Virus-resistant Plants Research Articles

Resistance of non-transgenic papaya plants to papaya ringspot virus (PRSV) mediated by intron-containing hairpin dsRNAs expressed in bacteria.

RNA-mediated virus resistance based on natural antiviral RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants. In this study, a conserved 3'-region (positions 9839-10117, 279 nt) of the capsid protein (CP) gene of papaya ringspot virus (PRSV), designated CP279, was used to generate an intron-containing hairpin RNA (ihpRNA) construct by one-step, zero-background ligation-independent cloning (OZ-LIC). The RNaseIII-deficient Escherichia coli strain M-JM109lacY was identified as the best choice for producing large quantities of specific ihpRNA-CP279. Resistance analyses and ELISA data verified that most papaya plants mechanically co-inoculated with TRIzol-extracted ihpRNA-CP279 and PRSV were resistant to PRSV, and resistance was maintained throughout the test period (>2 months post-inoculation). In contrast, a 1-2 day interval between sequential inoculation of PRSV and ihpRNA-CP279 did not result in complete protection against PRSV infection, but delayed the appearance of viral symptoms by 3 to 4 days. These findings indicate that direct mechanical inoculation of papaya plants with bacterially-expressed ihpRNA-CP279 targeting the PRSV CP gene can interfere with virus infection. This work lays a foundation for developing a non-transgenic approach to control PRSV by directly spraying plants with ihpRNA or crude bacterial extract preparations.

Allopolyploidy and the evolution of plant virus resistance.

BackgroundThe relationship between allopolyploidy and plant virus resistance is poorly understood. To determine the relationship of plant evolutionary history and basal virus resistance, a panel of Nicotiana species from diverse geographic regions and ploidy levels was assessed for resistance to non-coevolved viruses from the genus Nepovirus, family Secoviridae. The heritability of resistance was tested in a panel of synthetic allopolyploids. Leaves of different positions on each inoculated plant were tested for virus presence and a subset of plants was re-inoculated and assessed for systemic recovery.ResultsDepending on the host-virus combination, plants displayed immunity, susceptibility or intermediate levels of resistance. Synthetic allopolyploids showed an incompletely dominant resistance phenotype and manifested systemic recovery. Plant ploidy was weakly negatively correlated with virus resistance in Nicotiana species, but this trend did not hold when synthetic allopolyploids were taken into account. Furthermore, a relationship between resistance and geographical origin was observed.ConclusionThe gradients of resistance and virulence corresponded to a modified matching allele model of resistance. Intermediate resistance responses of allopolyploids corresponded with a model of multi-allelic additive resistance. The variable virus resistance of extant allopolyploids suggested that selection-based mechanisms surpass ploidy with respect to evolution of basal resistance to viruses.

植物ウイルス抵抗性の打破：ウイルス抵抗性の永続性を予測したり延長させたりできるだろうか？

Cultivars with introgressed natural resistance genes have been widely used for plant disease control, especially in the control of virus diseases, for which no effective chemical control agent is available. However, we often encounter virus mutants that break down or overcome the resistance. In this review, recent studies will be discussed with respect to breakdown of plant virus resistance.

Tomato yellow leaf curl Sardinia virus‐resistant tomato plants expressing the multifunctional N‐terminal domain of the replication‐associated protein show transcriptional changes resembling stress‐related responses

The N-terminal domain (amino acids 1-130) of the replication-associated protein (Rep130 ) of Tomato yellow leaf curl Sardinia virus (TYLCSV) retains the ability of full-length Rep to localize to the nucleus and to down-regulate C1 transcription when ectopically expressed in plants, both functions being required to inhibit homologous viral replication. In this study, we analysed the effect of Rep130 expression on virus resistance and the plant transcriptome in the natural and agronomically important host species of TYLCSV, Solanum lycopersicum. Tomato plants accumulating high levels of Rep130 were generated and proved to be resistant to TYLCSV. Using an in vitro assay, we showed that plant-expressed Rep130 also retains the catalytic activity of Rep, thus supporting the notion that this protein domain is fully functional. Interestingly, Rep130 -expressing tomatoes were characterized by an altered transcriptional profile resembling stress-related responses. Notably, the serine-type protease inhibitor (Ser-PI) category was over-represented among the 20 up-regulated genes. The involvement of Rep130 in the alteration of host mRNA steady-state levels was confirmed using a distinct set of virus-resistant transgenic tomato plants expressing the same TYLCSV Rep130 , but from a different, synthetic, gene. Eight genes were found to be up-regulated in both types of transgenic tomato and two encoded Ser-PIs. Four of these eight genes were also up-regulated in TYLCSV-infected wild-type tomato plants. Implications with regard to the ability of this Rep domain to interfere with viral infections and to alter the host transcriptome are discussed.

Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock

A major application of RNA interference (RNAi) is envisaged for the production of virus-resistant transgenic plants. For fruit trees, this remains the most, if not the only, viable option for the control of plant viral disease outbreaks in cultivated orchards, due to the difficulties associated with the use of traditional and conventional disease-control measures. The use of RNAi might provide an additional benefit for woody crops if silenced rootstock can efficiently transmit the silencing signal to non-transformed scions, as has already been demonstrated in herbaceous plants. This would provide a great opportunity to produce non-transgenic fruit from transgenic rootstock. In this review, we scrutinise some of the concerns that might arise with the use of RNAi for engineering virus-resistant plants, and we speculate that this virus resistance has fewer biosafety concerns. This is mainly because RNAi-eliciting constructs only express small RNA molecules rather than proteins, and because this technology can be applied using plant rootstock that can confer virus resistance to the scion, leaving the scion untransformed. We discuss the main biosafety concerns related to the release of new types of virus-resistant plants and the risk assessment approaches in the application of existing regulatory systems (in particular, those of the European Union, the USA, and Canada) for the evaluation and approval of RNAi-mediated virus-resistant plants, either as transgenic varieties or as plant virus resistance induced by transgenic rootstock.

A nucleic acid hydrolyzing recombinant antibody confers resistance to curtovirus infection in tobacco

A catalytic single chain variable antibody (scFv), 3D8 scFv, which has DNase activities, was functionally expressed in Nicotiana tabacum. The subcellular localization of the GFP-fused 3D8 indicated that the 3D8 protein was expressed in the cytosol of the N. tabacum protoplasts. Progenies of the transgenic tobacco plants exhibited complete resistance against two single stranded (ss) DNA geminiviruses, including the Beet curly top virus and the Beet severe curly top virus, without viral accumulation or disease symptoms. We presented a novel strategy for targeting the viral DNA itself in a sequence non-specific manner, rather than the viral proteins or RNAs, in order to generate virus-resistant transgenic plants. No noticeable adverse effects on the growth and reproduction of the transgenic plants were observed. Our results demonstrated that targeting viral DNA is an effective strategy for protecting plants from ssDNA viruses.

RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in transgenic Nicotiana tabacum

Plant viruses continue to cause diseases on economically important crops. Therefore, numerous attempts to produce virus resistant plants have been reported by using the mechanisms such as host mediated protection and virus mediated protection. Here, a novel strategy of targeting viral RNA itself, rather than viral gene products, is presented to generate virus-resistant transgenic plants. A catalytic single chain variable antibody, 3D8 scFv, which has RNase activities, was functionally expressed in the cytosol of Nicotiana tabacum. We found that progenies of the transgenic tobacco plant acquired complete resistances against four ss-RNA tobamoviruses and one cucumovirus tested without viral accumulation and delayed onset of disease symptoms. The results showed that the resistance observed in 3D8 scFv transgenic plants was caused by the RNase activity of 3D8 scFv itself, not by RNA-mediated gene silencing mechanism. Taken together, we suggested that newly gained resistance of the 3D8 scFv transgenic plants to five ss-RNA viruses most likely resulted from the RNase activity of 3D8 scFv.

Advances, Challenges and Prospects in Small RNA Mediated Approaches of Virus Resistance in Plants

RNA interference (RNAi) is a mechanism of small RNA-guided regulation of gene expression in which small RNAs inhibit the expression of genes with complementary nucleotide sequences. RNAi has emerged as a powerful modality for battling challenging viruses and provides a natural defense against viral pathogens. In plants, RNAi has been successfully used to express cognate dsRNAs for viral transcripts in order to initiate the process of viral gene silencing. Despite the wide applicability of RNAi for achieving viral resistance, there are some challenges and constraints that must to be addressed to develop RNAi as a more effective tool for virus resistance. The present review provides an update on different approaches of using RNAi for virus resistance development in various crop plants. The factors influencing RNAi-mediated virus resistance and the major constraints are also discussed in detail.

Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1

With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host–virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, like higher plants, may employ certain RNA silencing pathways to defend itself against virus infection. To our knowledge this is the first examination of RNA silencing genes in algae beyond core proteins, and the first analysis of their transcription during virus infection.

