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Cytokinin (CTK)-mediated pathways play essential roles in plant stress responses. How CTK receptors affect plant resistance to viral pathogens remains unclear. Tobacco mosaic virus [recombinant TMV encoding green fluorescent protein (TMV-GFP)] was used to investigate the role of Nicotiana tabacum CRE1 (CTK receptor 1) in modulating virus susceptibility in N. tabacum. CRE1 deficiency enhanced the susceptibility of N. tabacum to TMV-GFP. The cre1 RNAi mutants showed extensive green florescence and higher transcript levels of the TMV coat protein gene than the wild type (WT) plants. CRE1 deficiency affected the antioxidant response to TMV-GFP, and the RNAi mutants had depressed antioxidant enzyme activities compared to the WT plants under virus infection. In addition, endogenous salicylic acid (SA) accumulation and the expression levels of SA pathway-related defence genes induced by TMV-GFP were significantly repressed in the RNAi mutants compared with the WT plants. Furthermore, the presence of exogenous CTK could only partially reduce the susceptibility of tobacco to TMV caused by CRE1 deficiency. Taken together, these results suggest that CRE1 plays a positive role in the tobacco defence response to the virus and is required for the induction and development of complete defence responses.

This study uncovered a novel role for HCPro in ChiVMV pathogenicity by interacting with catalases and inhibiting their activities to counter plant immunity., Plant symptoms are derived from specific interactions between virus and host components. However, little is known about viral or host factors that participate in the establishment of systemic necrosis. Here, we showed that helper component proteinase (HCPro), encoded by Chilli veinal mottle virus (ChiVMV), could directly interact with catalase 1 (CAT1) and catalase 3 (CAT3) in the cytoplasm of tobacco (Nicotiana tabacum) plants to facilitate viral infection. In vitro, the activities of CAT1 and CAT3 were inhibited by the interaction between HCPro and CATs. The C-terminus of HCPro was essential for their interaction and was also required for the decrease of enzyme activities. Interestingly, the mRNA and protein level of CATs were up-regulated in tobacco plants in response to ChiVMV infection. Nicotiana tabacum plants with HCPro overexpression or CAT1 knockout were more susceptible to ChiVMV infection, which was similar to the case of H2O2-pre-treated plants, and the overexpression of CAT1 inhibited ChiVMV accumulation. Also, neither CAT1 nor CAT3 could affect the RNA silencing suppression (RSS) activity of HCPro. Our results showed that the interaction between HCPro and CATs promoted the development of plant systemic necrosis, revealing a novel role for HCPro in virus infection and pathogenicity.

As an important phytohormone, cytokinins confer resistance of plants to several fungal and bacterial pathogens. However, the discovery of cytokinins in the viral resistance of Nicotiana tabacum is seldom reported. In the current study, we obtained tobacco CRE1 (cytokinin receptor 1) deficient mutants by Agrobacterium-mediated transformation utilizing the recombinant pRNAi-LIC-CRE1. Our conclusions indicated that CRE1 deficiency increased the susceptibility of N. tabacum to Chilli veinal mottle virus (ChiVMV) infection. The CRE1 deficient mutant plants displayed both more evident ChiVMV-induced symptom and higher transcription level of viral coat protein gene compared to the wild type (WT) plants. The antioxidant metabolism was altered in CRE1 deficient plants under virus inoculation. Photosystem damages caused by virus were also more severe in CRE1 deficient mutant plants compared to that in the WT plants. Additionally, expressions of several defense-related genes induced by ChiVMV infection were repressed in CRE1 deficient plants. Taken all together, these results suggested that CRE1 is required for N. tabacum defense responses to ChiVMV infection.

Plants use multiple mechanisms to fight against pathogen infection. One of the major mechanisms involves the disease resistance (R) gene, which specifically mediates plant defense. Recent studies have shown that R genes have broad spectrum effects in response to various stresses. N gene is the resistance gene specifically resistant to Tobacco mosaic virus (TMV). However, the role of N gene in abiotic stress and other viral responses remains obscure. In this study, we investigated the mechanisms by which N regulates plant defense responses under Chilli veinal mottle virus (ChiVMV) infection and salt stress. Here, we monitored the physiological and molecular changes of tobacco plants under virus attack. The results showed that when tobaccoNN and tobacconn plants were exposed to ChiVMV, tobaccoNN plants displayed higher susceptibility at five days post infection (dpi), while tobacconn plants exhibited higher susceptibility at 20 dpi. In addition, accumulation of reactive oxygen species (ROS) and expression of HARPIN-INDUCED1(NtHIN1) were higher in tobaccoNN plants than in tobacconn plants at 5 dpi. Interestingly, the pathogenesis-related gene (NtPR1 and NtPR5), the activities of antioxidant enzymes, and the content of salicylic acid (SA) in tobaccoNN plants increased compared with those in tobacconn plants. It was suggested that the N gene induced a hypersensitive response (HR) and enhanced the systemic resistance of plants in response to ChiVMV via the SA-dependent signaling pathway. In addition, the N gene was also induced significantly by salt stress. However, tobaccoNN plants showed hypersensitivity toward increased salt stress, and this hypersensitivity was dependent on abscisic acid and jasmonic acid but not SA. Taken together, our results indicate that the N gene appears to be important in the plant response to ChiVMV infection and salt stress.

