Role of brassinosteroid signaling in modulating Tobacco mosaic virus


Plant steroid hormones, brassinosteroids (BRs), play essential roles in plant growth, development and stress responses. However, mechanisms by which BRs interfere with plant resistance to virus remain largely unclear. In this study, we used pharmacological and genetic approaches in combination with infection experiments to investigate the role of BRs in plant defense against Tobacco Mosaic Virus (TMV) in Nicotiana benthamiana. Exogenous applied BRs enhanced plant resistance to virus infection, while application of Bikinin (inhibitor of glycogen synthase kinase-3), which activated BR signaling, increased virus susceptibility. Silencing of NbBRI1 and NbBSK1 blocked BR-induced TMV resistance and silencing of NbBES1/BZR1 blocked Bikinin-reduced TMV resistance. Silencing of NbMEK2, NbSIPK and NbRBOHB all compromised BR-induced virus resistance and defense-associated genes expression. Furthermore, we found MEK2-SIPK cascade activated while BES1/BZR1 inhibited RBOHB-dependent ROS production, defense gene expression and virus resistance induced by BRs. Thus, our results revealed BR signaling had two opposite effects on viral defense response. On the one hand, BRs enhanced virus resistance through MEK2-SIPK cascade and RBOHB-dependent ROS burst. On the other hand, BES1/BZR1 inhibited RBOHB-dependent ROS production and acted as an important mediator of the trade-off between growth and immunity in BR signaling.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling pathways that transduce extracellular stimuli into intracellular responses in eukaryotes. MAPK cascades are composed of three protein kinase modules: MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs) and MAPKs, which are linked in various ways to upstream receptors and downstream targets. BR signaling pathway in modulating TMV resistance in N. benthamiana. Chemical treatment and VIGS approach demonstrated that BRI1, BSK1 and GSK3-like kinases positively while BES1/BZR1 negatively mediated BR-induced virus resistance. Loss-of-function analyses showed that MEK2-SIPK cascade and RBOHB played key roles in BR-induced virus resistance. We also showed that MEK2-SIPK cascade induced by BRs mediated RBOHB-dependent oxidative burst in N. benthamiana plants response to TMV.

We tested control and treated N. benthamiana plants for their resistance against infection of TMV, which was tagged with green fluorescent protein (GFP) . N. benthamiana plants were pretreated with water, brassinolide (BL, the most active BR) and brassinazole (BRZ, a specific inhibitor of BR biosynthesis) before TMV-GFP inoculation. Virus accumulation was confirmed by direct observation of GFP fluorescence, as well as by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis of viral replication, at 3, 5 and 7 days post-inoculation (dpi), respectively. N. benthamiana plants treated with BL showed weak GFP fluorescence as compared with water-treated plants conclusion is consistent with qRT-PCR and western blotting analysis of viral accumulation. However, plants treated with BRZ appeared to have the strongest GFP fluorescence and the highest viral replication level in comparison with water and BL treatment. These results indicate that BRs play a positive role in plant resistance to TMV.

Statistical analysis: Statistical analysis of the results from experiments with three or more mean values used a one-way analysis of variance (ANOVA) as dictated by the number of main effects. The difference was considered to be statistically significant when P < 0.05.

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