A single resistance factor to solve vineyard degeneration due to grapevine fanleaf virus.

Grapevine fanleaf disease, caused by grapevine fanleaf virus (GFLV), transmitted by the soil-borne nematode Xiphinema index, provokes severe symptoms and economic losses, threatening vineyards worldwide. As no effective solution exists so far to control grapevine fanleaf disease in an environmentally friendly way, we investigated the presence of resistance to GFLV in grapevine genetic resources. We discovered that the Riesling variety displays resistance to GFLV, although it is susceptible to X. index. This resistance is determined by a single recessive factor located on grapevine chromosome 1, which we have named rgflv1. The discovery of rgflv1 paves the way for the first effective and environmentally friendly solution to control grapevine fanleaf disease through the development of new GFLV-resistant grapevine rootstocks, which was hitherto an unthinkable prospect. Moreover, rgflv1 is putatively distinct from the virus susceptibility factors already described in plants.

Virus-Induced Gene Silencing in Sweet Basil (Ocimum basilicum).

Virus-induced gene silencing (VIGS) is a powerful reverse genetic tool for rapid functional analysis of plant genes. Over the last decade, VIGS has been widely used for conducting rapid gene knockdown experiment in plants and played a crucial role in advancing applied and basic research in plant science. VIGS was studied extensively in model plants Arabidopsis and tobacco. Moreover, several non-model plants such as Papaver (Hileman et al., Plant J 44:334-341, 2005), Aquilegia (Gould and Kramer, Plant Methods 3:6, 2007), Catharanthus (Liscombe and O'Connor, Phytochemistry 72:1969-1977, 2011), Withania (Singh et al., Plant Biol J 13:1287-1299, 2015), and Ocimum (Misra et al., New Phytol 214:706-720, 2017) were also successfully explored. We have recently developed a robust protocol for VIGS in sweet basil (Ocimum basilicum). Sweet basil, a popular medicinal/aromatic herb, is being studied for the diversity of specialized metabolites produced in it.

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein.

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. RESULTS: The epitope sequence was genetically inserted in the αB-αB" domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. CONCLUSIONS: The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

Expression and antiviral function of ARGONAUTE 2 in Nicotiana benthamiana plants infected with two isolates of tomato ringspot virus with varying degrees of virulence.

ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21-27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.

Nanobody-mediated resistance to Grapevine fanleaf virus in plants.

Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy-chain-only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode-transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell-to-cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.

The 50 distal amino acids of the 2AHP homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis.

Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2AHP , particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2AHP fused to an enhanced green fluorescent protein (EGFP) tag in N. occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N. occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression.

Detection of Nepovirus Vector and Nonvector Xiphinema Species in Grapevine.

Fanleaf degeneration is considered the most damaging viral disease of grapevine. The two major nepoviruses involved are Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) which are respectively and specifically transmitted by the dagger nematodes Xiphinema index and X. diversicaudatum. The methods described below are aimed at detecting four prevalent grapevine Xiphinema species: the vector species previously mentioned and two nonvector species X. vuittenezi and X. italiae.

Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.

Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.

A strain-specific segment of the RNA-dependent RNA polymerase of grapevine fanleaf virus determines symptoms in Nicotiana species.

Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1E(Pol)), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1E(Pol) was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1E(Pol) failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.

Antioxidative responses in Vitis vinifera infected by grapevine fanleaf virus.

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.

Tubule-guided cell-to-cell movement of a plant virus requires class XI myosin motors.

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells.

Comparisons of complete RNA-2 sequences, pathological and serological features among three Japanese isolates of Arabis mosaic virus.

Arabis mosaic virus lily and narcissus isolates (ArMV-Li and ArMV-Na) induced severe necrotic spots on Chenopodium quinoa, whereas ArMV butterbur isolate (ArMV-Bu) caused symptomless infection in the plant. The accumulation level of ArMV-Bu in upper non-inoculated leaves of C. quinoa was comparable to that of ArMV-Li or -Na. The agar gel double-diffusion test using an antiserum against ArMV-Li showed ArMV-Li was closely related to ArMV-Na, but not to ArMV-Bu. The RNAs-2 of ArMV-Li, -Na, and -Bu consist of 3707, 3709, and 3789 nucleotides, and they contain one open reading frame encoding a putative polyprotein of 1083, 1084, and 1122 amino acids, respectively. The overall identity of RNA-2 of ArMV-Li displayed more than 90% with ArMV-Na, but less than 70% with ArMV-Bu. A phylogenetic analysis of 2A sequences from ArMV isolates revealed ArMV-Bu was not categorized in any cluster. ArMV-Bu is a unique isolate from the point of view of pathological and serological features, and nucleotide sequence.