Small GTPases play critical roles in the regulation of plant growth and development. However, the mechanism of action of small GTPases in plant response to virus infection remains largely unknown. Here, the gene encoding a Rho-type GTPase, NtRHO1, was identified as one of the genes up-regulated after tobacco mosaic virus (TMV) infection. Subcellular localization of NtRHO1 showed that it was located in the cytoplasm, plasma membrane, and nucleus. Transient overexpression of NtRHO1 in Nicotiana benthamiana accelerated TMV reproduction and led to the production of reactive oxygen species. By contrast, silencing of NtRHO1 reduced the sensitivity of N. benthamiana to TMV-GFP. Further exploration revealed a direct interaction between NtRHO1 and NtWRKY50, a positive regulator of the N. benthamiana response to virus infection. Yeast one-hybrid and electrophoretic mobility shift assays showed that this regulation was related to the capacity of NtWRKY50 to bind to the WK-box of the PR1 promoter, which was weakened by the interaction between NtRHO1 and NtWRKY50. Thus, our results indicate that the small GTPase NtRHO1 plays a negative role in tobacco response to TMV infection by interacting with transcription factor NtWRKY50, resulting in reduced plant immunity.

Jasmonate (JA) plays a central role in plant response to wounding and is shown to have crosstalk with gibberellin (GA) and cytokinin (CTK). Nevertheless, a little attempt has been made in connecting JA with tolerant responses and determining its physiological role in such responses. Here, we investigated the possible role of JA in compensatory growth and photosynthesis of Lolium perenne under wounding stress. The results showed that wounding treatment successfully induced compensatory growth and photosynthesis. JA was induced upon wounding, while GA was downregulated. Meanwhile, CTK was upregulated in leaf blade, but downregulated in sheath. In wounded plants, the content of malondialdehyde (MDA) showed little increase, whereas antioxidant enzyme activities showed a statistically insignificant decrease except superoxide dismutase (SOD) in leaf blade and catalase (CAT) in leaf sheath, suggesting alternative antioxidant mechanisms upon wounding. Interestingly, JA treatment induced regrowth in clipped plants, but repressed growth in unclipped plants, and continuous treatment with JA also repressed regrowth in clipped plants, suggesting that the initial JA burst in plants upon wounding can induce growth in L. perenne response to wounding. In addition, JA and its inhibitor induced little changes in photosynthetic yield, displaying that JA was uncoupled from photosynthesis.

Twenty-six leaf samples from Capsicum annuum, Solanum lycopersicum, Brassica campestris and Pisum sativum var. saccharatum plants were collected from different locations in Sichuan (China) during 2008–2016. Using RT-PCR and sequencing of the coat protein (CP) genes, 11 cucumber mosaic virus (CMV) subgroup II isolates were detected. The sequences of the 11 isolates compared with each other showed 98.63 to 99.54% identity. Phylogenetic analysis of the CP sequences revealed that all isolates clustered in the subgroup II and showed no host range specificity. This is the first report of the rising frequency of occurrence of CMV subgroup II isolates in Sichuan. The information of host diversity and the epidemic tendency of these CMV isolates presented in this study will provide the basis for disease control.

