Xing-Guang Deng

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid-mediated systemic virus resistance in Nicotiana benthamiana.

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H2O2 and NO. Scavenging of H2O2 or NO in upper leaves blocked BR-induced systemic virus resistance. BR-induced systemic H2O2 accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite-dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR-triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR-activated H2O2 is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H2O2 generation blocked BR-induced systemic NO production, but BR-induced H2O2 production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR-induced systemic virus defense in NbRBOHB-silenced plants, but H2O2 did not reverse the effect of NbNR silencing on BR-induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR-mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR-induced H2O2 and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR-mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)-dependent H2O2 production and subsequent systemic NR-dependent NO generation.

Role of brassinosteroid signaling in modulating Tobacco mosaic virus resistance in Nicotiana benthamiana

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.

Ethylene is Involved in Brassinosteroids Induced Alternative Respiratory Pathway in Cucumber (Cucumis sativus L.) Seedlings Response to Abiotic Stress

Effects of brassinosteroids (BRs) on cucumber (Cucumis sativus L.) abiotic stresses resistance to salt, polyethylene glycol (PEG), cold and the potential mechanisms were investigated in this work. Previous reports have indicated that BRs can induce ethylene production and enhance alternative oxidase (AOX) pathway. The mechanisms whether ethylene is involved as a signal molecule which connected BR with AOX in regulating stress tolerance are still unknown. Here, we found that pretreatment with 1 μM brassinolide (BL, the most active BRs) relieved stress-caused oxidative damage in cucumber seedlings and clearly enhanced the capacity of AOX and the ethylene biosynthesis. Furthermore, transcription level of ethylene signaling biosynthesis genes including ripening-related ACC synthase1 (CSACS1), ripening-related ACC synthase2 (CSACS2), ripening-related ACC synthase3 (CSACS3), 1-aminocyclopropane-1-carboxylate oxidase1 (CSACO1), 1-aminocyclopropane-1-carboxylate oxidase2 (CSACO2), and CSAOX were increased after BL treatment. Importantly, the application of the salicylhydroxamic acid (SHAM, AOX inhibitor) and ethylene biosynthesis inhibitor aminooxyacetic acid (AOA) decreased plant resistance to environmental stress by blocking BRs-induced alternative respiration. Taken together, our results demonstrated that ethylene was involved in BRs-induced AOX activity which played important roles in abiotic stresses tolerance in cucumber seedlings.

Ethylene and hydrogen peroxide are involved in brassinosteroid-induced salt tolerance in tomato

Crosstalk between phytohormone pathways is essential in plant growth, development and stress responses. Brassinosteroids (BRs) and ethylene are both pivotal plant growth regulators, and the interaction between these two phytohormones in the tomato response to salt stress is still unclear. Here, we explored the mechanism by which BRs affect ethylene biosynthesis and signaling in tomato seedlings under salt stress. The activity of 1-aminocyclopropane-1-carboxylate synthase (ACS), an ethylene synthesis enzyme, and the ethylene signaling pathway were activated in plants pretreated with BRs. Scavenging of ethylene production or silencing of ethylene signaling components inhibited BR-induced salt tolerance and blocked BR-induced activities of several antioxidant enzymes. Previous studies have reported that BRs can induce plant tolerance to a variety of environmental stimuli by triggering the generation of H2O2 as a signaling molecule. We also found that H2O2 might be involved in the crosstalk between BRs and ethylene in the tomato response to salt stress. Simultaneously, BR-induced ethylene production was partially blocked by pretreated with a reactive oxygen species scavenger or synthesis inhibitor. These results strongly demonstrated that ethylene and H2O2 play important roles in BR-dependent induction of plant salt stress tolerance. Furthermore, we also investigated the relationship between BR signaling and ethylene signaling pathways in plant processes responding to salt stress.

Alternative oxidase pathway is involved in the exogenous SNP-elevated tolerance of Medicago truncatula to salt stress.

Exogenous application of sodium nitroprusside (SNP) would enhance the tolerance of plants to stress conditions. Some evidences suggested that nitric oxide (NO) could induce the expression of alternative oxidase (AOX). In this study, Medicago truncatula (Medicago) was chosen to study the role of AOX in the SNP-elevated resistance to salt stress. Our results showed that the expression of AOX genes (especially AOX1 and AOX2b1) and cyanide-resistant respiration rate (Valt) could be significantly induced by salt stress. Exogenous application of SNP could further enhance the expression of AOX genes and Valt. Exogenous application of SNP could alleviate the oxidative damage and photosynthetic damage caused by salt stress. However, the stress resistance was significantly decreased in the plants which were pretreated with n-propyl gallate (nPG). More importantly, the damage in nPG-pretreated plants could not be alleviated by application of SNP. Further study showed that effects of nPG on the activities of antioxidant enzymes were minor. These results showed that AOX pathway played an important role in the SNP-elevated resistance of Medicago to salt stress. AOX could contribute to regulating the accumulation of reactive oxygen (ROS) and protect of photosystem, and we proposed that all these were depend on the ability of maintaining the homeostasis of redox state.

The Chilli Veinal Mottle Virus Regulates Expression of the Tobacco Mosaic Virus Resistance Gene N and Jasmonic Acid/Ethylene Signaling Is Essential for Systemic Resistance Against Chilli Veinal Mottle Virus in Tobacco

Genetic, physiological, and molecular analyses have revealed that the stress-related phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are known to participate in defense responses to mitigate biotic stress in plants. Recent evidence suggests that N-gene (a typical resistance gene) transcription is upregulated by Tobacco mosaic virus (TMV) infection, which is specifically a TMV-related phenomenon. In this study, we investigated N-gene transcription in tobaccoNN infected with Chilli veinal mottle virus (ChiVMV). Furthermore, we used a virus-induced gene-silencing-based genetics approach to investigate the function of SA, JA, and ET biosynthesis or signaling genes in systemic resistance to ChiVMV. Northern blot and qRT-PCR analysis indicate that N-gene transcription is stimulated by ChiVMV. Hormone measurements demonstrate that JA and ET increase rapidly during the early stages of ChiVMV infection, whereas SA increases slightly at later stages. JA and ET biosynthetic, signaling, and marker genes are significantly activated after ChiVMV inoculation, whereas SA biosynthetic, signaling, and marker genes are increased slightly. Silencing of JA, ET biosynthetic and signaling genes strongly increase the plants’ susceptibility to ChiVMV, whereas silencing of SA biosynthetic and signaling genes only partly compromise systemic resistance. Extensive ROS accumulate in JA, ET biosynthetic and signaling gene-silenced plants after ChiVMV infection, whereas only slight ROS produce in SA biosynthetic and signaling gene-silenced plants. Taken together, our results indicate that N-gene transcription is upregulated by ChiVMV infection, and the JA/ET pathways play an important role in plant systemic resistance against ChiVMV, whereas the SA pathway is only minorly involved.

A critical domain of Sweet potato chlorotic fleck virus nucleotide‐binding protein (NaBp) for RNA silencing suppression, nuclear localization and viral pathogenesis

RNA silencing is an important mechanism of antiviral defence in plants. To counteract this resistance mechanism, many viruses have evolved RNA silencing suppressors. In this study, we analysed five proteins encoded by Sweet potato chlorotic fleck virus (SPCFV) for their abilities to suppress RNA silencing using a green fluorescent protein (GFP)-based transient expression assay in Nicotiana benthamiana line 16c plants. Our results showed that a putative nucleotide-binding protein (NaBp), but not other proteins encoded by the virus, could efficiently suppress local and systemic RNA silencing induced by either sense or double-stranded RNA (dsRNA) molecules. Deletion mutation analysis of NaBp demonstrated that the basic motif (an arginine-rich region) was critical for its RNA silencing suppression activity. Using confocal laser scanning microscopy imaging of transfected protoplasts expressing NaBp fused to GFP, we showed that NaBp accumulated predominantly in the nucleus. Mutational analysis of NaBp demonstrated that the basic motif represented part of the nuclear localization signal. In addition, we demonstrated that the basic motif in NaBp was a pathogenicity determinant in the Potato virus X (PVX) heterogeneous system. Overall, our results demonstrate that the basic motif of SPCFV NaBp plays a critical role in RNA silencing suppression, nuclear localization and viral pathogenesis.

