Infected Tobacco Plants Research Articles

CHARACTERIZATION OF A PETUNIA STRAIN OF TURNIP VEIN-CLEARING VIRUS

SUMMARY Virus-like symptoms were observed in February 2005 on Double Wave TM petunias (Petunia x hybrida Hort. Vilm.-Andr.) grown in a greenhouse in northeastern Mississippi. Electron microscope observations of leaf dips from symptomatic plants showed the presence of rodshaped virus-like particles. Infected petunia samples reacted weakly with an immunostrip test to Tobacco mosaic virus (TMV), but not in ELISA using a polyclonal antiTMV-c commercial kit. The dsRNA patterns from four infected petunias were identical and resembled those of tobamoviruses although they differed slightly in size from the replicative forms of TMV extracted from infected tobacco plants. Purified dsRNAs were used for random-primer cloning of the viral genome. A partial sequence of the viral 3’ end showed that the virus is a strain of Turnip vein-clearing virus (TVCV, reported also as Tobacco mosaic virus-crucifer strain) sharing 96% common nucleotides and 98% identical amino acids. To the best of our knowledge, TVCV has not been reported previously either from petunias or from Mississippi in general.

Occurrence of Potato Tuber Necrotic Isolates of Potato virus Y in a Commercial Tobacco Field in Southern Ontario, Canada.

Most strains of Potato virus Y (PVY) can infect tobacco plants (Nicotiana tabacum) and cause vein clearing followed by leaf mottling, except the PVYN strain, which induces severe vein necrosis. Some isolates within the PVYN strain also cause potato necrotic tuber ringspot disease, but these have not been reported from Canadian tobacco fields. PVYNTN isolates include European (EU) and North American (NA) types that are serologically identical to PVYN, but can be distinguished by nucleic acid-based assays and potato bioassay (1,2). Some PVY isolates, PVYN-Wi or PVYN:O, resulting from a recombination between RNA molecules of PVYN and the common strain, PVYO, are identified as PVYO in serological assays, but induce necrosis in tobacco (2). In August of 2007, two samples of tobacco (N. tabacum, unknown cultivar) leaves showing necrotic symptoms resembling those induced by PVYN, PVYNTN, or PVYN-Wi were collected from a tobacco field in southern Ontario, Canada and submitted to the Charlottetown Laboratory, Canadian Food Inspection Agency, Charlottetown, PE. Virus in both samples (PVY-204 and PVY-205) reacted with PVYN-specific antibodies 1F5 and 4E7 (3) and induced vein necrosis in tobacco (N. tabacum cv. Samsun). A multiplex reverse transcription (RT)-PCR assay (1) for the simultaneous detection and differentiation of various PVY strains amplified two fragments (181 and 452 bp) associated with EU-PVYNTN isolates. Restriction fragment length polymorphism (RFLP) analysis targeting the P1 and NIb gene (3) also indicated that PVY-204 and PVY-205 were EU-PVYNTN isolates. Known isolates of PVYO, PVYN, and NA-PVYNTN were used in all evaluations as references (3). Furthermore, the nucleotide sequences of the P1 and NIb genes of PVY-204 and PVY-205 determined by automated cycle sequencing (3) and subjected to phylogenetic analysis indicated that the nucleotide and deduced amino acid sequences of both isolates were 96 and 95% identical, respectively, to NA-PVYNTN isolates reported from Canada, but 99% identical (both nucleotide and amino acid) to EU-PVYNTN isolates from Europe and Mexico (3). Potato (cv. Yukon Gold) plants mechanically inoculated with leaf sap from tobacco (N. tabacum cv. Samsun) infected with PVY-204 and PVY-205 developed various leaf symptoms including severe local and systemic necrotic lesions, leaf wilting, and leaf death in 3 to 5 weeks postinoculation under greenhouse conditions. The infected plants recovered in 5 to 6 weeks. Potato (cv. Yukon Gold) plants inoculated with leaf sap from tobacco (N. tabacum cv. Samsun) infected with a PVYNTN isolate (HX8) (3) and healthy tobacco leaf sap were used as positive and negative controls. The number and yield of the tubers harvested from infected plants were significantly reduced (50%), and PVY-204 and PVY-205 induced typical potato tuber necrotic ringspot disease in 52.6% of the progeny tubers with an average disease index of 0.364 (C. Kerlan and K. Charlet-Ramage, EAPR Virology Meeting Proceedings, 1998). PVYNTN was detected by RT-PCR and RFLP in all necrotic tubers and 66.7% of the asymptomatic tubers. Some tubers (15.8%) harvested from the infected plants were negative in RT-PCR targeting either P1 protein gene or NIb gene and showed neither external nor internal necrotic symptom. To my knowledge, this is the first evidence of the occurrence of PVYNTN isolates in field-grown tobacco plants in Canada.