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29 kDa found in plants. This protein has been shown to be effective against a broad range of human viruses and also has anti-tumor activities. However, the mechanism by which α-MMC induces plant defense responses and regulates the N gene to promote resistance to the Tobacco mosaic virus (TMV) is still not clear. By using pharmacological and infection experiments, we found that α-MMC enhances TMV resistance of tobacco plants containing the N gene (tobaccoNN). Our results showed that plants pretreated with 0.5 mg/ml α-MMC could relieve TMV-induced oxidative damage, had enhanced the expression of the N gene and increased biosynthesis of jasmonic acid (JA) and salicylic acid (SA). Moreover, transcription of JA and SA signaling pathway genes were increased, and their expression persisted for a longer period of time in plants pretreated with α-MMC compared with those pretreated with water. Importantly, exogenous application of 1-Aminobenzotriazole (ABT, SA inhibitor) and ibuprofen (JA inhibitor) reduced α-MMC induced plant resistance under viral infection. Thus, our results revealed that α-MMC enhances TMV resistance of tobaccoNN plants by manipulating JA-SA crosstalk.

Citrus leaf blotch virus (CLBV) commonly occurs in some horticultural crops including kiwifruit. Sichuan Province is one of the major kiwifruit producing areas in southwestern China. In order to investigate the occurrence of CLBV infecting kiwifruit in Sichuan Province, an efficient reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with a lateral flow dipstick (LFD) was established as an effective diagnostic method for visually detecting the occurrence of CLBV. In the present study, a set of LAMP specific primers were designed based on the conserved region of coat protein gene of CLBV and the RT-LAMP-LFD assay could be accomplished in 60 min after extraction and cDNA synthesis. The optimized RT-LAMP-LFD method showed high specificity and sensitivity to CLBV, and displayed 1000 times higher sensitivity than that of conventional RT-PCR. Furthermore, 90 kiwifruit samples collected from Sichuan Province were screened by this developed method. The results showed that the incidence of CLBV in kiwifruit was 20% in Sichuan Province. Phylogenetic analysis revealed that all the CLBV Sichuan isolates were divided into different branches in group I and has some genetic distance with other host plants like citrus, rose and sweet cherry. Over all, this study provides a time saving and highly efficient diagnostic tool for monitoring CLBV infection status in kiwifruit.

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.

It is well known that light is involved in the plant immunity system. However, the mechanisms by which photoreceptors interfere with plant resistance to viruses remain largely unclear. In this study, we explored the defense response systems in different photoreceptor-defective mutants after cucumber mosaic virus (CMV) inoculation. We monitored the physiological and molecular changes of these mutants under virus attack. The results showed that mutants were more susceptible to CMV infection and suffered more serious oxidative damage compared with Col-0 plants. Furthermore, we found the transcription levels of defense-associated genes and the activities of some antioxidant enzymes in the mutants were much lower than those in Col-0 plants. Interestingly, the photosynthetic efficiency and respiratory rate in photoreceptor-defective mutants seemed to be abnormal compared with Col-0 plants after CMV infection. Furthermore, the integrities of chloroplasts and mitochondria of mutants were severely damaged after virus infection especially in phyB, phot2, and cry1 plants. In conclusion, our study demonstrated that the photoreceptors, PHYB, PHOT2, and CRY1, play important roles in affecting the defense of Arabidopsis thaliana against CMV infection and, to the best our knowledge, it is the first time that such comparison of the defense mechanisms of different photoreceptors against CMV stress has been made.

It has been reported that phytochrome A (phyA) and phytochrome B (phyB) are potent regulators of plant defense. However, the mechanisms that phytochromes use to interfere with plant resistance to viral infection remain largely unclear. In this study, Chilli veinal mottle virus (ChiVMV) was used to investigate the role of phytochromes in response to biotic stress. Our results showed that the phytochromes mutant phyAphyB28 plants displayed more serious necrosis and dwarf phenotypes compared to that of wild type plants (WT) after ChiVMV infection. qRT-PCR and Western blot analyses indicated that the expression and accumulation of ChiVMV were higher in phyAphyB28 mutants than that in WT plants. The leakage (EL) and the content of thiobarbituric acid-reactive substance (TBARS) suggested that phyAphyB28 mutants suffered more severe membrane damage than that of WT plants. In addition, extensive ROS accumulated in phyAphyB28 mutants after ChiVMV infection, whereas ROS production in WT plants were much less than mutant plants. The activities of antioxidant enzymes were down-regulated in phyAphyB28 mutants when compared with that in WT plants under ChiVMV infection. Besides, the contents of endogenous SA, JA and the expression of both hormones signaling related genes were lower in phyAphyB28 mutants compared to that in WT plants. Application of exogenous SA and JA could alleviate disease symptoms. Taken together, these results demonstrated that phyA and phyB positively regulated plant defense responses to ChiVMV infection and this process was dependent on the SA and JA defense pathways.

