Molecular Regulation Mechanism Of Callose Turnover During Soybean Resistance To Soybean Mosaic Virus Infection

Our previous work has proven that callose deposition at plasmodesmata is a critical factor in restricting the cell-to-cell movement of Soybean mosaic virus. We also found that in the incompatible combination the fluorescence spots due to callose formation at the inoculation point disappeared following the injection of 2-deoxy-D-glucose (DDG, an inhibitor of callose synthesis) and necrosis was observed and the expression of virus coat protein gene was detected in the upper leaf. These results showed that, during the defensive response of soybean to viral infection, callose deposition at Plasmodesmata (PD) was mainly responsible for restricting the movement of the virus between cells. In the present work, the regulation of individualβ-1,3-glucanase (BG) and callose synthase (CalS) were analyzed by immunohistochemistry, real-time RT-PCR, Western Blotting, activity determination and transmission electron microscopic immunolocalization in the incompatible combination between the Soybean mosaic virus (SMV) strain N3 and the soybean cv. Jidou 7 and in the compatible combination between the SMV strain SC-8 and the soybean cv. Jidou 7. The main results are as follows:1. CalS and BG were examined using immunohistochemistry in combination between the SMV and soybean. Results revealed that CalS and BG were located in the virus infected cells before necrotic spots appeared; CalS and BG were located in and around the necrotic spots after necrotic spots appeared.2. Changes in steady state levels of transcripts from CalS and BG were examined using real-time RT-PCR. Real-time RT-PCR analysis revealed that BG was upregulated in both incompatible combination and compatible combination; CalS was upregulated in the incompatible combination and was not upregulated in the compatible combination.3. Changes in steady state levels of posttranslation from CalS and BG were examined using Western Blotting. Western Blotting revealed that BG was upregulated in both incompatible combination and compatible combination; CalS was upregulated in the incompatible combination and was not upregulated in the compatible combination.4. Changes in steady state levels of activity from CalS and BG were examined. Our results showed that the activity of BG was upregulated in both incompatible combination and compatible combination; the activity of CalS was upregulated in the incompatible combination and was not upregulated in the compatible combination. 5. To directly test whether these enzymes target to PD, immunolocalization experiments were conducted with antibody raised against BG and CalS. Our results show that BG targeted to PD in the compatible combination and targeted to vacuole in the incompatible combination. CalS targeted to PD in both incompatible combination and compatible combination.Taken together, we get a basic model for mechanisms of callose turnover at plasmodesmata in SMV-soybean interaction. In the incompatible combination, BG was upregulated and it targeted to vacuole; CalS was upregulated and it targeted to PD. So there was lots of callose, which was responsible for restricting the movement of the virus between cells, deposited at PD in the incompatible combination. In the compatible combination, BG was upregulated and it targeted to PD; CalS was not upregulated and it targeted to PD. So there was no callose deposited at PD and viruses could transport between cells in the compatible combination.