Transgenic resistance to Bamboo mosaic virus by expression of interfering satellite RNA

Plant genetic engineering has broadened the options for plant virus resistance and is mostly based on pathogen-derived resistance. Previously, we have shown that interfering satellite RNA (satRNA) of Bamboo mosaic virus (satBaMV) greatly reduces Bamboo mosaic virus (BaMV) accumulation and BaMV-induced symptoms in co-inoculated plants. Here, we generated a nonviral source of virus-resistant transgenic Nicotiana benthamiana and Arabidopsis thaliana by introducing interfering satBaMV. Asymptomatic transgenic N. benthamiana lines were highly resistant to BaMV virion and viral RNA infection, and the expression of the transgene BSL6 was higher in asymptomatic than mildly symptomatic lines. In addition, BaMV- and satBaMV-specific small RNAs were detectable only after BaMV challenge, and their levels were associated with genomic viral RNA or satRNA levels. By transcriptomic analysis, the salicylic acid (SA) signalling pathway was not induced in satBaMV transgenic A. thaliana in mock conditions, suggesting that two major antiviral mechanisms, RNA silencing and SA-mediated resistance, are not involved directly in transgenic satBaMV-mediated BaMV interference. In contrast, resistance is associated with the level of the interfering satBaMV transgene. We propose satBaMV-mediated BaMV interference in transgenic plants by competition for replicase with BaMV.

Overexpression of a Single-Chain Variable Fragment (scFv) Antibody Confers Unstable Resistance to TuMV in Chinese Cabbage

A single-chain variable fragment (scFv) antibody is a recombinant protein composed of immunogloblin fused to a flexible linker protein. Recent studies have demonstrated that transfer of scFv to plants is effective in engineering virus resistance in plants. In this study, the utility of scFv in enhancing Turnip mosaic virus (TuMV) resistance in transgenic Chinese cabbage (Brassica rapa) was investigated. For this, a construct carrying scFv was transferred into Chinese cabbage and transgenic plant expressing scFv plants have been generated. DAS-ELISA and phenotypic data have revealed that not all transgenic plants carrying the scFv transgene were resistant to TuMV. Those transgenic lines resistant to TuMV had higher levels of scFv transcripts than transgenic lines susceptible to TuMV. Moreover, analysis of transgenic plants for methylation of the scFv transgene using the methylation-sensitive restriction enzyme HhaI has revealed that it was the non-methylated scFv transgene that was expressed in TuMV resistant lines whereas, the methylated scFv transgene was silenced, resulting in TuMV susceptibility in other transgenic lines. Bisulfate genomic sequencing, performed to further confirm methylation of the scFv transgene, has revealed that the promoter region of scFv in both resistant and susceptible transgenic was methylated but methylation was higher in susceptible plants than in resistant plants. In conclusion, overexpression of scFv confers TuMV resistance at low frequencies, and that the transgene is frequently silenced presumably due to cytotoxic effects of scFv in transgenic plants.

RNA Silencing and Plant Viral Diseases

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

Functional Differentiation in the Leucine-Rich Repeat Domains of Closely Related Plant Virus-Resistance Proteins That Recognize Common Avr Proteins

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.

A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance

To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40 days after sowing (20 days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.

Virus resistance obtained in transgenic tobacco and rice by RNA interference using promoters with distinct activity

To induce virus resistance in tobacco and rice we constructed hairpin RNA expression system harbouring inverted repeat fragments of coat protein cDNA of Potato virus Y (PVY) or Rice stripe virus (RSV). These structures were driven by three promoters [cauliflower mosaic virus 35S (CaMV 35S), polyubiqutin gene of maize (Ubi), and Pharbitis nil leucine zipper gene (PNZIP)] which have different tissue-specific activity. PVY resistance ratios were 65.18, 24.33 and 83.54 % in transgenic tobacco plants harboring p35S-PVY, pUbi-PVY and pPNZIP-PVY. RSV resistance was 16.21, 28.61 and 29.33 % in transgenic rice plants harboring p35S-RSV, pUbi-RSV and pPNZIP-RSV. Northern blotting and GUS assay demonstrated that virus resistance levels were related to promoter activity. Therefore, choice of the more effective and tissue-specific promoter to reinforce transcription of hpRNAs will favour the cultivation of highly virusresistant transgenic plants.

