Infected Transgenic Plants Research Articles

Facilitative and synergistic interactions between fungal and plant viruses

Plants and fungi are closely associated through parasitic or symbiotic relationships in which bidirectional exchanges of cellular contents occur. Recently, a plant virus was shown to be transmitted from a plant to a fungus, but it is unknown whether fungal viruses can also cross host barriers and spread to plants. In this study, we investigated the infectivity of Cryphonectria hypovirus 1 (CHV1, family Hypoviridae), a capsidless, positive-sense (+), single-stranded RNA (ssRNA) fungal virus in a model plant, Nicotiana tabacum CHV1 replicated in mechanically inoculated leaves but did not spread systemically, but coinoculation with an unrelated plant (+)ssRNA virus, tobacco mosaic virus (TMV, family Virgaviridae), or other plant RNA viruses, enabled CHV1 to systemically infect the plant. Likewise, CHV1 systemically infected transgenic plants expressing the TMV movement protein, and coinfection with TMV further enhanced CHV1 accumulation in these plants. Conversely, CHV1 infection increased TMV accumulation when TMV was introduced into a plant pathogenic fungus, Fusarium graminearum In the in planta F. graminearum inoculation experiment, we demonstrated that TMV infection of either the plant or the fungus enabled the horizontal transfer of CHV1 from the fungus to the plant, whereas CHV1 infection enhanced fungal acquisition of TMV. Our results demonstrate two-way facilitative interactions between the plant and fungal viruses that promote cross-kingdom virus infections and suggest the presence of plant-fungal-mediated routes for dissemination of fungal and plant viruses in nature.

Tomato Yellow Leaf Curl China Virus Impairs Photosynthesis in the Infected Nicotiana benthamiana with βC1 as an Aggravating Factor.

Tomato yellow leaf curl China virus is a species of the widespread geminiviruses. The infection of Nicotiana benthamiana by Tomato yellow leaf curl China virus (TYLCCNV) causes a reduction in photosynthetic activity, which is part of the viral symptoms. βC1 is a viral factor encoded by the betasatellite DNA (DNAβ) accompanying TYLCCNV. It is a major viral pathogenicity factor of TYLCCNV. To elucidate the effect of βC1 on plants’ photosynthesis, we measured the relative chlorophyll (Chl) content and Chl fluorescence in TYLCCNV-infected and βC1 transgenic N. benthamiana plants. The results showed that Chl content is reduced in TYLCCNV A–infected, TYLCCNV A plus DNAβ (TYLCCNV A + β)–infected and βC1 transgenic plants. Further, changes in Chl fluorescence parameters, such as electron transport rate, Fv/Fm, NPQ, and qP, revealed that photosynthetic efficiency is compromised in the aforementioned N. benthamiana plants. The presense of βC1 aggravated the decrease of Chl content and photosynthetic efficiency during viral infection. Additionally, the real-time quantitative PCR analysis of oxygen evolving complex genes in photosystem II, such as PsbO, PsbP, PsbQ, and PsbR, showed a significant reduction of the relative expression of these genes at the late stage of TYLCCNV A + β infection and at the vegetative stage of βC1 transgenic N. benthamiana plants. In summary, this study revealed the pathogenicity of TYLCCNV in photosynthesis and disclosed the effect of βC1 in exacerbating the damage in photosynthesis efficiency by TYLCCNV infection.

Fructan determination in transgenic nicotiana tabacum l. Plants harbouring human inf α2b gene infected by tobacco mosaic virus

Fructan is one of the main storage compounds in higher plants, their accumulation and synthesis is associated with the plant's adaptation. They take part in cryoprotection, osmoregulation and water balance and included in plant protection from pathogens (biotic stress). Fructan, anthocyanins, proline are involved in reduction of negative effects of biotic factors on plants.Utilizing of genetic engineering methods makes it possible to create stress resistant plants. However, the impact of the transformation still remains unclear, because transformation can be regarded as biotic stress factor for plants. Investigation of the biotic stress effects on metabolic pathways in transformed plants is still actual.The aim of our work was to identify changes in fructan accumulation caused by the presence of tobacco mosaic virus (TMV) in transgenic Nicotiana tabacum plants with human ifn - α2b gene. Control wild type plants contained 6.8 + 0.08 mg/g of fructan. In the transgenic line №1, the level of fructan accumulation was 4.68 ± 1.02 mg/g. In the transformed line №3, its content increased to 30+1.8 mg/g. The fructan content in control plants increased from 6.8 + 0.08 to 8.2 + 1 mg/g, after viral infection. For transgenic line №1, the content of fructan increased to 25 + 1 mg/g. The growth of fructan concentration was not observed in the line №3. The increase in fructan content in infected transgenic plants can be regarded as adaptive response to viral infection as biotic stress.

