Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus

To investigate the plant genes affected by Bamboo mosaic virus (BaMV) infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL) gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV) and Potato virus X (PVX); thus, NbSTKL might be a specific protein facilitating BaMV movement.     

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.     

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.     

An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication

One up-regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. The full length cDNA of this gene was cloned by 5′ and 3′-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up-regulated gene, NbUbE3R1 that plays a role in BaMV replication.     

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.     

NbRABG3f,a member of Rab GTPase,is involved in Bamboo mosaic virus infection in Nicotiana benthamiana

The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up‐regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up‐regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus‐induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane‐targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP‐NbRABG3f/T22N clearly co‐localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane‐targeting ability are crucial for BaMV accumulation at the cell level.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Alpha-momorcharin enhances Nicotiana benthamiana resistance to tobacco mosaic virus infection through modulation of reactive oxygen species

Alpha-momorcharin (α-MMC), a member of the plant ribosomal inactivating proteins (RIPs) family, has been proven to exhibit important biological properties in animals, including antiviral, antimicrobial, and antitumour activities. However, the mechanism by which α-MMC increases plant resistance to viral infections remains unclear. To study the effect of α-MMC on plant viral defence and how α-MMC increases plant resistance to viruses, recombinant DNA and transgenic technologies were employed to investigate the role of α-MMC in Nicotiana benthamiana resistance to tobacco mosaic virus (TMV) infection. Treatment with α-MMC produced through DNA recombinant technology or overexpression of α-MMC mediated by transgenic technology alleviated TMV-induced oxidative damage and reduced the accumulation of reactive oxygen species (ROS) during TMV-green fluorescent protein infection of N. benthamiana. There was a significant decrease in TMV replication in the upper leaves following local α-MMC treatment and in α-MMC-overexpressing plants relative to control plants. These results suggest that application or overexpression of α-MMC in N. benthamiana increases resistance to TMV infection. Finally, our results showed that overexpression of α-MMC up-regulated the expression of ROS scavenging-related genes. α-MMC confers resistance to TMV infection by means of modulating ROS homeostasis through controlling the expression of antioxidant enzyme-encoding genes. Overall, our study revealed a new crosstalk mechanism between α-MMC and ROS during resistance to viral infection and provides a framework to understand the molecular mechanisms of α-MMC in plant defence against viral pathogens.     

Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.     

Chloroplast clustering around the nucleus is a general response to pathogen perception in Nicotiana benthamiana

It is increasingly clear that chloroplasts play a central role in plant stress responses. Upon activation of immune responses, chloroplasts are the source of multiple defensive signals, including reactive oxygen species (ROS). Intriguingly, it has been described that chloroplasts establish physical contact with the nucleus, through clustering around it and extending stromules, following activation of effector-triggered immunity (ETI). However, how prevalent this phenomenon is in plant–pathogen interactions, how its induction occurs, and what the underlying biological significance is are important questions that remain unanswered. Here, we describe that the chloroplast perinuclear clustering seems to be a general plant response upon perception of an invasion threat. Indeed, activation of pattern-triggered immunity, ETI, transient expression of the Rep protein from geminiviruses, or infection with viruses or bacteria all are capable of triggering this response in Nicotiana benthamiana. Interestingly, this response seems non-cell-autonomous, and exogenous treatment with H₂O₂ is sufficient to elicit this relocalization of chloroplasts, which appears to require accumulation of ROS. Taken together, our results indicate that chloroplasts cluster around the nucleus during plant–pathogen interactions, suggesting a fundamental role of this positioning in plant defence, and identify ROS as sufficient and possibly required for the onset of this response.     

The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP⁺ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP⁺ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.     

A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana.