Effects of viral silencing suppressors on tobacco ringspot virus infection in two Nicotiana species.

This study investigated the effects of silencing suppressors derived from six different viruses (P1, P19, P25, HcPro, AC2 and 2b), expressed in transgenic Nicotiana tabacum and Nicotiana benthamiana plants, on the infection pattern of tobacco ringspot virus (TRSV) potato calico strain. In N. benthamiana, this virus produced an initial infection with severe systemic symptoms, but the infection was strongly reduced within a few weeks as the plant recovered from the infection. P25 and HcPro silencing suppressors effectively prevented recovery in this host, allowing continuous accumulation of the viral RNA as well as of the virus-specific small interfering RNAs, in the systemically infected leaves. In the P1-, P19-, AC2- or 2b-expressing transgenic N. benthamiana, the recovery was not complete. Susceptibility of N. tabacum to this virus was temperature sensitive. At lower temperatures, up to 25 degrees C, the plants became systemically infected, but at higher temperatures, the infections were limited to the inoculated leaves. In these preventative conditions, all silencing suppressor transgenes (except P25, which was expressed at very low levels) allowed the establishment of systemic infections. Very strong and consistent systemic infections were observed in HcPro- and AC2-expressing plants.

Virus diseases in the tobacco fields of Guilan and Western Azerbaijan provinces of Iran.

Tobacco (Nicotiana tabacum L.) is one of the important industrial plants in Iran. Viruses as an important group of plant pathogens cause many losses on the quality and quantity of tobacco crop. There was few information on the types of plant viruses infecting the tobacco fields of Guilan and almost no information for Western Azerbaijan province. During 2005-2007, leaf samples were taken from symptomatic plants in the growing areas of these two provinces. The observed symptoms on plants in the fields varied from mild mosaics to severe necrosis. The regions of sampling were including Rasht, Bazar-jomeh, Soumae-Sara, Talesh and Astara in Guilan and Ourmia, Sardasht and Ghara-Ziaeddin in Western Azerbaijan. The tobacco types and varieties from which the samples were taken included air-cured burley variety Burley 21 and to a lesser extent, oriental tobacco variety Basma Serres in W. Azerbaijan and flue-cured varieties Coker 347 and Virginia El in Guilan province. Samples were tested by DAS-ELISA method (Clark and Adams, 1977) using the polyclonal antibodies for a set of tobacco viruses. Some samples with positive reactions in DAS-ELISA were inoculated to indicator test plants such as Chenopodium amaranticolor, Datura metel, D. stramonium, Physalis floridana, Nicotiana rustica, N. glutinosa, and tobacco (varieties White burley and Samsun). The results of greenhouse experiments were consistent with serological tests. The following viruses which are listed in order of their overall abundance within the tested samples were detected: Tobacco streak virus (TSV), Tomato spotted wilt virus (TSWV), Tobacco etch virus (TEV), Tobacco ringspot virus (TRSV), Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In all samples more than one virus infection was detected. The most severe mosaic type symptoms including the deformation and blistering on leaves were mainly seen in the infections by CMV and TMV. The most severe necrotic type symptoms including necrosis of midribs or veins and in some cases stem necrosis were generally associated with the infections by PVY and TSWV. Except TMV infection which was not detected in the Burley 21 variety in W. Azerbaijan, the above mentioned viruses were present in all sampling regions. The lack of TMV infection on Burley 21 is due to the presence of N gene, conferring resistance in this variety.

Sequence analysis of grapevine isolates of Raspberry ringspot nepovirus.

The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.

Evaluating the efficiency of a TPIA method for the detection of two tobacco viruses.