Temperature is an important environmental factor controlling plant growth, development, and immune response. However, the role of temperature in plant disease resistance is still elusive. In the present study, the potential effects of temperature on the interaction between Nicotiana tabacum and Cucumber mosaic virus (CMV) were investigated. Our results indicated that N. tabacum plants displayed severe symptoms at early stage of post inoculation at high temperature (HT, 28°C), associated with higher viral replication level, more serious stress damages. By contrast, low temperature (LT, 18°C) effectively delayed the replication of CMV compared with elevated temperatures. Additionally, quantitative real-time PCR analyses revealed that lower temperatures (≤ 24°C) promote salicylic acid (SA) dependent responses, whereas higher temperatures (> 24°C) activate the genes expression of jasmonic acid (JA) pathway. Interestingly, the dark green islands (DGIs) appeared much earlier in CMV-inoculated plants grown at HT compared with those at LT and the accumulation of virus small interfering RNAs in plants were significantly up-regulated under elevated temperatures at early stage of post inoculation. Taken together, these results indicated that temperature changes had important effects on plant defence response, and different temperatures could induce different immune pathways of N. tabacum against CMV infection.

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.

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.

Light signaling and phytohormones play important roles in plant growth, development, and biotic and abiotic stress responses. However, the roles of phytochromes and cross-talk between these two signaling pathways in response to salt stress in tobacco plants remain underexplored. Here, we explored the defense response in phytochrome-defective mutants under salt stress. We monitored the physiological and molecular changes of these mutants under salt stress conditions. The results showed that phytochrome A (phyA), phytochrome B (phyB) and phyAphyB (phyAB) mutants exhibited improved salt stress tolerance compared with wild-type (WT) plants. The mutant plants had a lower electrolyte leakage (EL) and malondialdehyde (MDA) concentration than WT plants, and the effect was clearly synergistic in the phyAB double mutant plants. Furthermore, the data showed that the transcript levels of defense-associated genes and the activities of some antioxidant enzymes in the mutant plants were much higher than those in WT plants. Additionally, the results indicated that phytochrome signaling strongly modulates the expression of endogenous abscisic acid (ABA) and jasmonic acid (JA) of Nicotiana tobacum in response to salt stress. To illustrate further the relationship between phytochrome and phytohormone, we measured the expression of defense genes and phytochrome. The results displayed that salt stress and application of methyl jasmonate (MeJA) or ABA up-regulated the transcript levels of salt response-associated genes and inhibited the expression of NtphyA and NtphyB. Foliar application of inhibitors of ABA and JA further confirmed that JA co-operated with ABA in phytochrome-mediated salt stress tolerance.

Disease development in Nicotiana tabacum after inoculation with cucumber mosaic virus (CMV) was accelerated when plants were grown in high light (300 µmol m−2 s−1, HL) compared with those grown in low light (70 µmol m−2 s−1, LL). Several characteristic defense reactions, including release of reactive oxygen species, accumulation of salicylic acid (SA), expression of SA-mediated pathogenesis related (PR) proteins and the systemic development of disease, were proved to be light dependent. Plants exposed to HL or medium light (150 µmol m−2 s−1, ML) had serious oxidative damage caused by CMV infection compared with LL-plants. In addition, the levels of SA and mRNA transcripts for non-expressor of pathogenesis-related gene 1 in CMV-inoculated plants were the highest under LL with decreasing photon flux density. In HL-exposed plants, the expression of PR1 and PR2 were repressed, but the expression of PR5 was not significantly altered, revealing a complex cross talk between light intensity and the SA signaling pathway in regulating N. tabacum response to the infection of CMV. Taken together, our results revealed that increased light intensity reduced the resistance ability of N. tabacum to CMV, correlating with repression of the SA-dependent signaling pathway and a change in the cellular redox status in plants cells.

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.

Plants 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.

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.

