Strategies for antiviral resistance in transgenic plants

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.     

The mechanism(s) of coat protein-mediated resistance against tobacco mosaic virus

The resistance of transgenic plants express genes encoding viral coat proteins to infection by the viruses from which the genes are derived was termed coat protein-mediated resistance (CP-MR) and has been demonstrated for a variety of virus/host combinations. The mechanism of CP-MR is perhaps best understood in the tobacco/TMV system. CP-MR against TMV requires accumulation of CP and does not seem to involve the induction of plant defense mechanisms. The resistance appears to be mainly based on the inhibition of virion disassembly in transgenic cells although there is evidence that a later step of infection is also affected. CP-MR of tobacco to TMV shares some features with classical cross-protection and with CP-MR in some, but not all other host/virus combinations.     

Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.     

Field testing of virus resistant transgenic plants

A number of crop plants have been genetically modified for the purpose of resisting virus infection. Different resistance types have been observed in transgenic crops. The practical value of genetically modified, virus resistant, economically important crops can be evaluated only by field testing. The criteria for effective field resistance to viral disease can vary significantly depending on the crop and the virus. Furthermore, field testing is required to determine whether important agronomic properties of modified crops were changed by plant transformation and to confirm that the resistance observed under controlled environment is effective also under natural field conditions and to demonstrate the economical value of virus resistant, transgenic plants.     

Evaluation of DNA fragments covering the entire genome of a monopartite begomovirus for induction of viral resistance in transgenic plants via gene silencing

Tomato-infecting begomoviruses, a member of whitefly-transmitted geminivirus, cause the most devastating virus disease complex of cultivated tomato crops in the tropical and subtropical regions. Numerous strategies have been used to engineer crops for their resistance to geminiviruses. However, nearly all have concentrated on engineering the replication-associated gene (Rep), but not on a comprehensive evaluation of the entire virus genome. In this study, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting begomovirus in Taiwan, was subjected to the investigation of the viral gene fragments conferring resistance to geminiviruses in transgenic plants. Ten transgenic constructs covering the entire ToLCTWV genome were fused to a silencer DNA, the middle half of N gene of Tomato spot wilt virus (TSWV), to induce gene silencing and these constructs were transformed into Nicotiana benthamiana plants. Two constructs derived from IRC1 (intergenic region flanked with 5′ end Rep) and C2 (partial C2 ORF) were able to render resistance to ToLCTWV in transgenic N. benthamiana plants. Transgenic plants transformed with two other constructs, C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3′ end of the C1 ORF), significantly delayed the symptom development. Detection of siRNA confirmed that the mechanism of resistance was via gene silencing. This study demonstrated for the first time the screening of the entire genome of a monopartite begomovirus to discover viral DNA fragments that might be suitable for conferring virus resistance, and which could be potential candidates for developing transgenic plants with durable and broad-spectrum resistance to a DNA virus via a gene silencing approach.     

Engineered resistance against plant virus diseases

The development of genetic engineering techniques has enabled the production of transgenic plants that are resistant to viral diseases. Expressing the coat protein (CP) gene of a virus in Iransgenic plants confers resistance against the virus from which the gene was isolated, and to other closely related strains and viruses. This approach has been demonstrated to be effective in conferring protection against viruses from different virus groups including alfalfa mosaic virus, cucumovirus. ilarvirus, potex-virus, potyvirus, tobamovirus and tobravirus. The data available indicate that several factors may affect the efficiency of the protection obtained including the level of the CP in the transgenic plants, the plant in which the CP gene is expressed and enviromental conditions. These and other aspects of coat protein mediated resistance are discussed.     

Genetic characterization of measles virus strains isolated during an epidemic cluster in Puglia, Italy 2006–2007

Whitefly-transmitted geminiviruses (genusBegomovirus) are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2) to develop resistance againstTomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.     