NTH201, a Novel Class II KNOTTED1-Like Protein, Facilitates the Cell-to-Cell Movement of Tobacco mosaic virus in Tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.

Antiviral RNA Silencing Is Restricted to the Marginal Region of the Dark Green Tissue in the Mosaic Leaves of Tomato Mosaic Virus -Infected Tobacco Plants

Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue ( approximately 0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.

Pathogenicity of a naturally occurring recombinant DNA satellite associated with tomato yellow leaf curl China virus

Recombinant DNA beta molecules (RecDNA-Abeta) comprising parts of DNA A and DNA beta associated with tomato yellow leaf curl China virus (TYLCCNV) have been identified in naturally infected tobacco plants. Several examples of the recombinant DNA have been cloned and characterized by sequence analysis. All are approximately half the size of TYLCCNV genomic DNA, and all contain the betaC1 gene and the A-rich region from TYLCCNV DNA beta as well as intergenic region sequences and the 5' terminus of the AC1 gene from TYLCCNV DNA A. RecDNA-Abeta was detected by PCR in five of 25 TYLCCNV isolates. Co-inoculation of TYLCCNV DNA A and RecDNA-Abeta induced symptoms indistinguishable from those induced by TYLCCNV DNA A and DNA beta in Nicotiana benthamiana, Nicotiana glutinosa, Solanum lycopersicum and Petunia hybrida plants, and Southern blot hybridization results showed that RecDNA-Abeta could replicate stably in N. benthamiana plants.

Plant Protector Identified

Plants that survive an initial pathogen attack often develop enhanced resistance to subsequent infections. For example, previous infection of tobacco plants by tobacco mosaic virus (TMV) enhances resistance elsewhere in the plant to subsequent challenge by TMV or other pathogens, which is termed systemic acquired resistance (SAR). The development of SAR requires the movement of a signal made in the primary infected tissue through the phloem to the distal systemic tissue. Park et al . show that the mobile signal for SAR is a biologically inactive form of salicylic acid, methyl salicylate (MeSA), a key hormone for activating host defenses to many plant pathogens. S.-W. Park, E. Kaimoyo, D. Kumar, S. Mosher, D. F. Klessig, Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 318 , 113-116 (2007). [Abstract] [Full Text]

Transgenerational changes in the genome stability and methylation in pathogen-infected plants

Previously, we reported the generation of a virus-induced systemic signal that increased the somatic and meiotic recombination rates in tobacco mosaic virus (TMV)-infected tobacco plants. Here, we analyzed the progeny of plants that received the signal and found that these plants also have a higher frequency of rearrangements in the loci carrying the homology to LRR region of the gene of resistance to TMV (N-gene). Analysis of the stability of repetitive elements from Nicotiana tabacum loci and 5.8S ribosomal RNA loci did not show any changes. Further analysis of the changes in the progeny of infected plants revealed that they had substantially hypermethylated genomes. At the same time, loci-specific methylation analysis showed: (1) profound hypomethylation in several LRR-containing loci; (2) substantial hypermethylation of actin loci and (3) no change in methylation in the loci of repetitive elements from N. tabacum or 5.8S ribosomal RNA. Global genome hypermethylation of the progeny is believed to be part of a general protection mechanism against stress, whereas locus-specific hypomethylation is associated with a higher frequency of rearrangements. Increased recombination events combined with the specific methylation pattern induced by pathogen attack could be a sign of an adaptive response by plants.

