Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

International Trade and Local Effects of Viral and Bacterial Diseases in Ornamental Plants.

Since the 1950s, there have been major changes in the scope, value, and organization of the ornamental plant industry. With fewer individual producers and a strong trend toward consolidation and globalization, increasing quantities of diverse plant genera and species are being shipped internationally. Many more ornamentals are propagated vegetatively instead of by seed, further contributing to disease spread. These factors have led to global movement of pathogens to countries where they were not formerly known. The emergence of some previously undescribed pathogens has been facilitated by high-throughput sequencing, but biological studies are often lacking, so their roles in economic diseases are not yet known. Case studies of diseases in selected ornamentals discuss the factors involved in their spread, control measures to reduce their economic impact, and some potential effects on agronomic crops. Advances in diagnostic techniques are discussed, and parallels are drawn to the international movement of human diseases.

The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid (N) or the movement protein (NSm) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N-targeting dsRNAs, but not with NSm-targeting dsRNAs, remained asymptomatic until 40 (N. benthamiana) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N- and NSm-targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Arbuscular Mycorrhizal Symbiosis Primes Tolerance to Cucumber Mosaic Virus in Tomato.

Tomato plants can establish symbiotic interactions with arbuscular mycorrhizal fungi (AMF) able to promote plant nutrition and prime systemic plant defenses against pathogens attack; the mechanism involved is known as mycorrhiza-induced resistance (MIR). However, studies on the effect of AMF on viral infection, still limited and not conclusive, indicate that AMF colonization may have a detrimental effect on plant defenses against viruses, so that the term "mycorrhiza-induced susceptibility" (MIS) has been proposed for these cases. To expand the case studies to a not yet tested viral family, that is, Bromoviridae, we investigated the effect of the colonization by the AMF Funneliformis mosseae on cucumber mosaic virus (CMV) infection in tomato by phenotypic, physiological, biochemical, and transcriptional analyses. Our results showed that the establishment of a functional AM symbiosis is able to limit symptoms development. Physiological and transcriptomic data highlighted that AMF mitigates the drastic downregulation of photosynthesis-related genes and the reduction of photosynthetic CO2 assimilation rate caused by CMV infection. In parallel, an increase of salicylic acid level and a modulation of reactive oxygen species (ROS)-related genes, toward a limitation of ROS accumulation, was specifically observed in CMV-infected mycorrhizal plants. Overall, our data indicate that the AM symbiosis influences the development of CMV infection in tomato plants and exerts a priming effect able to enhance tolerance to viral infection.

ICTV Virus Taxonomy Profile: Alphaflexiviridae.

The family Alphaflexiviridae includes viruses with flexuous filamentous virions that are 470-800 nm in length and 12-13 nm in diameter. Alphaflexiviruses have a single-stranded, positive-sense RNA genome of 5.5-9 kb. They infect plants and plant-infecting fungi. They share a distinct lineage of alphavirus-like replication proteins that is unusual in lacking any recognized protease domain. With a single exception, cell-to-cell and long-distance movement is facilitated by triple gene block proteins in plant-infecting genera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Alphaflexiviridae, which is available at www.ictv.global/report/alphaflexiviridae.

Nondestructive Raman Spectroscopy as a Tool for Early Detection and Discrimination of the Infection of Tomato Plants by Two Economically Important Viruses.

Global population forecasts dictate a rapid adoption of multifaceted approaches to fulfill increasing food requirements, ameliorate food dietary value and security using sustainable and economically feasible agricultural processes. Plant pathogens induce up to 25% losses in vegetable crops and their early detection would contribute to limit their spread and economic impact. As an alternative to time-consuming, destructive, and expensive diagnostic procedures, such as immunological assays and nucleic acid-based techniques, Raman spectroscopy (RS) is a nondestructive rapid technique that generates a chemical fingerprinting of a sample, at low operating costs. Here, we assessed the suitability of RS combined to chemometric analysis to monitor the infection of an important vegetable crop plant, tomato, by two dangerous and peculiarly different viral pathogens, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato spotted wilt virus (TSWV). Experimentally inoculated plants were monitored over 28 days for symptom occurrence and subjected to RS analysis, alongside with measuring the virus amount by quantitative real-time PCR. RS allowed to discriminate mock inoculated (healthy) from virus-infected specimens, reaching an accuracy of >70% after only 14 days after inoculation for TYLCSV and >85% only after 8 days for TSWV, demonstrating its suitability for early detection of virus infection. Importantly, RS also highlighted spectral differences induced by the two viruses, providing specific information on the infecting agent.

