Transgenic Nicotiana Benthamiana Plants Research Articles

A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

RNAi mediated broad-spectrum transgenic resistance in Nicotiana benthamiana to chilli-infecting begomoviruses.

Two RNAi constructs were designed targeting chilli-infecting begomoviruses and associated betasatellites. Broad-spectrum resistance was achieved against multiple begomoviruses associated with leaf curl disease of chillies in India. Chilli leaf curl disease (ChiLCD) caused by begomoviruses (family: Geminiviridae) has emerged as one of the most devastating viral diseases of chilli, especially in the Indian sub-continent. The severity of disease incidence is expanding at an alarming rate due to the emergence of new begomoviruses with greater ability to infect this crop in almost all the major chilli producing regions of India. In this study, we applied the RNA interference (RNAi) based strategies to control infection of chilli-infecting begomoviruses (CIBs). For this, we have generated transgenic Nicotiana benthamiana plants harboring two different intron hairpin RNAi constructs [designated as TR1 (AC1/AC2) and TR2 (AC1/AC2/βC1)] using conserved regions of viral genome and associated betasatellite. During our study, we observed that, two lines harboring TR1 construct (13-1 and 2-4) and one line harboring TR2 construct (5-1) have shown resistance to the most predominant Indian CIBs like Chilli leaf curl virus-Pakistan isolate Varanasi, Tomato leaf curl New Delhi virus-isolate chilli, and a newly identified begomovirus species, Chilli leaf curl Vellanad virus. Resistant lines accumulated transgene-specific siRNAs, confirming RNAi-mediated resistance against these viruses. Furthermore, these resistant lines also displayed delayed symptom appearance and milder symptoms, as compared to virus-inoculated non-transgenic plants. Average viral DNA accumulation in the resistant lines was reduced up to 90% as compared to non-transgenic plants. Thus, our study demonstrated the application of RNAi-mediated approach in providing resistance against diverse monopartite and bipartite begomoviruses associated with ChiLCD.

The relationship between the plant-encoded RNA-dependent RNA polymerase 1 and alternative oxidase in tomato basal defense against Tobacco mosaic virus.

Salicylic acid (SA) plays a critical role in plant defense against pathogen attack. The SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense, which is pathogenesis-related protein-independent but involves an RNA-dependent RNA polymerase 1 (RDR1)-mediated RNA silencing mechanism and/or an alternative oxidase (AOX)-associated defense pathway. However, the relationship between these two viral defense-related pathways remains unclear. In this study, Tobacco mosaic virus (TMV) inoculation onto Solanum lycopersicum (tomato) leaves induced a rapid induction of the SlAOX1a transcript level as well as the total and CN-resistant respiration at 0.5dpi, followed by an increase in SlRDR1 gene expression at 1dpi in the upper uninoculated leaves. Silencing SlRDR1 using virus-induced gene silencing system significantly reduced SlRDR1 expression and tomato defense against TMV but had no evident effect on SlAOX1a transcription. Conversely, silencing SlAOX1a not only effectively reduced the AOX1a transcript level, but also blocked the TMV-induced SlRDR1 expression and decreased the basal defense against TMV. Furthermore, the application of an exogenous AOX activator on empty vector-silenced control plants greatly induced the accumulation of SlRDR1 and SlAOX1a transcript and reduced TMV viral RNA accumulation, but failed to have such effects on SlRDR1-silenced plants. Moreover, RDR1-overexpressed transgenic Nicotiana benthamiana plants enhanced defense against TMV than the empty vector-transformed plants, but these effects were not affected by the exogenous AOX activator or inhibitor. These results indicate that RDR1 is involved in the AOX-mediated defense pathway against TMV infection and plays a crucial role in enhancing RNA silencing to limit virus systemic spread.

Development of a VIGS vector based on the β-satellite DNA associated with bhendi yellow vein mosaic virus

Bhendi yellow vein mosaic virus (BYMV) is a monopartite begomovirus with an associated β-satellite. βC1 ORF encoded by the β-satellite is the symptom determinant and a strong suppressor of post transcriptional gene silencing. To create a virus induced gene silencing vector based upon the β-satellite associated with BYVMV the βC1 ORF was replaced with multiple cloning sites. GFP transgene and plant endogenous genes Su, PDS, PCNA and AGO1 were cloned into β-satellite based VIGS vector. GFP expression was silenced in the GFP expressing transgenic 16c Nicotiana benthamiana plants infiltrated with VIGS vector carrying GFP gene inside. N. benthamiana plants infiltrated with the VIGS vector harboring the endogenous genes Su, PDS, PCNA and AGO1 produced the phenotypic symptoms yellowing of the veins, photobleaching of the veins, stunting of the plant and upward leaf curling, respectively. Real time PCR analyses revealed a reduction in the levels of the corresponding transgene or endogenous target mRNA. The β-satellite based VIGS vector was able to silence the target genes effectively. Hence, BYVMV β-satellite based VIGS vector can be used in functional genomics studies.