Morphology and stability changes of recombinant TMV particles caused by a cysteine residue in the foreign peptide fused to the coat protein

In the studies of expressing various foreign peptides using a TMV-based vector, a portion of morphologically altered progeny viral particles from some recombinant TMV constructs were detected by transmission electron microscopy in the first systematically infected upper leaves, but not in the fully expanded inoculated leaves, from infected tobacco plants. Furthermore, in vitro stability of such recombinant TMV constructs were lower than those of the wild type and other recombinant TMV constructs able to form regular rod-shape virions, hence causing the lower yields of recombinant viral particles purified from the infected tobacco plants. Our studies revealed that the presence of a cysteine residue in the foreign peptides, regardless of its position and the peptide sequence, was directly related to changes in the morphology and stability of these TMV recombinants.

Transmission of Potato virus Y in Tobacco Plants by Myzus persicae nicotianae and M. persicae s.str.

The transmission efficiency of Potato virus Y N (PVYN) from and to tobacco plants by Myzus persicae nicotianae, a tobacco specialist, and M. persicae s.str. (Hemiptera: Aphididae), a generalist, was evaluated. In direct transmission tests, 1,004 spring migrants collected as nymphs from peach and 1,434 wingless females from 12 clonal lineages of both taxa were examined. Both the winged and wingless females were starved before being transferred individually for a 3-min acquisition period on infected plants and then moved to healthy plants for a 10-min inoculation access period. Little variation in transmission efficiency was observed. Although not statistically significant, M. persicae nicotianae was slightly more effective. This may suggest similar genetic properties (e.g., virion receptors in mouthparts) between the taxa in relation to virus transmission. M. persicae s.str. transmitted the virus less efficiently when a shorter inoculation period was given. This may indicate differences in probing behavior during the early phase of host selection. Nevertheless, behavioral differences related to host selection have been found using arena tests with infected and healthy tobacco plants where 180 winged females from each taxon were tested. M. persicae s.str. had significantly higher propensity to transmit PVYN than M. persicae nicotianae (31.9 versus 15.3%).

Effective Thermophilic Composting of Crop Residues for Inactivation of Tobacco Mosaic Virus

An effective thermophilic composting bioreactor, in which a homogenous distribution of temperature was maintained at 63-65°C by the addition of a bioavailable carbon and low mixing, was developed. The bioreactor operated on a mixture of tomato plant residues-wood shavings-municipal solid waste compost infected with tobacco mosaic virus (TMV). The initial C: N ratio and moisture content of the compost mixture were adjusted to 30:1 and 60%, respectively. The composting process was successful in destroying the tobacco mosaic virus. The results showed that the ability of the untreated virus (inoculum) to infect tobacco plants (150 LL L-1) was much higher than its ability to infect tomato plants (22 LL L-1). The TMV completely lost its ability to infect the leaves of susceptible hosts (tobacco and tomato plants) after 96 hrs of controlled thermophilic (63-65 oC) composting (or 126 h from the start of the composting process). Semilog plots of the ratio of the infection ability of the surviving virus to that of the initial inoculum (as measured by the number of local lesions) were developed. The decimal reduction time (the time necessary to reduce the infection ability of TMV by 1-log or 90%) was found to be 62.4 and 109.7 hrs for tobacco and tomato plants, respectively. The relatively short time required for complete inactivation of TMV in this study was achieved as a result of the extension of the thermophilic stage and maintaining a constant high temperature with a uniform temperature distribution by the continuous addition of the proper amount of bioavailable carbon (used cooking oil) and low mixing.

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.

First Report of Bean yellow mosaic virus (Pea Mosaic Strain) in Verbena × hybrida.

