the absence of immunogold particles at PD at 2, 12, 96 and 168 h post-inoculation in the mimic-inoculated plants. Immunogold particles were visible at cell walls and in cytoplasms of plants in the compatible combination (a) and from the mimic-inoculated plants (e) but these particles were not located at PD. The irregular immunogold particles were likely due to mechanical damage caused by inoculation or by cutting during sampling. CW = cell wall, PD = plasmodesmata. Bar = 500 nm (a and e), 330 nm (b and h), 800 nm (c), 380 nm (d and f) and 600 nm (g).
Fig. S5 Presence of sphincters at plasmodesmal entrances of cv. Jidou 7 leaves inoculated with SMV strain SC-8 and mimic-inoculated ones. a–d showing that electron-dense sphincters were not detected at PD entrances at 2, 12, 96 and 168 h post-inoculation from the compatible combination. e–h showing that electron-dense sphincters were not detected at PD entrances at 2, 12, 96 and 168 h post-inoculation in the mimic-inoculated plants. CW = cell wall, PD = plasmodesmata. Bar = 380 nm (a, d, e and g), 330 nm (b), 200 nm (c), 250 nm (f) and 450 nm (h).
Fig. S6 Virulence assays were conducted in the compatible combination between the soybean cv. Jidou 7 and SMV strain SC-8. Necrotic spots lengths were used as a measure of virulence. a–d showing necrotic spots at 96, 120, 144 and 168 h post-inoculation. Arrows point to the necrotic spots. a’–d’ showing necrotic spots at 96, 120, 144 and 168 h post-inoculation following the injection of 500 μM DDG. Arrows point to the necrotic spots. Bar = 2 mm.
Fig. S7 Necrotic spots lengths between injection of 500 μM DDG and without 500 μM DDG in the compatible combination between the soybean cv. Jidou 7 and SMV strain SC-8. There were no difference in necrotic spots lengths between injection of 500 μM DDG and without 500 μM DDG. In each experiment, 20 leaves were inoculated, and the mean ± standard deviations of data from three independent experiments are presented. Similar results were obtained in independent experiments.
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Li, W., Zhao, Y., Liu, C. et al. Callose deposition at plasmodesmata is a critical factor in restricting the cell-to-cell movement of Soybean mosaic virus . Plant Cell Rep 31, 905–916 (2012). https://doi.org/10.1007/s00299-011-1211-y
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DOI: https://doi.org/10.1007/s00299-011-1211-y