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing

In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFPendo). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFPendo (Nb-GFPendo) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFPendo coding region. Importantly, hpF-agroinfiltrated Nb-GFPendo plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFPcDNA) carrying a GFP cDNA transgene (GFPcDNA) were included in the analysis. HpF-agroinfiltrated Nb-GFPcDNA plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFPcDNA plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3′ of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFPcDNA coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.

Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S)lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions

Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens–mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process.

High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.

Effects of the virus satellite gene βC1 on host plant defense signaling and volatile emission

Tomato Yellow Leaf Curl China virus spreads together with its invasive vector, the silverleaf whitefly B biotype, which exhibits higher growth rates on infected plants. Previous studies indicate that the virus satellite gene βC1 accounts for the visible symptoms of infection and inhibits the constitutive expression of jasmonic acid (JA)—a phytohormone involved in plant defense against whiteflies—and of some JA-regulated genes. Here we present new details of the effects of on plant signaling and defense, obtained with (non-host) transgenic Arabidopsis thaliana and Nicotiana benthamiana plants. We found that JA induction in response to wounding was reduced in plants expressing βC1. This result implies that βC1 acts on conserved plant regulation mechanisms and might impair the entire JA defense pathway. Furthermore, transformed N. benthamiana plants exhibited elevated emissions of the volatile compound linalool, suggesting that βC1 also influences plant-derived olfactory cues available to vector and non-vector insects.

Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108-120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2 108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2 108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2 108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2 108-120 epitope were found after both methods of vaccine delivery.

Characterization and functional analysis of GhRDR6, a novel RDR6 gene from cotton (Gossypium hirsutum L.)

RDR6 (RNA-dependent RNA polymerase 6) is not only involved in virus resistance but also plays an important role in natural plant development. In the present study, a novel RDR gene, named GhRDR6 (Gossypium hirsutum RDR6), was isolated from cotton (G. hirsutum L.). Alignment and evolutionary relationship analyses showed that GhRDR6 was more closely related to RDR6 than to other RDRs. Expression analysis indicated that this single-copy gene is constitutively expressed in the roots, stems and leaves. Semi-quantitative RT-PCR (reverse transcription-PCR) showed that GhRDR6 was up-regulated by the application of various phytohormones, including MeJA [methyl JA (jasmonate)], ABA (abscisic acid), JA, α-naphthylacetic acid, gibberellins and ET (ethylene). In addition, GhRDR6 expression increased in response to wounding, cold (4°C) and NaCl treatments, but not by drought. Furthermore, overexpression of GhRDR6 in transgenic Nicotiana benthamiana plants resulted in root lengths longer than the wide-type during the seeding stage. Interestingly, the GhRDR6-overexpressing plants displayed reduced tolerance to oxidative damage, resulting in reduced ABA-sensitivity, but they tolerated freezing. Moreover, resistance to potato virus Y was enhanced in transgenic N. benthamiana plants. These results suggest that GhRDR6 may play an important role in plant defence responses and a pivotal role in plant development.

Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative‐sense single‐stranded RNA plant virus

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.

Genetic modification of alternative respiration in Nicotiana benthamianaaffects basal and salicylic acid-induced resistance to potato virus X

BackgroundSalicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.ResultsAntimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses) and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus.ConclusionsWe conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX.

The Solanum lycopersicum RNaseLER is a class II enzyme of the RNase T2 family and shows preferential expression in guard cells

Ribonucleases (RNases) occur in different gene families, functioning in RNA processing and degradation. In this study, we report on cloning and characterization of RNaseLER, the first class II gene of the RNase T2 family in tomato ( Solanum lycopersicum). The family also includes the class I members RNaseLE and RNaseLX, and the class III group of S-RNases acting in self incompatibility. The RNaseLER gene was cloned by polymerase chain reaction (PCR)-assisted methods. Structural analyses of RNaseLER and homologous genes revealed unique key features of class II RNase T2 genes. RNaseLER is a single copy gene in tomato and codes for a primary protein of 260 amino acids. Subcellular localization analyzed with a RNaseLER-eYFP fusion protein and co-localization experiments revealed an intracellular accumulation in the endoplasmic reticulum. Transgenic Nicotiana benthamiana plants carrying the uidA reporter gene under the control of a 900-bp RNaseLER promoter sequence express the reporter gene predominantly in guard cells and trichomes. This previously unknown spatial expression of a RNase T2 gene is consistent with ubiquitous detection of low RNaseLER transcript abundances in almost all parts of tomato plants. As revealed by quantitative real-time RT-PCR analysis treatments with abscisic acid, ethylene or other abiotic and biotic stress factors did not affect RNaseLER expression significantly. Unlike tomato class I genes, RNaseLER represents a constitutively expressed gene with a cell-specific role in stomata and trichomes and no involvement in stress responses.