CRISPR/Cas9-mediated resistance to cauliflower mosaic virus.

Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes (R gene) into elite crop cultivars has been the standard and most cost‐effective practice. However, R gene‐mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad‐spectrum antiviral strategies. The CRISPR‐Cas9 (clustered regularly interspaced palindromic repeat, CRISPR‐associated) editing system has been employed to confer resistance to human viruses and several plant single‐stranded DNA geminiviruses, pointing out the possible application of the CRISPR‐Cas9 system for virus control. Here, we demonstrate that strong viral resistance to cauliflower mosaic virus (CaMV), a pararetrovirus with a double‐stranded DNA genome, can be achieved through Cas9‐mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNAs (siRNA) are produced and mostly map to the 3′ end of single‐guide RNAs (sgRNA), although very low levels of siRNAs map to the spacer region as well. However, these siRNAs are not responsible for the inhibited CaMV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9‐induced DNA breaks at the sgRNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, nonfunctional coat proteins. We also recovered wild‐type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild‐type coat proteins. Our data demonstrate that the CRISPR‐Cas9 system can be used for virus control against plant pararetroviruses with further modifications.

RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite.

BackgroundTomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels.ResultsA hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants.ConclusionsThese results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0263-y) contains supplementary material, which is available to authorized users.

Disruption of Vector Transmission by a Plant-Expressed Viral Glycoprotein

Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus-vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.

Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

Use of Tomato leaf curl virus (TYLCV) truncated Rep gene sequence to engineer TYLCV resistance in tomato plants

Tomato yellow leaf curl disease causes severe losses in tomato production throughout Mediterranean countries including Tunisia. In order to generate engineered resistance to this disease, an intron-hairpin RNA construct harboring a Tomato yellow leaf curl Sardinia virus (TYLCSV) truncated replication-associated protein (Rep) gene was used to transform genotype of tomato plants. Prepared transgenic plants were agro-inoculated with Tunisian infectious strain of TYLCSV and screened for the resistance to infection. The infected transgenic plants were divided into 3 different groups according to their specific symptoms. Only one of them contained transgenic plants fully resistant to the tomato yellow leaf curl disease.

No recombination detected in artificial potyvirus mixed infections and between potyvirus derived transgenes and heterologous challenging potyviruses

Risk-assessment studies of virus-resistant transgenic plants (VRTPs) focussing on recombination of a plant virus with a transgenic sequence of a different virus should include a comparison of recombination frequencies between viruses in double-infected non-transgenic plants with those observed in singly infected transgenic plants to estimate recombination incidence in VRTPs. In this study, the occurrence of recombination events was investigated in non-transgenic plants double-infected with two different potyviruses, as well as in potyviral genomes in singly infected transgenic plants expressing potyvirus sequences. Different potyviruses, namely Potato virus A (PVA), Tobacco vein mottling virus (TVMV), two strains of Potato virus Y (PVY-O, PVY-H) and two strains of Plum pox virus (PPV-NAT, PPV-SK68), were used in three combinations for double infection of a common host. Furthermore, transgenic plants expressing either potyviral coat protein (CP), helicase (CI) or polymerase (NIb) coding sequences (PPV-NAT-CP, PVY-CI, PVY-NIb) were singly-infected with a heterologous potyvirus, which was not targeted by the respective transgenic resistance. To identify recombinant potyviral sequences, a sensitive RT-PCR was developed to detect up to one recombinant molecule out of 10(6) parental molecules. In 304 mixed infected non-transgenic plants, 92 mixed and 164 single infected transgenic plants screened for recombinant sequences no recombinant potyviral sequence was found. These results indicate that recombination events between different potyviruses in mixed infections and between a potyvirus infecting a potyvirus-resistant transgenic plant are likely to be very infrequent.