植物のRNAサイレンシングとウイルスの病徴誘導

RNA silencing plays an important role in plant resistance against viruses. As a counter-defense against RNA silencing, plant viruses have evolved RNA silencing suppressors (RSSs). RNA silencing is likely to play a major role in disease development. For example, RSSs have been found to disturb the gene expression controlled by miRNAs in plant tissue and organ development, resulting in plant malformation. Mosaic symptoms, which are typical in virus-infected plants, are actually a consequence of local arms race between host RNA silencing and viral RSSs. In addition, recent studies revealed that viral siRNAs could induce RNA silencing even against a certain host gene and thus a disease symptom through a complementary (homologous) sequence coincidentally found between virus and host gene. RNA silencing is the principal mediator of viral pathogenicity and disease induction and therefore should be exploited as a powerful tool for engineering virus resistance in plants as well as in animals.

Genetic transformation and development of Cucumber mosaic virus resistant transgenic plants of Chrysanthemum morifolium cv. Kundan

Chrysanthemum morifolium Ramat. cv. Kundan has been found susceptible to infections of Cucumber mosaic virus (CMV) which drastically affects the quality and quantity of blooms and poses significant constraints in commercial cultivation of chrysanthemum in India. Therefore, development of inbuilt resistance in C. morifolium against CMV seems to be essential. Agrobacterium-mediated transformation of petiole explants of C. morifolium was attempted using p RoK2 binary vector harbouring coat protein (CP) gene of CMV under the control of CaMV 35S promoter. A total of 257 explants were transformed and 73 putative transgenic plants from seven independent co-cultivation events were obtained with ∼6% transformation efficiency. Molecular analysis of these plants confirmed the successful integration of CP transgene in 63% plants, of which 12.3% plants were able to transcript and translate the transgene. Expression of coat protein did not evoke any abnormal phenotype. Transgenic plants showed delayed resistance when challenged by CMV-chrysanthemum strain which produced good quality blooms as compared to the susceptible ones.

Antiviral strategies in plants based on RNA silencing

One of the challenges being faced in the twenty-first century is the biological control of plant viral infections. Among the different strategies to combat virus infections, those based on pathogen-derived resistance (PDR) are probably the most powerful approaches to confer virus resistance in plants. The application of the PDR concept not only revealed the existence of a previously unknown sequence-specific RNA-degradation mechanism in plants, but has also helped to design antiviral strategies to engineer viral resistant plants in the last 25 years. In this article, we review the different platforms related to RNA silencing that have been developed during this time to obtain plants resistant to viruses and illustrate examples of current applications of RNA silencing to protect crop plants against viral diseases of agronomic relevance. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

Transgenic Tobacco Plants Resistant to Two Viruses via RNA Silencing

RNA silencing is a phenomenon of homologous RNA degradation induced by dsRNA, which is an effective strategy to obtain virus resistant plants so far. By means of this strategy, higher bio-security immune transgenic plants were obtained, avoiding recombining and transcapsidating with other virus genes. In this study, the recombinant plant expression vector pBIN438-MPRep (i/r) consisting the inverted repeat of TMV-ΔMP and CMV-ΔRep fusion fragment was transformed into tobacco cultivar K326 via Agrobacterium-mediated. The transformants were selected in the culture medium with 100 mg L^(-1) Kan. One hundred and ninety-six transgenic plants were obtained, one hundred and twenty-eight of which were positive plants, and the resistance to CMV and TMV was tested at the degree of virus. PCR-Southern blot and RT-PCR analysis of the transgenic plants demonstrated that the exogenous DNA was integrated into the tobacco genomic DNA and was expressed in transcriptional level. Resistance assay indicated that about 20.3% transgenic plants were immune to the co-infection with TMV and CMV. This result will provide to crops an important reference for plant anti-viral breeding and for preventing the viral co-infection.

Virus resistance of transgenic potato plants (Solanum tuberosum L.) bearing PVY NTN -CP gene of potato virus Y coat protein

The PVY NTN -CP coat protein gene from a necrotic strain of potato virus Y (PVY NTN ) has been transferred into two potato Solanum tuberosum L. cultivars, Mindenes and Somogyi kifli, via agrobacterial transformation. Expression of the PVY NTN -CP gene was revealed in 33 (89%) of 37 and 3 (75%) of 4 kanamycin-resistant regenerates of these cultivars, respectively. The level of virus resistance against two virus strains (PVY O , PVY NTN ) of independent lines of transgenic potatoes varied from extreme resistance to full susceptibility. The three independent lines of transgenic potatoes proved to be extreme resistant to both PVY strains.