Verbena × hybrida is an ornamental annual used in rock gardens as an edging plant and hanging baskets. It comes in a variety of colors and grows approximately 1.5 to 2.5 cm (6 to 10 inches) high. In the spring of 2002, verbena cv. Lavender Shades plants from California showing leaf mosaic symptoms tested positive for potyvirus using an antigen-coated plate enzyme-linked immunosorbent assay with our genus Potyvirus broad spectrum reacting PTY-1 monoclonal as the detecting antibody (3). The virus was transmitted mechanically to Nicotiana benthamiana by sap inoculation from infected verbena plants. Infected tobacco showed systemic mild mosaic symptoms. Total RNA extractions from infected verbena and tobacco leaves were used in reverse transcription-polymerase chain reaction (RT-PCR) assays with generic potyvirus-specific primers that amplify highly conserved 700-bp or 1,600-bp fragments from the 3' terminus of most potyviruses. This region includes the 3' noncoding region (3'NCR) and the potyviral coat protein (CP). The PCR-amplified fragments were cloned by using standard TA cloning procedures and sequenced using dye-terminator chemistry. The cloned nucleotide and putative coat protein amino acid sequences from the infected verbena and tobacco plants were compared with the corresponding regions of other potyviruses. Amino acid comparison of the CP region of the verbena po-tyvirus showed 95 to 96% identity to four pea mosaic strains (PMV) of Bean yellow mosaic virus (BYMV), 85 to 89% identity to 20 other strains of BYMV, 74 to 76% identity with six strains of Clover yellow vein virus (CYVV), and only 50 to 64% identity with 28 other potyviruses. Pairwise comparisons among and between the CP sequences of PMV, BYMV, CYVV, and other potyviruses revealed identities of 92 to 99% for BYMV∷ BYMV, PMV∷PMV, and CYVV∷CYVV; 84 to 89% for BYMV∷ PMV, 69 to 78% for BYMV∷CYVV and PMV∷CYVV, and 50 to 64% for all other potyvirus combinations. Additionally, similar pairwise analysis of the 3'NCR of the verbena potyvirus revealed 98 to 99% identity to PMV strains, 81 to 94% to other BYMVs, 68 to 75% to CYVVs, and 52 to 64% with other potyviruses. Other 3'NCR pairwise comparisons generally revealed the same identity trend as described for the CP. Further serological analysis with our panel of BYMV-specific, BYMV-subgroup, and potyvirus cross-reactive monoclonal antibodies (3) confirmed the designation of the verbena potyvirus isolate as a pea mosaic strain of BYMV. To our knowledge this is the first confirmed report of BYMV-pea mosaic strain in Verbena (1,2).

Electrospray ionization of a whole virus: analyzing mass, structure, and viability.

Staying together: Electrospray ionization has been shown to generate intact gas-phase viral ions that can be used to examine mass and structure of whole viruses such as the tabac mosaic virus. That these particles are viable can be demonstrated by the infection of tobacco plants, which then develop characteristic lesions (see picture).

Systemic plant signal triggers genome instability.

Previously, we have shown that infection of tobacco plants with a viral pathogen triggers local and systemic induction of homologous recombination (HR). Here, we have tested the hypothesis of whether free radicals are potentially involved in the induction of the systemic effect. We report a significant induction of HR in tobacco plants treated with radical-generating agents, UVC or rose Bengal (RB). Importantly, the recombination increase was observed in local (treated) as well as systemic (non-treated) tissue. The systemic increase in recombination implies the existence of a signal that is transmitted to non-treated tissue. Several sets of grafting experiments proved the generation of said signal by both RB and UVC exposure. A statistically significant increase in HR was observed in tissue that received a systemic signal via a grafted leaf. Similar data were obtained from transgenic plants naphthalene degrading salicylate 1-hydroxylase (NahG) unable to accumulate salicylic acid (SA). Interestingly, pre-treatment of plants with the radical-scavenging compound N-acetyl-l-cysteine (NAC) led to a significantly lower recombination increase upon grafting after treatment with UVC and RB. Moreover, leaves taken for grafting from NAC-pre-treated plants exhibited a lower level of oxidized organic compounds. Our data suggest the involvement of free radical production in either generation or maintenance of the recombination signal. We discuss potential mechanisms for generation of the signal and possible adaptive advantages of enhanced genomic flexibility following exposure to DNA-damaging agents.