Cucurbit biotechnology-the importance of virus resistance

Summary The cucurbit family includes a number of valuable crop species (melon, cucumber, squash/pumpkin, watermelon). Much of this review is concerned with transgenic resistance to viruses, shown to be the major application of biotechnology in the cucurbit family. Progress made with the production of transgenic cucurbit crops is discussed. Published data on field tests of transgenic cucurbits are reviewed, showing that much progress has been made with multiple virus-resistant cucurbit crops which can be productive without chemical control of insect virus vectors. Modes of virus resistance in trangenic cucurbits are discussed, as is the bio-safety of such crops. For the first time a detailed analysis has been made of world-wide and US field test applications for cucurbit crops. World-wide, most field test applications were for melon (54%), followed by squash (32%). World-wide most field test applications were for virus resistance (84%), and most applications (77%) were in the USA. Two transgenic multiple virus-resistant squash crops have been deregulated (released for sale). Additionally, the analysis shows that there are transgenic multiple virus-resistant crops in all major cucurbit species already available, for which several different companies have applied for field tests. This would imply that such crops are ready to be marketed should conditions permit, which would have an impact world-wide in reduction of ecological damage due to chemical control of the insect viral vectors.     

转基因RNAi技术在动物抗病毒育种中的应用

病毒对动物和人类健康都是极大的威胁,抗病毒疫苗虽然能在一定程度上预防病毒病,但目前几乎还不能对变异的传染病进行抗病毒治疗。尽管RNA干扰研究到目前才短短十几年,其作用机理已基本清楚。RNAi能够非常有效的抑制病毒体内复制,其介导的抗病转基因动物的研究相继取得了阶段性进展,抗疯牛病转基因羊和牛,抗内源性逆转录病毒猪以及抗核型多角体病毒病的转基因家蚕已经成功获得。尽管如此,目前的研究主要还是集中在细胞水平及小鼠模型方面,获得的转基因动物种类和数量有限,但为培育动物抗病毒品种提供了理论依据和技术支撑。随着转基因技术的不断的进步和成熟, RNA干扰技术将成为动物抗病毒育种中最有应用前景的方法之一。     

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

     

Constraints to virus infection in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants transformed with a potyvirus amplicon

Background  

Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression.     

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.     

昆虫抗药性分子机制研究的新进展

昆虫抗性机制的研究对于抗性监测、治理及新农药的研制具有重要意义。在过去几十年中,人们对与昆虫杀虫剂抗性有关的昆虫行为、生理代谢活动以及作用靶标等进行了广泛的研究。已经证实,昆虫的抗药性与行为改变、生理功能改变、解毒功能增强以及靶标不敏感性有关。近年来,随着分子生物学以及昆虫基因组学的发展,昆虫抗药性的分子机理有了突破性进展,已发现并克隆了一些靶标基因,与抗药性相关的基因突变也得到广泛验证。本文综述了昆虫的抗药性机理在分子生物学上的研究最新进展,重点阐述了与昆虫抗性相关基因的扩增、表达及基因结构的改变等相关内容。     

Mechanisms of plant resistance to viruses

Plants have evolved in an environment rich with microorganisms that are eager to capitalize on the plants' biosynthetic and energy-producing capabilities. There are approximately 450 species of plant-pathogenic viruses, which cause a range of diseases. However, plants have not been passive in the face of these assaults, but have developed elaborate and effective defence mechanisms to prevent, or limit, damage owing to viral infection. Plant resistance genes confer resistance to various pathogens, including viruses. The defence response that is initiated after detection of a specific virus is stereotypical, and the cellular and physiological features associated with it have been well characterized. Recently, RNA silencing has gained prominence as an important cellular pathway for defence against foreign nucleic acids, including viruses. These pathways function in concert to result in effective protection against virus infection in plants.     

Peptide inhibitors against herpes simplex virus infections

Herpes simplex virus (HSV) is a significant human pathogen causing mucocutaneous lesions primarily in the oral or genital mucosa. Although acyclovir (ACV) and related nucleoside analogs provide successful treatment, HSV remains highly prevalent worldwide and is a major cofactor for the spread of human immunodeficiency virus. Encephalitis, meningitis, and blinding keratitis are among the most severe diseases caused by HSV. ACV resistance poses an important problem for immunocompromised patients and highlights the need for new safe and effective agents; therefore, the development of novel strategies to eradicate HSV is a global public health priority. Despite the continued global epidemic of HSV and extensive research, there have been few major breakthroughs in the treatment or prevention of the virus since the introduction of ACV in the 1980s. A therapeutic strategy at the moment not fully addressed is the use of small peptide molecules. These can be either modeled on viral proteins or derived from antimicrobial peptides. Any peptide that interrupts protein–protein or viral protein–host cell membrane interactions is potentially a novel antiviral drug and may be a useful tool for elucidating the mechanisms of viral entry. This review summarizes current knowledge and strategies in the development of synthetic and natural peptides to inhibit HSV infectivity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

DNA Virus Replication Compartments

Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication.