Nitric oxide as a signaling molecule in brassinosteroid-mediated virus resistance to Cucumber mosaic virus in Arabidopsis thaliana.

Brassinosteroids (BRs) are growth-promoting plant hormones that play a crucial role in biotic stress responses. Here, we found that BR treatment increased nitric oxide (NO) accumulation, and a significant reduction of virus accumulation in Arabidopsis thaliana. However, the plants pre-treated with NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxyl-3-oxide (PTIO)] or nitrate reductase (NR) inhibitor (tungstate) hardly had any NO generation and appeared to have the highest viral replication and suffer more damages. Furthermore, the antioxidant system and photosystem parameters were up-regulated in brassinolide (BL)-treated plants but down regulated in PTIO- or tungstate-treated plants, suggesting NO may be involved in BRs-induced virus resistance in Arabidopsis. Further evidence showed that NIA1 pathway was responsible for BR-induced NO accumulation in Arabidopsis. These results indicated that NO participated in the BRs-induced systemic resistance in Arabidopsis. As BL treatment could not increase NO levels in nia1 plants in comparison to nia2 plants. And nia1 mutant exhibited decreased virus resistance relative to Col-0 or nia2 plants after BL treatment. Taken together, our study addressed that NIA1-mediated NO biosynthesis is involved in BRs-mediated virus resistance in A. thaliana.

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 Transcription Factor HAT1 in Modulating Arabidopsis thaliana Response to Cucumber mosaic virus.

Arabidopsis thaliana homeodomain-leucine zipper protein 1 (HAT1) belongs to the homeodomain-leucine zipper (HD-Zip) family class II that plays important roles in plant growth and development as a transcription factor. To elucidate further the role of HD-Zip II transcription factors in plant defense, the A. thaliana hat1, hat1hat3 and hat1hat2hat3 mutants and HAT1 overexpression plants (HAT1OX) were challenged with Cucumber mosaic virus (CMV). HAT1OX displayed more susceptibility, while loss-of-function mutants of HAT1 exhibited less susceptibility to CMV infection. HAT1 and its close homologs HAT2 and HAT3 function redundantly, as the triple mutant hat1hat2hat3 displayed increased virus resistance compared with the hat1 and hat1hat3 mutants. Furthermore, the induction of the antioxidant system (the activities and expression of enzymatic antioxidants) and the expression of defense-associated genes were down-regulated in HAT1OX but up-regulated in hat1hat2hat3 when compared with Col-0 after CMV infection. Further evidence showed that the involvement of HAT1 in the anti-CMV defense response might be dependent on salicylic acid (SA) but not jasmonic acid (JA). The SA level or expression of SA synthesis-related genes was decreased in HAT1OX but increased in hat1hat2hat3 compared with Col-0 after CMV infection, but there were little difference in JA level or JA synthesis-related gene expression among HAT1OX or defective plants. In addition, HAT1 expression is dependent on SA accumulation. Taken together, our study indicated that HAT1 negatively regulates plant defense responses to CMV.

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.

Induction of plant virus defense response by brassinosteroids and brassinosteroid signaling in Arabidopsis thaliana.

MAIN CONCLUSION: Our study demonstrated that CMV resistance was upregulated by brassinosteroids (BRs) treatment, and BR signaling was needed for this BRs-induced CMV tolerance. Plant steroid hormones, brassinosteroids (BRs), play essential roles in variety of plant developmental processes and adaptation to various biotic and abiotic stresses. BR signal through plasma membrane-localized receptor and other components to modulate several transcription factors that modulate thousands of target genes including certain stress-responsive genes. To study the effects of BRs on plant virus defense and how BRs induce plant virus stress tolerance, we manipulated the BRs levels in Arabidopsis thaliana and found that BRs levels were positively correlated with the tolerance to Cucumber mosaic virus (CMV). We also showed that BRs treatment alleviated photosystem damage, enhanced antioxidant enzymes activity and induced defense-associated genes expression under CMV stress in Arabidopsis. To see whether BR signaling is essential for the plant virus defense response, we made use of BR signaling mutants (a weak allele of the BRs receptor mutant bri1-5 and constitutive BRs response mutant bes1-D). Compared with wild-type Arabidopsis plants, bri1-5 displayed reversed tolerance to CMV, but the resistance was enhanced in bes1-D. Together our results suggest that BRs can induce plant virus defense response through BR signaling.

The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in Nicotiana benthamiana.

Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana.

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.

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.

Mitochondrial alternative oxidase is involved in both compatible and incompatible host-virus combinations in Nicotiana benthamiana.

The alternative oxidase (AOX) functions in the resistance to biotic stress. However, the mechanisms of AOX in the systemic antiviral defense response and N (a typical resistance gene)-mediated resistance to Tobacco mosaic virus (TMV) are elusive. A chemical approach was undertaken to investigate the role of NbAOX in the systemic resistance to RNA viruses. Furthermore, we used a virus-induced gene-silencing (VIGS)-based genetics approach to investigate the function of AOX in the N-mediated resistance to TMV. The inoculation of virus significantly increased the NbAOX transcript and protein levels and the cyanide-resistant respiration in the upper un-inoculated leaves. Pretreatment with potassium cyanide greatly increased the plant's systemic resistance, whereas the application of salicylhydroxamic acid significantly compromised the plant's systemic resistance. Additionally, in NbAOX1a-silenced N-transgenic Nicotiana benthamiana plants, the inoculated leaf collapsed and the movement of TMV into the systemic tissue eventually led to the spreading of HR-PCD and the death of the whole plant. The hypersensitive response marker gene HIN1 was significantly increased in the NbAOX1a-silenced plants. Significant amounts of TMV-CP mRNA and protein were detected in the NbAOX1a-silenced plants but not in the control plants. Overall, evidence is provided that AOX plays important roles in both compatible and incompatible plant-virus combinations.

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.

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 (C S ACS1), ripening-related ACC synthase2 (C S ACS2), ripening-related ACC synthase3 (C S ACS3), 1-aminocyclopropane-1-carboxylate oxidase1 (C S ACO1), 1-aminocyclopropane-1-carboxylate oxidase2 (C S ACO2), and C S AOX 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.

The capsid protein p38 of turnip crinkle virus is associated with the suppression of cucumber mosaic virus in Arabidopsis thaliana co-infected with cucumber mosaic virus and turnip crinkle virus.

Infection of plants by multiple viruses is common in nature. Cucumber mosaic virus (CMV) and Turnip crinkle virus (TCV) belong to different families, but Arabidopsis thaliana and Nicotiana benthamiana are commonly shared hosts for both viruses. In this study, we found that TCV provides effective resistance to infection by CMV in Arabidopsis plants co-infected by both viruses, and this antagonistic effect is much weaker when the two viruses are inoculated into different leaves of the same plant. However, similar antagonism is not observed in N. benthamiana plants. We further demonstrate that disrupting the RNA silencing-mediated defense of the Arabidopsis host does not affect this antagonism, but capsid protein (CP or p38)-defective mutant TCV loses the ability to repress CMV, suggesting that TCV CP plays an important role in the antagonistic effect of TCV toward CMV in Arabidopsis plants co-infected with both viruses.