Virus-Mediated Targeted DNA Methylation Illuminates the Dynamics of Methylation in an Endogenous Plant Gene.

DNA methylation maintains genome stability and regulates gene expression in plants. RNA-directed DNA methylation (RdDM) is critical for appropriate methylation. However, no efficient tools are available for the investigation of the functions of specific DNA methylation. In this study, the cucumber mosaic virus vector was used for targeted DNA methylation. Methylation was rapidly induced but gradually decreased from the 3' end of the target endogenous sequence in Nicotiana benthamiana, suggesting a mechanism to protect against the ectopic introduction of DNA methylation. Increasing 24-nt siRNAs blocked this reduction in methylation by down-regulating DCL2 and DCL4. RdDM relies on the sequence identity between RNA and genomic DNA; however, this identity does not appear to be the sole determinant for efficient DNA methylation. The current findings provide new insight into the regulation of DNA methylation and promote additional effort to develop efficient targeted DNA methylation in plants.

Role of salicylic acid glucosyltransferase in balancing growth and defence for optimum plant fitness.

Salicylic acid (SA), an essential secondary messenger for plant defence responses, plays a role in maintaining a balance (trade-off) between plant growth and resistance induction, but the detailed mechanism has not been explored. Because the SA mimic benzothiadiazole (BTH) is a more stable inducer of plant defence than SA after exogenous application, we analysed expression profiles of defence genes after BTH treatment to better understand SA-mediated immune induction. Transcript levels of the salicylic acid glucosyltransferase (SAGT) gene were significantly lower in BTH-treated Nicotiana tabacum (Nt) plants than in SA-treated Nt control plants, suggesting that SAGT may play an important role in SA-related host defence responses. Treatment with BTH followed by SA suppressed SAGT transcription, indicating that the inhibitory effect of BTH is not reversible. In addition, in BTH-treated Nt and Nicotiana benthamiana (Nb) plants, an early high accumulation of SA and SA 2-O-ß-d-glucoside was only transient compared to the control. This observation agreed well with the finding that SAGT-overexpressing (OE) Nb lines contained less SA and jasmonic acid (JA) than in the Nb plants. When inoculated with a virus, the OE Nb plants showed more severe symptoms and accumulated higher levels of virus, while resistance increased in SAGT-silenced (IR) Nb plants. In addition, the IR plants restricted bacterial spread to the inoculated leaves. After the BTH treatment, OE Nb plants were slightly larger than the Nb plants. These results together indicate that SAGT has a pivotal role in the balance between plant growth and SA/JA-mediated defence for optimum plant fitness.

Rapid transient protein production by the coat protein-deficient cucumber mosaic virus vector: non-packaged CMV system, NoPaCS.

KEY MESSAGE: We developed a non-packaged CMV system (NoPaCS) for CMV-agroinfection with a virus-inescapable transgenic plant platform, enabling rapid, high production of a large-sequence target protein. For rapidly producing high levels of a desirable protein, many plant virus vectors have been developed. However, there is always a concern that such recombinant viruses may escape into the environment. Especially for insect-transmissible viruses, certain measures must be taken. We here developed a new cucumber mosaic virus (CMV) RNA 3-based vector that is not transmitted by aphids because we deleted the coat protein (CP) gene responsible for aphid transmission and replaced it with a foreign gene. Transgenic Nicotiana benthamiana plants expressing CMV RNA 1 (CR1Tg) were found to be the most suitable platform for producing a recombinant protein using the CMV vector. By agroinfiltrating CR1Tg plants with the RNA 2 construct and the CMV vector harboring the green fluorescence protein (GFP) gene instead of the CP gene, we achieved a high yield of GFP (e.g., ~ 750 mg/kg FW) throughout the bacteria-infiltrated tissues at 2-3 days after infiltration. Furthermore, with this CMV-agroinfection system, a large gene such as the ß-glucuronidase (GUS) gene can be expressed because the viral RNAs are not necessarily encapsidated for replication. The system is designated "non-packaged CMV system (NoPaCS)".

A simple agroinfiltration method for transient gene expression in plant leaf discs.

In the present study, we developed a simple transient gene expression system based on Agrobacterium-mediated transformation. Vacuum infiltration was applied to leaf discs from Nicotiana benthamiana plants with Agrobacterium suspension solution under conventional vacuum conditions in a needleless plastic syringe. Model proteins, green fluorescent protein, ß-glucuronidase, mouse granulocyte-macrophage colony-stimulating factor, and human fibroblast growth factor 1 were successfully expressed in leaf discs within 4 days after infiltration. In addition, the functional evaluation of viral RNA silencing suppressors, Artichoke mottled crinkle virus p19 protein, was also performed. Using this method, the contamination and diffusion of genetically modified bacterium to the environment and important transgenic plants were prevented. This method can be conducted without specialized apparatuses or large amounts of Agrobacterium suspension solutions; thus, the simultaneous evaluation of multiple vectors will be easily possible.

NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana.

KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.

Risk-managed production of bioactive recombinant proteins using a novel plant virus vector with a helper plant to complement viral systemic movement.