Tobacco EXPA4 plays a role in Nicotiana benthamiana defence against virus attack and affects antioxidative metabolism and phytohormone-mediated immunity responses in tobacco. Expansins are cell wall-loosening proteins known for their endogenous functions in cell wall extensibility during plant growth. The effects of expansins on plant growth, developmental processes and environment stress responses have been well studied. However, the exploration of expansins in plant virus resistance is rarely reported. In the present study, virus-induced gene silencing (VIGS) and Agrobacterium-mediated transient overexpression were conducted to investigate the role of Nicotiana tabacum alpha-expansin 4 (EXPA4) in modulating Tobacco mosaic virus (TMV-GFP) resistance in Nicotiana benthamiana. The results indicated that silencing of EXPA4 reduced the sensitivity of N. benthamiana to TMV-GFP, and EXPA4 overexpression accelerated virus reproduction on tobacco. In addition, our data suggested that the changes of virus accumulation in response to EXPA4 expression levels could further affect the antioxidative metabolism and phytohormone-related pathways in tobacco induced by virus inoculation. EXPA4-silenced plants with TMV-GFP have enhanced antioxidant enzymes activities, which were down-regulated in virus-inoculated 35S:EXPA4 plants. Salicylic acid accumulation and SA-mediated defence genes induced by TMV-GFP were up-regulated in EXPA4-silenced plants, but depressed in 35S:EXPA4 plants. Furthermore, a VIGS approach was used in combination with exogenous phytohormone treatments, suggesting that EXPA4 has different responses to different phytohormones. Taken together, these results suggested that EXPA4 plays a role in tobacco defence against viral pathogens.

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29 kDa found in plants. This protein has been shown to be effective against a broad range of human viruses and also has anti-tumor activities. However, the mechanism by which α-MMC induces plant defense responses and regulates the N gene to promote resistance to the Tobacco mosaic virus (TMV) is still not clear. By using pharmacological and infection experiments, we found that α-MMC enhances TMV resistance of tobacco plants containing the N gene (tobacco

Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various viruses that induce leaf mosaic in infected plants. The inoculation of Cucumber mosaic virus (CMV) in Nicotiana tabacum produced a commonly occurring sequence of classic patterns of DGIs and LGTs. Previous studies confirmed that there are significant differences between DGIs and LGTs in terms of physiology, biochemistry and molecular biology, but the mechanisms by which DGIs form remain unclear. To investigate the global gene expression changes that occur in these special tissues, individual differential gene expression tag libraries were constructed from three total RNA samples isolated from DGIs, LGTs and control plants (CK) and were sequenced using an Illumina HiSeqTM 2000. An analysis of differentially expressed genes (DEGs) and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. These analyses revealed the differences between DGIs, LGTs and CK. GO enrichment and KEGG pathway analyses suggested that several pathways related to photosynthesis and chlorophyll metabolism were enriched in DGIs compared to LGTs and CK. Several pathways related to apoptosis were significantly up-regulated in LGTs compared to DGIs. Additionally, we identified sets of DEGs that may be related to the formation or development of DGIs and LGTs. Our systematic analyses provide comprehensive transcriptomic information regarding DGIs and LGTs in CMV-infected N. tabacum. These data will help characterize the detailed mechanisms of DGI and LGT formation.

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.

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.

An improved multiplex RT-PCR assay combined with magnetic nanobeads (MNB-RT-PCR) was developed for simultaneous detection of four sweet potato viruses, Sweet potato virus G (SPVG), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC) and Sweet potato chlorotic fleck virus (SPCFV). Four primer pairs specific for each virus were designed and the corresponding PCR products were 169, 357, 516 and 900 bp in length for SPVG, SPFMV, SPVC and SPCFV, respectively. The specificity of the method was tested using different combinations of virus templates, and the identities of the amplification products were confirmed by sequencing. The limits of detection for all four viruses by single and multiplex MNB-RT-PCR assays were comparable. The assay was further evaluated using laboratory and field samples compared with a conventional CTAB-RT-PCR assay, and the comparative results showed that the MNB-RT-PCR assay was more rapid and sensitive. These results suggest that the multiplex MNB-RT-PCR assay is an effective and preferable method for virus detection in sweet potato.

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.

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29kDa that is found in Momordica charantia, and has been shown to be effective against a broad range of human viruses as well as having anti-tumor activities. However, the role of endogenous α-MMC under viral infection and the mechanism of the anti-viral activities of α-MMC in plants are still unknown. To study the effect of α-MMC on plant viral defense and how α-MMC increases plant resistance to virus, the M. charantia–cucumber mosaic virus (CMV) interaction system was investigated. The results showed that the α-MMC level was positively correlated with the resistance of M. charantia to CMV. α-MMC treatment could alleviate photosystem damage and enhance the ratio of glutathione/glutathione disulfide (GSH/GSSH) in M. charantia under CMV infection. The relationship of α-MMC and defense related phytohormones, and their roles in plant defense were further investigated. α-MMC treatment led to a significant increase of jasmonic acid (JA) and vice versa, while there was no obvious relevance between salicylic acid (SA) and α-MMC. In addition, reactive oxygen species (ROS) were induced in α-MMC-pretreated plants, in a similar way to the ROS burst in JA-pretreated plants. The production of ROS in both ibuprofen (JA inhibitor) and (α-MMC+ibuprofen)-pretreated plants was reduced markedly, leading to a greater susceptibility of M. charantia to CMV. Our results indicate that the anti-viral activities of α-MMC in M. charantia may be accomplished through the JA related signaling pathway.

