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Previous literature indicates that both light and photoreceptors may play key roles in host susceptibility to viruses. Our previous studies have suggested that phytochromeB is essential for the defense strategies of -Nicotiana tabacum against Cucumber mosaic virus. Here, we further investigated the roles of phytochromes in N. tabacum against CMV. The recombinant vector pRNAi-LIC-PHYA/PHYB was used to produce tobacco phyA and phyAphyB mutants via the Agrobacterium-mediated transformation method. The results suggested that both phyA and phyB deficiency increased the susceptibility of N. tabacum to CMV infection and showed more obvious CMV-induced symptoms and higher transcriptional levels of the CMV-coat protein gene than wild plant, and the effect was clearly synergistic in the phyAphyB double mutant. Furthermore, our data showed that mutations in phyA/B up-regulated the sensitivity of N. tabacum to CMV by repressing the SA-mediated defence pathway in tobacco plants. Additionally, the results in the present study indicated that phytochrome signalling strongly modulates the response of N. tabacum to endogenous SA but not to endogenous JA. Taken together, these results reinforced the idea that an intact phytochrome signalling plays a positive role in the ability of N. tabacum to resist CMV and is required for the induction and development of SA-mediated defence responses in CMV-inoculated N. tabacum. This article is protected by copyright. All rights reserved.

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.

Light is an important environment factor controlling plant growth, development, and nutritional quality and is also one of the most important factors inducing plant defence. In this study, we assayed the potential effects of light quality on the interaction between Nicotiana tabacum and cucumber mosaic virus (CMV). Our results indicated that white light-treated N. tabacum plants displayed obvious symptoms at early stage postinoculation, while the symptoms were significantly inhibited under red light and blue light. Western blotting and quantitative real-time PCR (qRT-PCR) analyses showed that blue light and red light can effectively delay the replication of CMV compared with white light. The activities of various reactive oxygen species (ROS)-scavenging enzymes and reducing substances [reduced glutathione (GSH) and ascorbic acid (ASA)] were increased under blue light and red light. In addition, hormone measurements and qRT-PCR analyses revealed that salicylic acid (SA)-mediated signalling pathway plays positive role in the related regulation, and cytokinin (CTK) may also participate in them. Furthermore, we found that the formation of dark green islands (DGIs) was significantly suppressed in plants under red light and blue light at 30 days postinoculation (Dpi). However, the accumulation of virus in plants under different light conditions had no notable differences at later stage of postinoculation. Taken together, these results indicated that red light and blue light could effectively delay symptom expression and replication of CMV on N. tabacum at the relatively earlier stage postinoculation.

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.

Cross-protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor-defective mutant, TCVCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVCP provides some protection against wild-type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross-protection. However, similar cross-protection afforded by TCVCP is not observed in Nicotiana benthamiana plants. As expected, TCVCP pre-infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross-protection afforded by TCVCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross-protection afforded by TCVCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process.

The interaction between Cucumber mosaic virus (CMV) and Turnip crinkle virus (TCV) in Arabidopsis thaliana is reported. Although TCV coat protein accumulates to a similar level in singly or doubly infected plants, CMV coat protein is significantly decreased in doubly infected plants, which develop symptoms similar to those of TCV infection alone. TCV thus strongly interfers with infection by CMV. A significant reduction in CMV 2b gene expression also occurs in co-infected plants. A defence signalling initiator, salicylic acid (SA), further inhibits CMV accumulation during CMV-TCV co-infection. This interference is correlated with both enhanced virus gene silencing and defence signalling.

Mixed infections of Nicotiana benthamiana plants by Tobacco necrosis virus (TNV) and Turnip crinkle virus (TCV) exhibited an interference interaction. Accumulation of TNV (+)RNA as well as capsid protein in mixed infection were considerably lower than that of singly infected plants. There were also a slight reduction in the levels of TCV (+)RNA and capsid protein in doubly infected plants, which displayed the concentration of both viruses decreased in dually infected plants. Tissue immunoblot analysis of systemic N. benthamiana leaves infected by TNV and TCV singly or doubly showed the interference between the two viruses in situ, which exhibited the decrease of both viruses in doubly infected leaves although the distribution of them did not change remarkably. These results were consistent with the hybridization analysis of viral genomic RNA and coat protein. Both cross-protection test and mixed infection of the two viruses confirmed TCV had relatively stronger interference to the infection of TNV. Interference infection by TNV and TCV induced higher increase in the levels of cytochrome pathway respiration and alternative pathway respiration in host plants, especially the latter. Interference often occurred in different strains of one kind of virus or two different closely related viruses in one genus. Our results showed that interference could also occur in different viruses belonging to different genera.

Mixed infections of Nicotiana benthamiana plants by Cucumber mosaic virus (CMV) and Tobacco necrosis virus (TNV) exhibit a synergistic interaction and result in symptom enhancement. Accumulation of CMV(+) RNA as well as capsid protein (CP) in mixed infection was considerably higher than that of singly-infected plants. There was also a slight increase in TNV(+) RNA and CP levels in doubly infected plants. Synergistic infection by CMV- and TNV-induced higher increase in the levels of malonyldialdehyde, hydrogen peroxide (H 2 O 2 ) and more decline in the activities of catalase than singly infected ones. Both peroxidase and superoxide dismutase activities increased rapidly for the first 10 days post inoculation (dpi) in doubly-infected plants and then declined, whereas the enzyme activities continued to increase after 10 dpi in singly infected plants and had higher enzyme activities in the late stages than that of co-infected plants. These results suggest that synergistic infection by CMV and TNV produced severes oxidative stress in N. benthamiana plants and the synergy between the two viruses was mutual.