Pathogen-induced systemic DNA rearrangement in plants

Infection of tobacco plants with tobacco mosaic virus and oilseed rape mosaic virus was shown to induce a threefold increase in homologous DNA recombination in non-infected tissues. Grafting experiments by Igor Kovalchuk et al. demonstrated that this increase was mediated by a systemic recombination signal that traveled ahead of the virus. A similar increase in DNA recombination was also observed in the progeny of the infected plants, indicating that pathogen-induced recombination can lead to heritable adaptations to environmental stresses.

First Report of Zucchini yellow mosaic virus in Cucumber in Poland.

In June 2002, mosaic and interveinal chlorosis were observed on two cucumber plants (Cucumis sativus) grown in one commercial greenhouse in the western region of Poland. Electron microscopic examination of leaf-dip preparations from infected plants showed flexuous filamentous virus particles typical of potyviruses (720 to 750 nm long). Chenopodium amaranticolor, Chenopodium quinoa, Citrullus lanatus, C. melo, C. sativus, Cucurbita maxima, Cucurbita pepo, Cucurbita pepo cv. Giromontiina, Cucurbita pepo cv. Patissoniana, Nicotiana benthamiana, and N. tabacum were mechanically inoculated with sap from symptomatic cucumber leaves. The virus caused local chlorotic lesions on Chenopodium amaranticolor and Chenopodium quinoa and systemic infection in all tested cucurbits but it did not infect tobacco plants. Reverse transcription-polymerase chain reaction (RT-PCR) amplification of the 3' end of the genomic RNA was done by using P9502 as a downstream primer and degenerate CPUP as an upstream primer to amplify a highly conserved region of the potyviral coat protein (1). The PCR products were directly sequenced with the CEQ DTCS dye terminator cycle sequencing kit (Beckman Coulter, Inc., Fullerton, CA), and the analysis of dideoxy terminated fragments was conducted by capillary electrophoresis using a CEQ 2000 DNA Analysis System (Beckman Coulter, Inc.). The obtained 684 nt sequence (GenBank Accession No. AY347476) was almost identical with sequences of Zucchini yellow mosaic virus (ZYMV) isolates from Austria (GenBank Accession Nos. AJ420012-AJ420019 and AJ420027) and Hungary (GenBank Accession Nos. AJ459954 and AJ459955). The above suggested that the Polish isolate of ZYMV belonged to the Central European branch of the phylogenetic tree (2). To our knowledge, this is the first report of ZYMV in Poland.

Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector

We expressed two immunogenic dominant epitopes of foot-and-mouth disease virus (FMDV) serotype O in tobacco plant using a vector based on a recombinant tobacco mosaic virus (TMV). The recombinant viruses TMVF11 and TMVF14 contained peptides of 11 and 14 amino acid residues, respectively, from FMDV VP 1 fused to the open reading frame of TMV coat protein (CP) gene between amino acid residues 154 and 155. TMVF11 and TMVF14 systemically infected tobacco plant and produced large quantities of stable progeny viral particles assembled with the modified CP subunits. Guinea pigs, mice and swine were used to test the protective effects of the recombinant viruses against FMDV infection. Most guinea pigs were protected against FMDV challenge after parenteral injection with TMVF11, TMVF14, or the mixture TMVF11/TMVF14, but not wtTMV. The TMVF11/TMVF14 mixture protected all animals when challenged with 150 guinea pig 50% infection dosage (GPID 50) FMDV. Oral administration of the TMVF11/TMVF14 mixture (3 mg total) protected 3/8 guinea pigs against the same FMDV challenge. Most of the suckling mice parenterally injected with antiserum from guinea pigs immunized with the TMVF11/TMVF14 mixture, but not with wtTMV, were also protected against FMDV challenge with 10 suckling mouse 50% lethal dosage (SMLD 50), indicating that antibodies produced in guinea pigs immunized with the TMVF11/TMVF14 mixture specifically neutralized FMDV. Western blot analysis indicated that antiserum from those guinea pigs reacted with the FMDV VP1 protein. The protective effect of TMVF11 was also demonstrated in swine, where preliminary tests showed that nine pigs immunized with TMVF11 in three experiments were protected against FMDV challenge with 20 minimal infecting dose (MID).