China is the origin of Actinidia spp. (kiwifruit) and has largest kiwifruit production around the world. Although kiwifruit was domesticated only in the 20th century, it has become an important horticultural crop. A total of 17 viruses have been found in kiwifruit growing areas including China, Italy, and New Zealand since 2002 (Blouin et al. 2013, 2018; Veerakone et al. 2018). Sichuan Province has a unique geographical condition and climate for growing kiwifruit. It is the second largest area after Shanxi Province for commercial production of kiwifruit in China. With an increasing number of years of kiwifruit cultivation, suspect viral symptoms such as leaf mottle, chlorosis, and malformation are increasing. To confirm which viruses are infecting Actinidia spp. and to determine the extent of their distribution in Sichuan Province, a survey was conducted from April 2016 to May 2018. Fifty-six kiwifruit samples that showed virus-like symptoms such as feathery mottle, chlorotic and necrotic spots, and yellowing on leaves were collected from five different varieties of Actinidia chinensis at seven different geographic locations in Sichuan Province. Electron microscopy examination by negative staining for crude extracts of the symptomatic leaves revealed flexuous filaments of 9 to 10 nm × 1,350 to 1,400 nm. The particle size suggested a species of Closteroviridae. To identify the virus, total RNA of the respective samples was extracted with the EASYspin RNA Plant Mini Kit (Aidlab Biotechnologies Co., China) and reverse transcribed using random hexamers (Fermentas, Thermo Scientific). Actinidia virus 1 (AcV-1) coat protein specific primers, AcV1-CP F (5′- CGCGGATCCATGACTACCAAAGAAACGAA-3′) and AcV1-CP R (5′- ACGCGTCGACTCAATGATAACCTGAAGTGA-3′), were used to examine the presence of AcV-1, a new Closteroviridae virus recently identified in kiwi vines in Italy (Blouin et al. 2018). The results showed that 22 of the 56 kiwifruit samples generated bands of the expected size (732 bp) for AcV-1. One polymerase chain reaction amplicon from one sample from each of the seven different geographic locations was cloned into a pEASY-Blunt Zero vector (TransGen Biotech, China) and Sanger sequenced by Chengdu Tsingke BioTech Co. DNAMAN sequence alignment showed that across the seven clones, there were five different sequences, which shared 87.4 to 99.7% nucleotide sequence identity and 92.6 to 100% amino acid sequence identity (GenBank accession nos. MH557852 to MH557856). The variations were mainly concentrated in the middle domains of the coat protein genes. Further analysis showed that the five AcV-1 isolates shared 89.8 to 90.2% nucleotide sequence identity and 94.7 to 95.9% amino acid sequence identity to the corresponding gene of AcV-1 isolate K75 from Italy (KX857665, Blouin et al. 2018). Our results confirmed the widespread presence of AcV-1 in all kiwifruit planting areas that were investigated and in different varieties of A. chinensis (Hongyang, Donghong, Honghua, Jinguo, and Jinyan). Our sequence data also showed that there was genetic variability among different AcV-1 isolates in Sichuan Province. However, the role of AcV-1 as the causal agent for the symptoms observed in Actinidia is unknown, because plants were coinfected with other Actinidia-infecting viruses. To our knowledge, this is the first report of AcV-1 infecting A. chinensis in China. The apparent expansion of this virus is worthy of further attention.

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.

Plant-virus interactions are affected by environmental factors, including temperature. Plant defenses are often inhibited by high or low temperature. In this study, oxidative damage and gene expression were detected in Arabidopsis thaliana infected with cucumber mosaic virus (CMV) at different temperatures. Before virus inoculation, plants were treated with jasmonic acid (JA) and salicylic acid (SA), both of which are important signaling molecules in plant defense responses. The levels of MDA and hydrogen peroxide (H2O2), and electrolyte leakage were significantly higher in CMV-infected leaves at 15 and 37°C. The accumulation of H2O2 and superoxide radical (O2·−) was obviously suppressed by spraying with JA followed by SA (JA → SA) at different temperatures. The CMV-CP expression analysis showed that virus replication was inhibited efficiently in the (JA → SA) treatment. Therefore, many JA- and SA-responsible resistance genes were quantified; MPK4 was expressed highly and steadily in the (JA → SA) treatment. To further confirm the role of MPK4, the CMV-CP gene expression was evaluated in wild-type Arabidopsis and its mpk4 mutant infected with CMV. The results suggested that MPK4 might play an important role in the antagonism between JA and SA at temperature fluctuation.