A plant viral vector has the potential to efficiently produce recombinant proteins at a low cost in a short period. Although recombinant proteins can be also produced by transgenic plants, a plant viral vector, if available, may be more convenient when urgent scale-up in production is needed. However, it is difficult to use a viral vector in open fields because of the risk of escape to the environment. In this study, we constructed a novel viral vector system using a movement-defective Cucumber mosaic virus (CMV) vector, which is theoretically localized in the inoculated cells but infects systemically only with the aid of the transgenic helper plant that complements viral movement, diminishing the risk of viral proliferation. Interestingly, the helper plant systemically infected with the vector gave strong cross-protection against challenge inoculation with wild-type CMVs. Using CMV strains belonging to two discrete CMV groups (subgroups I and II), we also improved the system to prevent recombination between the vector and the transgene transcript in the helper plant. We here demonstrate the expression of an anti-dioxin single chain variable fragment (DxscFv) and interleukin-1 receptor antagonist (IL1-Ra) in Nicotiana benthamiana by this viral vector confinement system, which is applicable for many useful high-quality recombinant proteins.

Production of biologically active Atlantic salmon interferon in transgenic potato and rice plants.

Interferons (IFNs) are cytokines that induce an antiviral state in vertebrate cells. The Atlantic salmon (Salmo salar) IFN gene (SasaIFN-alpha1) was introduced in potato and rice plants by Agrobacterium-mediated transformation to produce a biologically active fish IFN in these plants. The transgenes and their transcripts were detected by PCR and Northern blot analysis. Western blot analysis showed the existence of SasaIFN-alpha1in transgenic plants. The antiviral activity of the SasaIFN-alpha1 protein expressed in these plants was determined by the survival rates of pre-treated cultured fish cells against pancreatic necrosis virus infection. The survival rate of cells pre-treated with transgenic samples was up to 95% but was reduced to 30-47% when cells were pre-treated with non-transgenic samples. These results demonstrated an antiviral effect of the SasaIFN-alpha1 protein derived from transgenic plants. Plant-derived IFNs may be suitable as components of functional feeds because such IFNs are free of animal pathogens and can be produced at a lower cost compared with those from transgenic mammalian and bacterial cells. This is the first study describing the production of a biologically active fish IFN using transgenic plants.

HC-Pro, a potyvirus RNA silencing suppressor, cancels cycling of Cucumber mosaic virus in Nicotiana benthamiana plants.

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses--Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)--synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.

Comparative contribution of the aryl hydrocarbon receptor gene to perinatal stage development and dioxin-induced toxicity between the urogenital complex and testis in the mouse.

TCDD (2,3,7,8-tetrachlorodebenzo-p-dioxin) requires the presence of the aryl hydrocarbon receptor (Ahr) gene for its toxic effects, such as reproductive disorders in male offspring of maternally exposed rats and mice. To study the involvement of the Ahr gene in producing the toxic phenotype with respect to testicular development, we administered a relatively high dose of TCDD to mice with three different maternally derived Ahr genotypic traits, and then compared several Ahr-dependent alterations among male reproductive systems on Postnatal Day 14. Reduction in anogenital distance and expression of prostatic epithelial genes in the urogenital complex (UGC) were detected in Ahr(+/+) and Ahr(+/-) mice exposed to TCDD, whereas no difference was observed in Ahr(-/-) mice. In situ hybridization revealed the absence of probasin mRNA expression in the prostate epithelium, despite the obvious development of prostatic lobes in TCDD-exposed mice. In contrast to obvious prostatic dysfunction and induction of cytochrome P450 (CYP) family genes in the UGC by TCDD, no alterations in testicular functions were observed in germ cell/Sertoli cell/interstitial cell marker gene expression or CYP family induction. No histopathological changes were observed among the three genotypes and between control and TCDD-exposed mice. Therefore, mouse external genitalia and prostatic development are much more sensitive to TCDD treatment than testis. Further, the Ahr gene, analyzed in this study, does not significantly contribute to testicular function during perinatal and immature stages, and the developing mouse testis appears to be quite resistant to TCDD exposure.

Testicular cytochrome P450scc and LHR as possible targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the mouse.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult animals has been reported to perturb the regulation of steroidogenesis in the testis, possibly by arylhydrocarbon receptor (AhR). To clarify how AhR is involved in the testicular steroidogenesis, we carried out comparative experiments using wild-type and AhR-null male mice that were intraperitoneally administered TCDD. The TCDD administration to wild-type mice showed significant reduction of P450scc and LHR in the testis, whereas the levels in the AhR-null mouse testis were unchanged. To compare anti-androgenic properties on hypothalamo-pituitary-gonadal (HPG) axis, estradiol-3-benzoate (EB), a synthetic estrogen agonist, was administered to mice, the expression of the LHalpha/FSHalpha, LHbeta, FSHbeta and GnRHR genes was severely impaired in the pituitary gland, in contrast to no observed effects in the TCDD-treated mice. In addition, the expression of the LHR gene was increased in the testis of the EB-treated mice. These observations suggest that the target of TCDD is different from that of EB on HPG axis and that TCDD treatment suppresses the P450scc and LHR genes in the testis in an AhR-dependent manner.

Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat.

ABSTRACT. To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.