Expression of recombinant trichosanthin, a ribosome-inactivating protein, in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.

A New Genotype within Cucumber Mosaic Virus Subgroup I: Molecular Characterization of the Polymerase Encoding RNA 2 and its Capability to Overcome Replicase‐mediated Resistance

A number of cucumber mosaic virus subgroup I strains originating from Asia were collected and tested for their ability to overcome replicase‐mediated resistance. Strains were inoculated to replicase‐mediated resistant plants transformed with a defective RNA 2 construct from Fny‐CMV. Most of the strains were able to overcome resistance to some extent. The resistance‐breaking capability of MB‐CMV, PRC‐CMV, C6‐CMV and 30RS‐CMV was higher in comparison with the other strains tested, based on the number of infected transgenic plants. The resistance‐breaking capability of MB‐CMV and PRC‐CMV was mapped to RNA 2 of these strains. The nucleotide sequences of MB‐CMV RNA 2 was determined and compared with other CMV subgroup I strains. Data obtained showed that MB‐CMV differs from all other strains by more than 7% in nucleotide sequence identity. This indicates a high genetic variability within CMV subgroup I strains from Asia based on RNA 2.

Transformation of tobacco and potato with cDNA encoding the full-length genome of Potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication

A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.

Unusual Mitochondrial Aggregation with Virus in Infected Transgenic Plants

Abstract Viruses grouped in the family Potyviridaeare long flexious rod-shaped viruses and cause the most damage of all plant vims groups. They possess a single stranded positive sense RNA genome that encodes a single polyprotein product which yields eight or more proteins by secondary processing. Considerable efforts have been made to transform plants with DNA or RNA sequences of these proteins in hopes of obtaining pathogen-derived resistant plants. Hull has suggested that a close assessment of the risks involved in field release of transformed plants would be desirable. However, to our knowledge, there have been no published reports regarding properties of viruses that do infect some transformed plants. We describe here an ultrastructural appearance of transgenic plant cells infected by potato virus Y (PVY) (ATCC PV No. 50; PVY-50) and unusual mitochondrial aggregation with the virus.

Efficacy of Endogenous Satellite Expression to Confer Resistance to CMV in Satellite Transgenic Tomato under Field Conditions

Viral satellite RNA associated with cucumber mosaic virus (CMV) is know to modulate CMV symptomology. Virulent CMV associated RNA 5 (CARNA 5) satellites may intensify crop disease. Naturally occurring variants of these satellites, however, attenuate CMV symptoms. Satellite transgenic tomato plants expressing the S-CARNA 5 or 1-CARNA 5 ameliorating forms of the satellite were evaluated under simulated CMV epidemic conditions in USDA–APHIS approved field trials. Trials conducted at Beltsville, Md., in 1994 and 1995 demonstrated that CMV can be effectively controlled under field conditions in satellite transgenic plants. Yields of transgenic lines infected with CMV were 50%–65% greater than that of non-transgenic infected controls. Yields of noninfected transgenic lines ranged from 5% greater than, to 33% less than, noninfected nontransgenic controls. Expression of CARNA 5 in inoculated transgenic plants greatly reduced CMV foliar symptoms and virus titers when compared to inoculated control plants. Levels of CARNA 5 were detected at varying levels in infected transgenic plants throughout the growing season. Virus or satellite was not detected in samples collected from tomato border plants and weeds growing inside and outside a nonhost crop border surrounding the test plot. Field tests conducted in 1996 will evaluate transgenic tomato plants with a double construct coding for the CMV coat protein gene and 1-CARNA 5 satellite.

Accumulation kinetics of CMV RNA 3-encoded proteins and subcellular localization of the 3a protein in infected and transgenic tobacco plants.