Stable expression of Rice dwarf virus Pns10 suppresses the post-transcriptional gene silencing in transgenic Nicotiana benthamiana plants

RNA silencing is a conserved mechanism that defends against viral infection and retrotransposon activity for protection of the genome. Segment 10 (S10) of Rice dwarf virus (RDV) encodes Pns10 protein, a viral suppressor of RNAi that suppresses the host RNA silencing machinery. In this study, we obtained stable transgenic RDV-S10 Nicotiana benthamiana plants expressing Pns10. Suppression of post-transcriptional gene silencing (PTGS) by Pns10 supported the conclusion that this protein exhibited the RNA silencing suppressor activity. In particular, the transgenic plants stably expressing a viral suppressor of RNAi (VSR) provide a model system for investigating the mechanism of RNA silencing.<br /> RNA silencing; VSR; Rice dwarf virus; Pns10; transgenic plant.

Differential resistance to Citrus psorosis virus in transgenic Nicotiana benthamiana plants expressing hairpin RNA derived from the coat protein and 54K protein genes

Citrus psorosis virus (CPsV), genus Ophiovirus, family Ophioviridae, is the causal agent of a serious disease affecting citrus trees in many countries. The viral genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of plant defence against viruses, can be induced by transgenic expression of virus-derived sequences encoding hairpin RNAs. Since the production of transgenic citrus lines and their evaluation would take years, a herbaceous model plant, Nicotiana benthamiana, was used to test hairpin constructs. The expression of self-complementary hairpin RNA fragments from the coat protein (cp) and 54K genes of the Argentine CPsV 90-1-1 isolate conferred resistance on N. benthamiana plants, indicating that these constructs are good candidates for the transformation of citrus plants. The degree of resistance obtained varied depending on the viral sequence chosen. The analysis of the levels of small interfering RNA accumulation and viral RNAs indicated that the construct derived from cp gene was better at inducing PTGS than that originating from the 54K gene. The dependence of PTGS induction on the degree of identity between the target and the inducer transgene sequences was tested using sequences derived from CPV4, a more distant isolate of CPsV, as PTGS targets. Efficient silencing induction was also obtained to this isolate through the expression of the cp-derived hairpin. This is the first report of transgenic-resistant plants within the context of this serious citrus disease.

RNAi-Mediated Transgenic Tospovirus Resistance Broken by IntraspeciesSilencing Suppressor Protein Complementation

Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants additionally resistant to this strain but not to the soybean strain of this Tospovirus sp. (TYRV-s), both strains exhibiting 14.4% nucleotide sequence divergence in their N genes. Surprisingly, coinoculation of the TYRV-t-resistant transgenic lines with both TYRV-t and TYRV-s resulted in rescue of the former. Mass-spectrometric analysis of the viral ribonucleocapsids accumulating in the transgenic plants showed the presence of the N proteins of both strains excluding hetero-encapsidation as rescue mechanism and indicating suppression of TYRV-t N gene transcript breakdown by RNA interference. Prior (Potato virus X [PVX]-vector-mediated) expression of the TYRV-s silencing suppressor (NS(s)) gene also allowed TYRV-t to break the resistance. This phenomenon was also observed when the homologous (TYRV-t) NS(s) gene was provided from a PVX replicon, demonstrating that TYRV can break RNA-mediated host resistance upon a priori expression of its NS(s) protein. Remarkably, mixed inoculation of TYRV-t with other Tospovirus spp. or nonrelated viruses did not result in resistance breaking, indicating that the rescuing activity of NS(s)-though based on suppressing RNA silencing-is species-dependent.

Biochemical and immunological characterization of the plant‐derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB–MPR649–684

Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.

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 FL(3), 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 T(1) and T(2) 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.

Hairy-root organ cultures for the production of human acetylcholinesterase

BackgroundHuman cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.ResultsAs a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4°C for up to 5 months.ConclusionOur results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.