Brome mosaic virus (BMV) is a positive-strand RNA virus with a multipartite genome that causes symptomless infection in Nicotiana benthamiana. We have isolated and characterized a strain of BMV that produced uniform vein chlorosis in systemically infected N. benthamiana. Analysis of pseudorecombinants constructed by exchanging RNA 1 and 2 and RNA 3 components between wild-type (non-symptom-inducing) and vein chlorosis-inducing strains of BMV indicated that the genetic determinant for the induction of the chlorotic phenotype is located on RNA 3. Sequence analysis of progeny RNA 3 recovered from symptomatic N. benthamiana plants revealed that vein chlorosis is due to the single nucleotide transition 887G-->887A, which changes the codon for Val-266 to Ile-266 in the movement protein gene. The mutation had no detectable effect on the accumulation of virus in either inoculated or systematically infected leaves of N. benthamiana. The vein chlorosis phenotype is the manifestation of the substitution of Ile-266 for Val-266 in the movement protein gene, since additional alterations in this region (a silent mutation, i.e., 887GUU889-->GUC, and an alteration of valine to phenylalanine, i.e., 887GUU889-->887UUU889) resulted in symptomless infections on N. benthamiana. The modulation of the symptom phenotype by the substitution of Ile-266 for Val-266 is specific for N. benthamiana, since neither movement nor the symptom phenotype in barley plants was affected.     

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis

Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.     

A variant of the satellite RNA of groundnut rosette virus that induces brilliant yellow blotch mosaic symptoms in Nicotiana benthamiana

Some Malawian cultures of groundnut rosette virus (GRV) give rise to variants that, although still causing symptoms of the chlorotic type of rosette in groundnut, induce brilliant yellow blotch mosaic symptoms, instead of the usual veinal chlorosis and mild mottle, in Nicotiana benthamiana. One such isolate (YB) induced the formation in infected plants of a 0.9 kbp dsRNA having extensive sequence homology with molecules of similar size in other naturally occurring isolates of GRV. These dsRNA molecules were shown to be double-stranded forms of single-stranded satellite RNA molecules. Experiments in which the satellite was removed from and restored to isolate YB, or exchanged with those from other GRV isolates, showed that it carries the determinant for yellow blotch mosaic symptoms. Plants inoculated with the 0.9 kbp dsRNA (denatured or undenatured) developed yellow blotch mosaic even when the satellite-free GRV helper was not inoculated until 11 days later. The satellite RNA is therefore a very stable molecule. Prior infection of N. benthamiana with a GRV isolate containing a normal form of the satellite protected against expression of yellow blotch mosaic symptoms when the plants were later inoculated with isolate YB, whereas prior infection with satellite-free isolates did not. This provides a simple method of determining whether a GRV isolate has an associated satellite RNA. The YB satellite seems to be a newly recognised variant additional to those known to cause the chlorotic, green and other forms of groundnut rosette disease.     

Grapevine fanleaf virus (GFLV)-specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana

Grapevine fanleaf virus (GFLV) is one of the most destructive pathogens of grapevine. In this study, we generated monoclonal antibodies binding specifically to the coat protein of GFLV. Antibody FL₃, which bound most strongly to GFLV and showed cross-reactivity to Arabis mosaic virus (ArMV), was used to construct the single-chain antibody fragment scFvGFLVcp-55. To evaluate the potential of this single-chain variable fragment (scFv) to confer antibody-mediated virus resistance, transgenic Nicotiana benthamiana plants were generated in which the scFv accumulated in the cytosol. Recombinant protein levels of up to 0.1% total soluble protein were achieved. The T₁ and T₂ progenies conferred partial or complete protection against GFLV on challenge with the viral pathogen. The resistance to GFLV in transgenic plants was strictly related to scFvGFLVcp-55 accumulation levels, confirming that the antibody fragment was functional in planta and responsible for the GFLV resistance. In addition, transgenic plants conferring complete protection to GFLV showed substantially enhanced tolerance to ArMV. We demonstrate the first step towards the control of grapevine fanleaf degeneration, as scFvGFLVcp-55 could be an ideal candidate for mediating nepovirus resistance.