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.

Cross protection is a common phenomenon among closely related strain viruses in co-infected plants. However, unrelated viruses, Turnip crinkle virus (TCV) and Cucumber mosaic virus (CMV), also show an antagonistic effect in co-infected Arabidopsis plants. In many cases, viral suppressors of RNA silencing (VSRs) have important roles in the interactions between viruses in mixed infections. CMV 2b and TCV p38 are multifunctional proteins and both of them are well characterized VSRs and have important roles in operation synergistic interactions with other viruses. Here, we demonstrated antagonistic effects of TCV toward CMV and showed that RNA silencing-mediated resistance protein, RCY1 and TCV-interacting protein (TIP) of Arabidopsis plants did not affect this antagonism effect. We further showed that TCV p38 and CMV 2b could interact with each other in vivo but not in vitro. Further mutational analysis showed that C-terminal of 2b and middle domains of p38 had more important roles in the interaction between the two viruses.

Members of the plant mitochondrial energy-dissipation pathway (MEDP) coordinate cellular energy metabolism, redox homeostasis and the balance of ROS production. However, the roles of MEDP members, particularly uncoupling protein (UCP), in resistance to turnip crinkle virus infection (TCV) are poorly understood. Here, we showed that disrupting some MEDP genes compromises plant resistance to TCV viral infection and this is partly associated with damaged photosynthetic characteristics, altered cellular redox and increased ROS production. Experiments using mutant plants with impaired cellular compartment redox poising further demonstrated that impaired chloroplast/mitochondria and cystosol redox increases the susceptibility of plants to viral infection. Our results illustrate a mechanism by which MEDP and cellular compartment redox act in concert to regulate plant resistance to viral infections.

Alpha-momorcharin (α-MMC) is type-1 ribosome inactivating proteins (RIPs) with molecular weight of 29 kDa and has lots of biological activity. Our recent study indicated that the α-MMC purified from seeds of Momordica charantia exhibited distinct antiviral and antifungal activity. Tobacco plants pre-treated with 0.5 mg/mL α-MMC 3 days before inoculation with various viruses showed less-severe symptom and less reactive oxygen species (ROS) accumulation compared to that inoculated with viruses only. Quantitative real-time PCR analysis revealed that the replication levels of viruses were lower in the plants treated with the α-MMC than control plants at 15 days post inoculation. Moreover, the coat protein expression of viruses was almost completely inhibited in plants which were treated with the α-MMC compared with control plants. Furthermore, the SA-responsive defense-related genes including non-expressor of pathogenesis-related genes 1 (NPR1), PR1, PR2 were up-regulated and activities of some antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were increased after the α-MMC treatment. In addition, the α-MMC (500 μg/mL) revealed remarkable antifungal effect against phytopathogenic fungi, in the growth inhibition range 50.35-67.21 %, along with their MIC values ranging from 100 to 500 μg/mL. The α-MMC had also a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of Sclerotinia sclerotiorum. The α-MMC showed a remarkable antiviral and antifungal effect and hence could possibly be exploited in crop protection for controlling certain important plant diseases.

Systemic acquired resistance is a form of inducible resistance that is triggered in systemic healthy tissues of local-infected plants. Several candidate signaling molecules emerged in the past two years, including the methylated derivatives of well-known defense hormones salicylic acid (SA) and jasmonic acid (JA). In our present study, the symptom on Cucumber mosaic virus (CMV) infected Arabidopsis leaves in 0.1 mM SA or 0.06 mM JA pre-treated plants was lighter (less reactive oxygen species accumulation and less oxidative damages) than that of the control group. JA followed by SA (JA → SA) had the highest inhibitory efficiency to CMV replication, higher than JA and SA simultaneous co-pretreatment (JA+SA), and higher than a JA or a SA single pretreatment. The crosstalk between the two hormones was further investigated at the transcriptional levels of pathogenesis-related genes. The time-course measurement showed JA might play a more important role in the interaction between JA and SA.

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.