The complete nucleotide sequence of RNA 3 of a Spanish isolate of cucumber mosaic virus (CMV-24) has been determined. The encoded putative cell-to-cell movement protein (3a protein) and the coat protein are 279 and 218 amino acids long, respectively. The 3a protein was expressed in Escherichia coli using the vector pT7-7 and was used to raise an immunoserum. We have followed the time course of accumulation of the 3a protein, in parallel to that of the coat protein, and its subcellular localization as a function of time after CMV-24 infection on tobacco plants. The maximum accumulation level of the 3a protein was reached at early stages of infection, being detected in the cytosolic and the cell wall fractions. At later stages of infection, a decline in accumulation levels of the 3a protein was observed, and the protein was essentially associated with the cell wall fractions. These data were corroborated by immunocytochemistry performed in both infected and 3a-expressing transgenic tobacco plants.

Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid‐transmissible strain of plum pox potyvirus (PPV‐D) were infected with an aphid non‐transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV‐NAT). Non‐viruliferous Myzus persicae could acquire and transmit ZYMV‐NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV‐NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV‐NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.

Cytology of infection of 35S‐bean chitinase transgenic canola plants by Rhizoctonia solani: cytochemical aspects of chitin breakdown in vivo

Transgenic canola plants containing high, constitutive levels of bean endochitinase have been shown to be more resistant to infection by the soil‐borne pathogen, Rhizoctonia solani, than are wild‐type plants that lack the chimeric chitinase gene. To determine whether the resistance of the 35S‐chitinase plants to Rhizoctonia infection results from an antimicrobial activity of the bean chitinase in planta, an ultrastructural and cyto‐chemical study was performed on infected control and transgenic canola plants. Analysis of root tissues of infected wild‐type canola plants revealed that R. solani was capable of extensive tissue colonization including the xylem vessels. Pathogen ingress towards the vascular system was associated with marked host cell wall alterations such as disruption of middle lamella matrices that occurred in advance of fungal penetration. Fungal hyphae colonizing these tissues appeared metabolically active as judged by their typical morphological features and their extensive multiplication. In infected transgenic plants, however, the pattern of fungal colonization was different to that observed in wild‐type plants. Penetration of the host cuticle and epidermis was frequently observed, but fungal colonization was usually restricted to the cortex although, in a few cases, some fungal cells could be seen in xylem vessels. In all samples examined, severe hyphal alterations ranging from increased vacuolization to cell lysis were seen. Hyphae occasionally seen in xylem vessels were markedly damaged and often reduced to convoluted wall fragments. Cytochemical labeling of chitin using the WGA/ovomucoid‐gold complex showed that hyphal alterations correlated with extensive chitin degradation. Thus, reduction in fungal biomass, increase in hyphal alterations leading to fungal lysis and chitin breakdown appear to be typical features observed in transgenic canola plants. Because these features were not seen in infected wild‐type plants, it is likely that constitutive expression of the bean endochitinase gene is, at least in part, responsible for the enhanced protection against fungal attack observed in these plants. It is not known, however, if other components of the host defense response contribute to the resistance phenotype.

Biologically active cymbidium ringspot virus satellite RNA in transgenic plants suppresses accumulation of DI RNA

A full-length DNA copy of cymbidium ringspot virus (CyRSV) satellite RNA was cloned downstream of the bacteriophage T7 RNA polymerase promoter. In vitro transcripts were biologically active in plants when coinoculated with the helper virus or its RNA. Although the transcripts contained 7 or 29 extra nucleotides at the 3′ end, the proper 3′ terminus was restored in the satRNA progeny. Full-length cDNA clones of CyRSV satRNA under the control of the cauliflower mosaic virus 35S promoter and terminator were used to transform Nicotiana benthamiana plants. Integration of CyRSV satRNA sequence in the plant genome was tested by PCR amplification of DNA extracts from transformed plants and by detection of satRNA-related transcripts in total RNA extracts. Inoculation of transgenic plants with the helper virus induced replication of satRNA of the same size as the native molecule. Sequence analysis of the satRNA progeny showed that it was identical to natural CyRSV satRNA. Infected transgenic plants were not protected from apical necrosis and death by the presence of satRNA sequences. Rather, replication of satRNA was found to suppress accumulation of defective interfering RNA, which acts in the absence of satRNA as an attenuator of virus replication and disease.