Arbuscular Mycorrhizal Symbiosis: Plant Friend or Foe in the Fight Against Viruses?

Plant roots establish interactions with several beneficial soil microorganisms including arbuscular mycorrhizal fungi (AMF). In addition to promoting plant nutrition and growth, AMF colonization can prime systemic plant defense and enhance tolerance to a wide range of environmental stresses and below-ground pathogens. A protective effect of the AMF against above-ground pathogens has also been described in different plant species, but it seems to largely rely on the type of attacker. Viruses are obligate biotrophic pathogens able to infect a large number of plant species, causing massive losses in crop yield worldwide. Despite their economic importance, information on the effect of the AM symbiosis on viral infection is limited and not conclusive. However, several experimental evidences, obtained under controlled conditions, show that AMF colonization may enhance viral infection, affecting susceptibility, symptomatology and viral replication, possibly related to the improved nutritional status and to the delayed induction of pathogenesis-related proteins in the mycorrhizal plants. In this review, we give an overview of the impact of the AMF colonization on plant infection by pathogenic viruses and summarize the current knowledge of the underlying mechanisms. For the cases where AMF colonization increases the susceptibility of plants to viruses, the term "mycorrhiza-induced susceptibility" (MIS) is proposed.

Virus-mediated export of chromosomal DNA in plants.

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species.

Deep Sequencing Data and Infectivity Assays Indicate that Chickpea Chlorotic Dwarf Virus is the Etiological Agent of the "Hard Fruit Syndrome" of Watermelon.

Chickpea chlorotic dwarf virus (CpCDV), a polyphagous mastrevirus, family Geminiviridae, has been recently linked to the onset of the "hard fruit syndrome" of watermelon, first described in Tunisia, that makes fruits unmarketable due to the presence of white hard portions in the flesh, chlorotic mottling on the rind, and an unpleasant taste. To investigate the etiological agent of this disease, total RNA extracted from symptomatic watermelon fruits was subjected to small RNA sequencing through next generation sequencing (NGS) techniques. Data obtained showed the presence of CpCDV and two other viral species. However, following validation through polymerase chain reaction (PCR), CpCDV was the only viral species consistently detected in all samples. Watermelon seedlings were then challenged by an agroinfectious CpCDV clone; several plants proved to be CpCDV-infected, and were able to produce fruits. CpCDV infected and replicated in watermelon fruits and leaves, leading to abnormality in fruits and in seed production, similar to those described in field. These results indicate that CpCDV is the etiological agent of the "hard fruit syndrome" of watermelon.

ICTV Virus Taxonomy Profile: Ophioviridae.

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein.

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CPSP) with that from AltMV-Po (CP(Po)) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP(Po) [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CPSP but not CP(Po) interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CPSP than CP(Po) in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN.

From immunity to susceptibility: virus resistance induced in tomato by a silenced transgene is lost as TGS overcomes PTGS.

Tomato line 30.4 was obtained engineering the nucleocapsid (N) gene of tomato spotted wilt virus into plant genome, and immunity to tomato spotted wilt virus infection of its self-pollinated homozygous progeny was observed. Despite the presence of a high amount of transgenic transcripts, transgenic proteins have not been detected, suggesting a mechanism of resistance mediated by RNA. In the present study, we identify post-transcriptional gene silencing as the main mechanism of resistance, which is able to spread systemically through grafting, and show that the line 30.4 resistant plants produce both 24 and 21-22 nt N-gene specific siRNA classes. The transgenic locus in chromosome 4 shows complex multiple insertions of four T-DNA copies in various orientations, all with 3' end deletions in the terminator and part of the N gene. However, for three of them, polyadenylated transcripts are produced, due to flanking tomato genome sequences acting as alternative terminators. Interestingly, starting at the fifth generation after the transformation event, some individual plants show a tomato spotted wilt virus-susceptible phenotype. The change is associated with the disappearance of transgene-specific transcripts and siRNAs, and with hyper-methylation of the transgene, which proceeds gradually through the generations. Once it reaches a critical threshold, the shift from post-transcriptional gene silencing to transcriptional silencing of the transgene eliminates the previously well established virus resistance.

Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue.

The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportions. The CP ORF contains two 5'-proximal AUGs encoding Met 1 and Met 49, respectively promoting translation of the 33 and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast transit peptide, leading to protein cleavage and alternative derivation of the approximately 28 kDa CP. Mutational analysis of the two in-frame start codons and of the putative proteolytic-cleavage site showed that the N-terminal sequence is crucial for efficient cell-to-cell movement, functional systemic movement, homologous CP interactions and particle formation, but is not required for virus replication. Blocking production of the 28 kDa CP by internal initiation shows no major outcome, whereas additional mutation to prevent proteolytic cleavage at the chloroplast membrane has a dramatic effect on virus infection.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement.

Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.

Pathogenicity of Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of silencing suppression in a movement-competent TGB1.

Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 degrees C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 degrees C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the 'core' Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P(1110),R(1121),K(1255)]+TGB1(L(88))] always induced systemic necrosis at 15 degrees C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1(P(88)) significantly reducing silencing suppression. The difference in silencing suppression between TGB1(P(88)) and TGB1(L(88)) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1(P(88)) and TGB1(L(88)) resulted in interference in the suppression of silencing by TGB1(L(88)). Yeast two-hybrid analysis confirmed that TGB1(P(88)) and TGB1(L(88)) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.

Subcellular localization of the barley stripe mosaic virus triple gene block proteins.

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.

Molecular characterization of Lolium latent virus, proposed type member of a new genus in the family Flexiviridae.

Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrids (Lolium perenne x Lolium multiflorum). The genome of one USA isolate, LoLV-US1, has now been fully sequenced. The positive strand genomic RNA is 7674 nucleotides (nt) long and is organized in five open reading frames (ORFs) encoding the replication-associated protein, the movement-associated triple gene block proteins and the coat protein (CP). The genome organization is similar to that of viruses in the genera Potexvirus and Foveavirus; however, analysis of the complete LoLV genomic sequence, phylogenetic analyses of the deduced amino acid (aa) sequences of the polymerase and the CP, presence of a putative ORF 6, and the in vivo detection of two CPs in equimolar amounts, highlight features peculiar to LoLV. These characteristics indicate that LoLV forms a monotypic group separate from existing genera and unassigned species within the family Flexiviridae, for which we propose the genus name Lolavirus. One-step RT-PCR was developed for quick and reliable LoLV detection.

Field Evaluation of Tomato Hybrids Engineered with Tomato spotted wilt virus Sequences for Virus Resistance, Agronomic Performance, and Pollen-Mediated Transgene Flow.

ABSTRACT Tomato hybrids obtained from homozygous progeny of line 30-4, engineered for Tomato spotted wilt virus (TSWV) resistance, were tested under field conditions in two locations with their corresponding nontransgenic hybrids. No transgenic hybrid became infected, but 33 to 50% of plants of each nontransgenic hybrid became infected with a severe reduction of marketable fruit production. The transgenic hybrids conformed to the standard agronomic characteristics of the corresponding nontransgenic ones. Fruit were collected from the nontransgenic plots included in the experimental field and from border rows, and seed were used to estimate the flow of the transgene via pollen. No transgene flow was detected in the protected crops; however, in the open field experiment, 0.32% of tomato seedlings were found to contain the genetic modification. Immunity to TSWV infection in 30-4 hybrids was confirmed in laboratory conditions using mechanical inoculation and grafting. Thrips inoculation in leaf discs of line 30-4 demonstrated that TSWV replication was inhibited at the primary infection site but not in leaf discs of a commercial hybrid containing the naturally occurring resistance gene Sw-5. Due to the high economic value of tomato crops worldwide and the importance of TSWV, the engineered resistance described here is of practical value for breeding into cultivars of commercial interest, because it could be combined with naturally occurring resistance, thus greatly reducing the ability of the virus to develop resistance-breaking strains.