Nitric oxide is involved in brassinosteroid‐induced alternative respiratory pathway in Nicotiana benthamiana seedlings' response to salt stress

Recent studies reported that brassinosteroids (BRs) can induce plant tolerance to different environmental stresses via the nitric oxide (NO) signaling pathway. Previous reports have indicated that alternative oxidase (AOX) plays an important role in plants under various stresses. The mechanisms governing how NO is involved as a signal molecule which connects BR with AOX in regulating stress tolerance are still unknown. Recently, we found that Nicotiana benthamiana seedlings which were pretreated with BR have more tolerance to salt stress, accompanied with an increase of CN-resistant respiration. Our results suggested that pretreatment with 0.1 μM brassinolide (BL, the most active brassinosteroid) alleviated salt-induced oxidative damage and increased the NbAOX1 transcript level. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxyl-3-oxide (cPTIO, an NO scavenger) or virus-induced gene silencing of nitrate reductase (NR) and nitric oxide synthase (NOS)-like enzyme compromised the BRs-induced alternative respiratory pathway. Furthermore, pretreatment with specific chemical inhibitors of NR and NOS or gene silencing experiments decreased plant resistance to salt stress which also compromised BRs-induced salt stress tolerance. In conclusion, NO is involved in BRs-induced AOX capability which plays essential roles in salt tolerance in N. benthamiana seedlings.

Role of plastid signals in modulating Arabidopsis responses to Cucumber mosaic virus

AbstractChloroplasts can serve as sensors for detecting perturbations in the subcellular environment and actively communicate these signals to other organelles. They also play a critical role in plant immunity. Previous studies have indicated that some plastid-signaling deficient mutants have impaired abiotic stresses tolerance. However, whether plastid signals are involved in biotic stress response is rarely reported. In this study, the relationship between plastid signals and Cucumber mosaic virus (CMV) stress response was investigated in Arabidopsis thaliana. Our results showed that plastid-signaling defective mutants were more susceptible to CMV infection and showed more serious stress damages [higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation] compared with the wild-type plants. Furthermore, the induction of defense-associated genes and antioxidant enzymes activity in plastid-signaling defective mutants gun1 and abi4 were lesser than that observed in the wild-type plants after CMV infection. Collectively, our reports provide evidences that plastid signals are necessary for defense responses to CMV in A. thaliana. Moreover, the present study contributes to the understanding of the signaling pathway mediated by chloroplast in response to virus pathogens.

Involvement of PHYB in resistance to Cucumber mosaic virus in Nicotiana tabacum

AbstractPlants are exposed to a diversity of environmental stresses throughout their life cycles and need to constantly regulate their developmental and physiological processes to respond various external stimuli. It is well known that the interaction between light and plant defence is multifaceted and complicated. Here, the recombinant vector pRNAi-LIC-PHYB was used for achieving tobacco (Nicotiana tabacum) PHYB defective mutant via the Agrobacterium-mediated transformation method. Furthermore, we investigated molecular and physiological principles in PHYB defective N. tabacum plants in response to Cucumber mottle virus (CMV) infection. Our results indicated that PHYB defective plants were more susceptible to CMV infection. There were higher viral replication level, more impetuous viral symptoms and more serious stress damages in PHYB defective mutants along the time going when compared with wild-type (wt) plants. Moreover, the transcripts of defence-related genes and activities of antioxidant enzymes in phyB mutants were down-regulated when compared with wt plants after CMV infection. Overall, our results provided new information about the molecular link between PHYB and plant immune system in N. tabacum.