Field experiments are usually necessary for analyzing the efficiency of control methods, the resistance of varieties and many other virological studies. Such experiments generally include a large number of samples to be tested serologically. DAS-ELISA is a very accurate technique that has been widely used for identifying viral infections. For large numbers of plant samples, it takes a long time for the sample preparation, plate washing and other procedures. In this study, the efficiency of a simple and laborsaving TPIA (Tissue Print Immunoassay) method was evaluated for the identification of two important aphid-transmitted viruses (CMV and PVY) of tobacco fields in comparison with DAS-ELISA as the standard method. The leaf samples were collected from the fields of three commercial tobacco types (flue-cured, burley and oriental). Each sample was divided to two parts and each part was examined by one of the methods. DAS-ELISA was done based on the method described by Clark and Adams (1977). In TPIA, small parts of the leaf samples were rolled and then cut by a sterile sharp blade. The cut surface was gently printed on 1 cm2 blocks drawn on a nitrocellulose paper. Air dried paper was located first in 1% BSA for blocking the empty sites on paper, then in the buffer containing AP-conjugated polyclonal antibody for 3 h and finally in NBT-BCIP solution for color development. Between these stages, the paper was washed thoroughly (three times) by shaking in fresh washing buffer. The results of each sample were recorded and compared with those of DAS-ELISA. By considering DAS-ELISA as the reference method, the sensitivity of TPIA for the detection of PVY and CMV was 96.1% and 92.7%, respectively. The positive results by TPIA which were not detected positive by DAS-ELISA were regarded as false positive. These were 8 (out of 316 tested samples) for CMV and 6 (out of 204 samples) for PVY. Although the results of TPIA were not completely consistent with DAS-ELISA, it seems that this method can be used for some general studies. The most important advantages of this method were that it didn't need sample extraction and was done using only one antibody which was the conjugated antibody of each virus. This method gives more rapid results (within a day) in comparison with DAS-ELISA that needs more time.

Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix.

The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.

Comparative development and impact of transgenic papayas in Hawaii, Jamaica, and Venezuela.

We present data concerning the creation of transgenic papayas resistant to Papaya ringspot virus (PRSV) and their adoption by three different countries: the United States (e.g., Hawaii), Jamaica, and Venezuela. Although the three sets of transgenic papayas showed effective resistance to PRSV, the adoption rate in each country has varied from full utilization in Hawaii to aggressive testing but delay in deregulating of the product in Jamaica to rejection at an early stage in Venezuela. Factors that contributed to the rapid adoption in Hawaii include a timely development of the transgenic product, PRSV causing severe damage to the papaya industry, close collaboration between researchers and the industry, and the existence of procedures for deregulating a transgenic product. In Jamaica, the technology for developing the initial field-testing of the product progressed rather rapidly, but the process of deregulation has been slowed down owing to the lack of sustained governmental efforts to complete the regulatory procedures for transgenic crops. In Venezuela, the technology to develop and greenhouse test the transgenic papaya has moved abreast with the Jamaica project, but the field testing of the transgenic papaya within the country was stopped very early on by actions by people opposed to transgenic products. The three cases are discussed in an effort to provide information on factors, other than technology, that can influence the adoption of a transgenic product.

The specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index is solely determined by the viral coat protein.

The viral determinants involved in the specific transmission of Grapevine fanleaf virus (GFLV) by its nematode vector Xiphinema index are located within the 513 C-terminal residues of the RNA2-encoded polyprotein, that is, the 9 C-terminal amino acids of the movement protein (2BMP) and contiguous 504 amino acids of the coat protein (2CCP) [Virology 291 (2001) 161]. To further delineate the viral determinants responsible for the specific spread, the four amino acids that are different within the 9 C-terminal 2BMP residues between GFLV and Arabis mosaic virus (ArMV), another nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index, were subjected to mutational analysis. Of the recombinant viruses derived from transcripts of GFLV RNA1 and RNA2 mutants that systemically infected herbaceous host plants, all with the 2CCP of GFLV were transmitted by X. index unlike none with the 2CCP of ArMV, regardless of the mutations within the 2BMP C-terminus. These results demonstrate that the coat protein is the sole viral determinant for the specific spread of GFLV by X. index.