De-Hui Xi

Salicylic Acid and Jasmonic Acid Are Essential for Systemic Resistance Against Tobacco mosaic virus in Nicotiana benthamiana

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.

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

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

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

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

Effects of Salicylic Acid on Alternative Pathway Respiration and Alternative Oxidase Expression in Tobacco Calli

The alternative pathway (AP) respiration of plants is a cyanide-resistant and non-phosphorylating electron transport pathway in mitochondria. Alternative oxidase (AOX) is the terminal oxidase of the AP and exists in plant mitochondria as two states: the reduced, noncovalently linked state or the oxidized, covalently cross-linked state. In the present study, the effects of 20 μm exogenous salicylic acid (SA) on both AP activity and AOX expression in mitochondria of tobacco (Nicotiana rustica L. cv. yellow flower) calli were investigated. The results showed that SA treatment enhanced the AP activity. During the process of SA treatment, the AP activity increased dramatically and achieved the peak value after 8 h of treatment. Then it declined until 16 h, and maintained a steady level between 16 and 24 h. Changes in both the total AOX protein level and the reduced state were in accordance with the AP activity, but the oxidized state changed differently. The aox1 gene transcript level also showed a similar change as the AP activity and AOX protein level. The induction of AOX expression by low concentrations of SA was inferred through a reactive oxygen species (ROS)-independent pathway. These results indicate that the enhancement of AP activity in response to SA is correlated to the expression of AOX, and the reduced, non-covalently linked state of AOX plays an important role during this process.

Variation analysis of two cucumber mosaic viruses and their associated satellite RNAs from sugar beet in China

Two cucumber mosaic virus (CMV) isolates XJ1 and XJ2 were obtained from sugar beet showing yellow mosaic symptom in Shihezi, Xinjiang Uigur municipality of China. The coat protein gene of the two CMV isolates and their associated satellite RNAs were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and were cloned and sequenced. Comparison of CP gene sequences showed that XJ1 and XJ2 have the highest sequence identity with that of CMV-Danshen (97.8%) and CMV-SD (98.7%), respectively. Two types of satellite RNAs (XJs1 and XJs2) were found to be associated with the two CMV isolates consisting of 384 nucleotides and 336 nucleotides, respectively. Sequence comparisons revealed that XJs1 and XJs2 were most closely related to CS2-sat and CS1-sat, respectively, with 98.9% and 98.5% nucleotide sequence identity. Phylogenetic analysis of nucleotide sequence and deduced amino acid sequence of coat protein gene revealed that XJ1 and XJ2 belong to subgroup IB but there exist some variation between them. Parallel analyses of nucleotide sequence of XJsl and XJs2 suggested that these two satellite RNAs probably originated from China.

The dual effects of salicylic acid on dehydrin accumulation in water-stressed barley seedlings

Dehydrins are a group of plant proteins that usually accumulate in response to environmental stresses. They are proposed to play specific protective roles in plant cells. Present study showed that the accumulation of dehydrins in water-stressed barley (Hordeum vulgare L.) seedlings was influenced by their treatment with salicylic acid (SA). The level of dehydrin proteins was increased by 0.20 mM SA, but decreased by 0.50 mM SA treatment. Both mRNA expression and protein accumulation of a typical barley dehydrin, DHN5, were enhanced by SA treatment when SA concentrations were lower than 0.25 mM. However, the higher SA concentrations significantly decreased the protein level of DHN5 despite of a stable mRNA level. Our results also showed that low SA concentrations (less than 0.25 mM) decreased the electrolyte leakage and malondialdehyde (MDA) and H2O2 contents in water-stressed barley seedlings. But high SA concentrations (more than 0.25 mM) enhanced H2O2 accumulation, tended to cause more electrolyte leakage, and increase MDA content. These data indicated that SA could up-regulate the dehydrin gene expression and protein accumulation. Since the protective role of dehydrins in plant cells, such effect could be an important reason for the SA-mediated alleviation on water stress injury. But excessive SA could suppress the accumulation of dehydrin proteins and aggravate the oxidative damage.

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

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

Difference of Physiological Characters in Dark Green Islands and Yellow Leaf Tissue of Cucumber mosaic Virus (CMV)-Infected Nicotiana tabacum Leaves

Dark green islands (DGIs) are a common symptom of plants systemically infected with the mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow leaf tissue that is full of virus particles. In Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves, the respiration and photosynthesis capabilities of DGIs and yellow leaf tissues were measured. The results showed that the cyanide-resistant respiration was enhanced in yellow leaf tissue and the photosynthesis was declined, while in DGIs they were less affected. The activities of the oxygen-scavenging enzymes catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in infected leaves were signifi cantly higher than those in the healthy leaves, and the enzyme activities in DGIs were always lower than in the yellow leaf tissues. Reactive oxygen species (ROS) staining showed that the hydrogen peroxide content in yellow leaf tissues was apparently higher than that in DGIs, while the superoxide content was on the contrary. Formation of DGIs may be a strategy of the host plants resistance to the CMV infection.

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.

Application of Jasmonic Acid Followed by Salicylic Acid Inhibits Cucumber mosaic virus Replication

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.