BAK1 and BKK1 are receptor-like protein kinases (RLKs) involved in brassinosteroid signal transduction and plant resistance to bacteria and fungi. Here we report that loss-of-function mutants of BAK1 or BKK1 in Col-0 showed enhanced susceptibility to Turnip crinkle virus (TCV) infection. Cell death and chlorosis occurred much earlier in bak1-4 and bkk1-1 mutants than in wild-type plants, suggesting an important role of BAK1 and BKK1 in controlling cell death associated with TCV infection. The bak1-4 and bkk1-1 mutants showed the elevated transcription levels of pathogenesis genes, such as PR1, PR2, PR5, PDF1.2. Enhanced expression of these genes, however, failed to resist the transport of TCV virus. At 9 days postinoculation, TCV levels were the highest in the systemic leaves of bak1-4, but were undetected in BAK1 or BKK1 overexpressing plants. These results signify that both BAK1 and BKK1 are important components in controlling TCV infection in Arabidopsis plants.

Two types of satellite RNAs (XJs1 and XJs2) were found to be associated with the two Cucumber mosaic virus (CMV) beet isolates XJ1 and XJ2 consisting of 384 nucleotides and 336 nucleotides, respectively. Our work showed that XJs1 and XJs2 could attenuate symptoms on Nicotiana glutinosa induced by CMV and the effect of XJs1 on symptoms was stronger than XJs2. Co-inoculation of CMV with XJs1 produced lighter oxidative stress than that with XJs2. Oxygen uptake rates through the alternative pathway in leaves infected with CMV and XJs2 were always higher than that with CMV and XJs1 and lower than infection with CMV alone. The non-photochemical fluorescence quenching (qN) increased more in N. glutinosa infected with CMV and XJs2 than that with CMV and XJs1. The relationship among helper virus, satRNAs and the host plants is discussed. Two closely-related satRNAs from the same plant has not been reported so far. The effects of these two satRNAs on virus-infected N. glutinosa were significantly different. The difference may be caused by deletion and mutation of the satRNAs.

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.

There are significant differences between the DGIs and LGTs. Additionally, most of the characteristics indicate that the DGIs are more similar to recovered tissue and can resist viral attacks. Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various mosaic viruses. We performed cytological, physiological and molecular biological analyses of the DGIs and LGTs in cucumber mosaic virus-infected Nicotiana tabacum leaves. Our results indicated that the DGIs contained less virus than did the LGTs. Compared to the LGTs, the DGIs contained higher levels of the metabolites involved in plant defence. The contents of reduced glutathione and ascorbic acid were increased in the DGIs to reach levels that were even higher than those of control plants. Moreover, hormone measurements and quantitative real-time PCR analysis revealed that the endogenous salicylic acid, ethylene and defence genes mediated these elevations by playing positive roles in the regulation of the DGIs responses to viral infection. The accumulation of cytokinin was also much greater in the DGIs than in the LGTs. Finally, northern blotting analysis indicated that the accumulation of viral small interfering RNAs was decreased in the DGIs compared to the LGTs. Taken together, these results suggest that DGIs might represent leaf areas that have recovered from viral infection due to locally enhanced defence responses.

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.

Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing–based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography–mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

The nucleotide sequence of the pathogenesis-related protein 1(PR-1) gene was obtained from Nicotiana benthamiana using RT-PCR. Restriction enzyme cutting sites of EcoRI and NotI were introduced to the ORF fragments of PR-1, they were then linked together with pET-30a (+) and transformed into E. coli BL21 (DE3). The target protein was induced by 1.5 mM IPTG, at 37°C for 4 h. The expressed protein was purified by Ni-NTA and an anti-NbPR-1 polyclonal antibody was prepared using rabbits. The antibody could detect the expression of PR-1 in N. benthamiana and other Nicotiana plants. NbPR-1 protein has four α-helices and two β-sheets by homology modeling. Furthermore, the purified NbPR-1 protein exhibited a broad-spectrum antifungal activity.

Salicylic acid (SA) is a critical signal for activation of both local and systemic resistance responses. However, SA-deficient plants adapt to RNA virus infections better, which show a less-severe symptom and less reactive oxygen species (ROS) accumulation. The higher levels of reduced glutathione (GSH) and reduced ascorbic acid (AsA) in SA-deficient plants may contribute to their alleviated symptoms, which are consistent with their higher expression levels of dehydroascorbate reductase gene (DHAR) and glutathione reductase gene (GR). High-dose AsA or GSH treatment could alleviate the symptom and inhibit virus replication after 20 days, but ROS eliminators could not imitate the effect of AsA or GSH. The data show a new link between SA and AsA/GSH-mediated redox homeostasis.