Virulence determines beneficial trade‐offs in the response of virus‐infected plants to drought via induction of salicylic acid

It has been hypothesized that plants can get beneficial trade‐offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus‐induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought‐tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus‐infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid‐independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non‐infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.     

Identification of differentially expressed genes induced by <Emphasis Type="Italic">Bamboo mosaic virus</Emphasis> infection in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>by cDNA-amplified fragment length polymorphism

Background  

The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP).     

Heat shock protein 70 is necessary for Rice stripe virus infection in plants

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive‐stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative‐stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull‐down assays, the N‐terminus of RSV RNA‐dependent RNA polymerase (RdRp) interacted and co‐localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N‐terminus of RdRp when expressed alone was not obviously different from when it was co‐expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.     

High Temperature,High Ambient CO2 Affect the Interactions between Three Positive-Sense RNA Viruses and a Compatible Host Differentially,but not Their Silencing Suppression Efficiencies

We compared infection of Nicotiana benthamiana plants by the positive-sense RNA viruses Cucumber mosaic virus (CMV), Potato virus Y (PVY), and by a Potato virus X (PVX) vector, the latter either unaltered or expressing the CMV 2b protein or the PVY HCPro suppressors of silencing, at 25°C vs. 30°C, or at standard (~401 parts per million, ppm) vs. elevated (970 ppm) CO₂ levels. We also assessed the activities of their suppressors of silencing under those conditions. We found that at 30°C, accumulation of the CMV isolate and infection symptoms remained comparable to those at 25°C, whereas accumulation of the PVY isolate and those of the three PVX constructs decreased markedly, even when expressing the heterologous suppressors 2b or HCPro, and plants had either very attenuated or no symptoms. Under elevated CO₂ plants grew larger, but contained less total protein/unit of leaf area. In contrast to temperature, infection symptoms remained unaltered for the five viruses at elevated CO₂ levels, but viral titers in leaf disks as a proportion of the total protein content increased in all cases, markedly for CMV, and less so for PVY and the PVX constructs. Despite these differences, we found that neither high temperature nor elevated CO₂ prevented efficient suppression of silencing by their viral suppressors in agropatch assays. Our results suggest that the strength of antiviral silencing at high temperature or CO₂ levels, or those of the viral suppressors that counteract it, may not be the main determinants of the observed infection outcomes.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

Characterization of Synergy between Cucumber mosaic virus and Tobacco necrosis virus in Nicotiana benthamiana

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 (H₂O₂) 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.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Mitogen‐activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana

In plants, the mitogen‐activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen‐associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo mosaic virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV‐encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity‐dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail mosaic virus but decreased Potato virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad‐spectrum resistance through genetically manipulating a single player in MAPK cascades.     

Translationally controlled tumour protein (TCTP) from tomato and Nicotiana benthamiana is necessary for successful infection by a potyvirus

Translationally controlled tumour protein (TCTP) is a ubiquitously distributed protein in eukaryotes, involved in the regulation of several processes, including cell cycle progression, cell growth, stress protection, apoptosis and maintenance of genomic integrity. Its expression is induced during the early stages of tomato (Solanum lycopersicum) infection by the potyvirus Pepper yellow mosaic virus (PepYMV, a close relative of Potato virus Y). Tomato TCTP is a protein of 168 amino acids, which contains all the conserved domains of the TCTP family. To study the effects of TCTP silencing in PepYMV infection, Nicotiana benthamiana plants were silenced by virus‐induced gene silencing (VIGS) and transgenic tomato plants silenced for TCTP were obtained. In the early stages of infection, both tomato and N. benthamiana silenced plants accumulated less virus than control plants. Transgenic tomato plants showed a drastic reduction in symptoms and no viral accumulation at 14 days post‐inoculation. Subcellular localization of TCTP was determined in healthy and systemically infected N. benthamiana leaves. TCTP was observed in both the nuclei and cytoplasm of non‐infected cells, but only in the cytoplasm of infected cells. Our results indicate that TCTP is a growth regulator necessary for successful PepYMV infection and that its localization is altered by the virus, probably to favour the establishment of virus infection. A network with putative interactions that may occur between TCTP and Arabidopsis thaliana proteins was built. This network brings together experimental data of interactions that occur in other eukaryotes and helps us to discuss the possibilities of TCTP involvement in viral infection.     

The influence of the N- and C- terminal modifications of Potato virus X coat protein on virus properties

The Potato virus X (PVX)-based vector was used for the construction of N- and C-terminally modified PVX coat protein (XCP) chimeras. N-terminal XCP modifications do not influence the viral life cycle, whereas the simple XCP C-terminal fusion impedes the viral replication. We designed several C-terminally modified XCP chimeras and tested their viabilities in various Nicotiana benthamiana genotypes. Our results showed the negative impact of 3′-terminal modification of XCP on the chimera’s life cycle. To ensure chimeric constructs stability, the second copy of the last 60 nucleotides of XCP followed by the 3′-untranslated region (UTR) was added downstream of the recombinant sequence. Simultaneously, the first copy of the last 60 nucleotides of XCP was mutated in order to prevent recombination between the two identical sequences. The movement protein of Tobacco mosaic virus expressed in transgenic N. benthamiana plants positively affected the cell-to-cell spread of C-terminally modified XCP chimeras.     

Interference Between Tobacco necrosis virus and Turnip crinkle virus in Nicotiana benthamiana

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.     

TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8‐kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV‐p3), and we noted the up‐regulation of SKP1 and several endoplasmic reticulum (ER)‐resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV‐p3, but not TMV or PVX. Such lesions were the result of TGBp3‐elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR‐related gene expression occurred within 8 h of TMV‐p3 inoculation and declined before the onset of PCD. TGBp3‐mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro‐survival mechanism. Anti‐apoptotic genes Bcl‐xl, CED‐9 and Op‐IAP were expressed in transgenic plants and suppressed N gene‐mediated resistance to TMV, but failed to alleviate TGBp3‐induced PCD. However, TGBp3‐mediated cell death was reduced in SKP1‐silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.     

A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

An up‐regulated gene derived from Bamboo mosaic virus (BaMV)‐infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single‐stranded, positive‐sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active‐site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus‐induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post‐inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2‐knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2‐knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2‐OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co‐immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.     

The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases

Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus‐encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro‐Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2‐green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2‐GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)‐C2 displayed chlorotic lesions and stunted growth. PVX‐C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host‐defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3–2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3–2 gene and pNbCMT3–2::GUS (β‐glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation.     

Disruption of the methionine cycle and reduced cellular gluthathione levels underlie potex–potyvirus synergism in Nicotiana benthamiana

Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex–potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S‐adenosyl‐l ‐methionine synthetase (SAMS) and S‐adenosyl‐l ‐homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post‐infiltration (dpi) and upregulation of (–)‐strand gRNA accumulation at 9 dpi were observed in the SAHH‐silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX‐infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX‐infected plants caused an even stronger reduction in GSH levels than did SAMS + SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase‐silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex–potyvirus mixed infection.