Plant Pathogens Trigger DNA Recombination

In plants, DNA rearrangement can be stimulated by various environmental and pathogenic stresses. Kovalchuk et al. designed a luciferase reporter assay that depended on DNA recombination events to visualize stimulation of DNA rearrangement in virally infected tobacco plants. The recombination events occurred in leaves that did not yet contain detectable virus. Stimulation of DNA rearrangement preceded spread of the virus to uninfected leaves and could be transmitted to uninfected plants by grafting of uninfected leaves from plants exposed to virus on other leaves. These results indicated that the pathogen itself was not causing DNA rearrangement directly, but rather that the virus activated a signaling cascade that triggered the increased recombination rate. The results were confirmed by following recombination frequencies of an endogenous gene. Increased frequency of recombination in plants grown from seeds of plants grafted with leaves from infected plants was also noted. The authors propose that a mobile signal, the systemic recombination signal, is generated in response to viral infection that stimulates DNA rearrangement throughout the plant. This signal did not appear to be the same as that involved in systemic acquired resistance. I. Kovalchuk, O. Kovalchuk, V. Kalck, V. Boyko, J. Filkowski, M. Heinlein, B. Hohn, Pathogen-induced systemic plant signal triggers DNA rearrangements. Nature 423 , 760-762 (2003). [Online Journal]

Amino acid substitutions within the Cys-rich domain of the tobacco etch potyvirus HC-Pro result in loss of transmissibility by aphids

We examined the role of several amino acid residues located at the N-terminus of the tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro) in virus transmissibility by aphids. Site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. The TEV HC-Pro amino acid residues Gly343, Val345, Ala346, Ile348, Pro355, Lys358, and Ile359 were arranged within a Cys-rich domain in a region dispensable for TEV infectivity. Two HC-Pro mutants (TEV-P355R and -K358N) exhibited a drastically reduced rate of aphid transmission whereas other mutants (TEV-G343D, -V345E, -A346H, -I348D, and -P355L) were completely unable to be aphid transmitted. In contrast, the I359M mutation had no effect on aphid transmissibility of TEV. This lack of transmissibility did not appear to be due to large differences in the amounts of both coat protein (CP) and HC-Pro in infected tobacco plants. Our results indicated that these amino acid residues likely play a highly conserved role in aphid transmission among potyviruses.

The Efficiency by Which Thrips tabaci Populations Transmit Tomato spotted wilt virus Depends on Their Host Preference and Reproductive Strategy

ABSTRACT Arrhenotokous and thelytokous populations of Thrips tabaci from tobacco or leek plants were evaluated for their ability to transmit Tomato spotted wilt virus (TSWV) and for their host preference. Transmission efficiencies were comparatively studied using leaf disks of Petunia hybrida, Datura stramonium, and Nicotiana tabacum cv. Basmas. Adults of arrhenotokous populations collected on infected tobacco plants in the field were efficient transmitters (up to 48.5% transmission) and remained so when maintained on tobacco for several generations. Arrhenotokous T. tabacipopulations from leek plants were poor transmitters (up to 3.1% transmission), whereas no transmission was obtained with thelytokous populations from leek. All populations could infest leek, however none of the arrhenotokous and thelytokous populations from leek plants was able to infest tobacco. TSWV could be acquired by both first and second larval instars of a T. tabacipopulation from tobacco. However, the transmission by adults decreased with the age at which the virus was acquired by larvae. The highest efficiencies (61% of males and 51% of females transmitted) were obtained when newborn (0- to 24-h old) larvae acquired the virus. The majority of thrips started to transmit after becoming adult and rates were positively correlated with the temperature at which the thrips were kept. The median latent period values found for adults decreased with increasing temperature. The median acquisition access period (AAP50) of the population was 41 min, whereas the AAP(50) was 65 min for males and 35 min for females. The median inoculation access period of males was 246 and 365 min on tobacco and petunia, respectively, and 96 and 345 min for females. The results show that T. tabaci forms a complex in terms of host preference, reproductive strategy, and ability to transmit TSWV. The transmission parameters show that the thrips of arrhenotokous populations infesting tobacco are highly efficient vectors.