Bottlenecks and complementation in the aphid transmission of citrus tristeza virus populations.

Aphid transmission is a major factor in the formation of citrus tristeza virus (CTV) populations. Here, we examined the effect of population interaction on aphid transmissibility of different CTV genotypes. We found that there was no correlation between the proportion of viral genotypes in the source population and what was transmitted. We next examined the transmission of a poorly transmitted infectious cDNA clone (T36) in mixture with other CTV genotypes. T36 transmission increased from 0.5% alone, to up to 35.7%, depending on the coinfecting genotype. These results suggest that interaction between CTV genotypes affects the transmission of this virus.

Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida.

Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-ß-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. IMPORTANCE: Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere with the CTV binding to its insect vector to block the transmission.

Some like it hot: citrus tristeza virus strains react differently to elevated temperature.

Viruses often infect plants as a mixed population. The dynamics of viral populations dictate the success of the infection, yet there is little understanding of the factors that influence them. It is known that temperature can affect individual viruses; could it also affect a virus population? In order to study this, we observed citrus tristeza virus (CTV) populations in different hosts under winter and summer conditions (25 versus 36 °C). We found that only some CTV strains were affected by a higher summer temperature, which lead to a change in CTV population structure, and that this effect was host dependent.

Sequence variation in two genes determines the efficacy of transmission of citrus tristeza virus by the brown citrus aphid.

Vector transmission is an important part of the viral infection cycle, yet for many viruses little is known about this process, or how viral sequence variation affects transmission efficacy. Here we examined the effect of substituting genes from the highly transmissible FS577 isolate of citrus tristeza virus (CTV) in to the poorly transmissible T36-based infectious clone. We found that introducing p65 or p61 sequences from FS577 significantly increased transmission efficacy. Interestingly, replacement of both genes produced a greater increase than either gene alone, suggesting that CTV transmission requires the concerted action of co-evolved p65 and p61 proteins.

Not all GMOs are crop plants: non-plant GMO applications in agriculture.

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.

A real-time RT-qPCR assay for the detection of Citrus tatter leaf virus.

Citrus tatter leaf virus (CLTV) is globally distributed wherever citrus is grown, and, given the extensive use of CTLV sensitive rootstock, has the potential to be a significant threat to the citrus industry. In order to facilitate fast and reliable detection of this virus, we have developed a CTLV-specific real-time RT-qPCR assay. The optimized assay was found to be more reliable and sensitive compared to ELISA and end-point RT-PCR, detecting CTLV in up to 70% more plants. The real-time RT-qPCR is also specific, as it did not cross-react with the closely related Apple stem grooving virus or with the host itself; robust, being able to detect CTLV in young and mature host tissue types; and rapid.

Isolate fitness and tissue-tropism determine superinfection success.

The mechanism of cross-protection, the deliberate infection of plants with a "mild" virus isolate to protect against "severe" isolates, has long been a topic of debate. In our model system, Citrus tristeza virus (CTV), this appears to be genotype-specific superinfection-exclusion, suggesting a simple recipe for cross-protection. However, this concept failed in field trials, which led us to examine the process of superinfection-exclusion more closely. We found that exclusion relies on the relative fitness of the primary versus the challenge isolates, and the host infected, and that significant differences in superinfection success could occur between isolates that differ by as few as 3 nucleotides. Furthermore, we found that exclusion was not uniform throughout the plant, but was tissue-specific. These data suggest that cross-protection is not a simple like-for-like process but a complex interaction between the primary and challenge isolates and the host.

The structure of viral coat protein and its in disease.

Because the structure of the tobacco mosaic virus capsid protein is known, mutations that alter the phenotype of the virus-plant interaction can be correlated with structural changes in this protein. These mutations affect the disease symptoms caused by the virus and recognition of the virus by a plant resistance gene product. Recognition-host interaction is analogous to the gene-for-gene interactions of bacteria and fungi.

Structure-function relationship between tobacco mosaic virus coat protein and hypersensitivity in Nicotiana sylvestris.

Alterations in the structure of the tobacco mosaic virus (TMV) coat protein affect the elicitation of the N' gene hypersensitive response (HR) in Nicotiana sylvestris. To investigate this structure-function relationship, amino acid substitutions with predicted structural effects were created throughout the known structure of the TMV coat protein. Substitutions that resulted in the elicitation of the HR resided within and would predictably interfere with interface regions located between adjacent subunits in ordered aggregates of coat protein. Substitutions that did not result in the elicitation of the HR were either conservative or located outside these interface regions. In vitro analysis of coat protein aggregates demonstrated HR-eliciting coat proteins to have reduced aggregate stability in comparison with non-HR-eliciting coat proteins and a correlation existed between the strength of the elicited HR and the ability of a substitution to interfere with ordered aggregate formation. This finding corresponded with the predicted structural effects of HR-eliciting substitutions. Radical substitutions that predictably disrupted coat protein tertiary structure were found to prevent HR elicitation. These findings demonstrate that structural alterations that affect the stability of coat protein quaternary structure but not tertiary structure lead to host cell recognition and HR elicitation. A model for HR elicitation is proposed, in which disassembly of coat protein aggregates exposes a host "receptor" binding site.

Finding balance: Virus populations reach equilibrium during the infection process.

Virus populations, mixtures of viral strains or species, are a common feature of viral infection, and influence many viral processes including infection, transmission, and the induction of disease. Yet, little is known of the rules that define the composition and structure of these populations. In this study, we used three distinct strains of Citrus tristeza virus (CTV) to examine the effect of inoculum composition, titer, and order, on the virus population. We found that CTV populations stabilized at the same equilibrium irrespective of how that population was introduced into a host. In addition, both field and experimental observations showed that these equilibria were relatively uniform between individual hosts of the same species and under the same conditions. We observed that the structure of the equilibria reached is determined primarily by the host, with the same inoculum reaching different equilibria in different species, and by the fitness of individual virus variants.

With a little help from my friends: complementation as a survival strategy for viruses in a long-lived host system.

In selective host species, the extent of Citrus tristeza virus (CTV) infection is limited through the prevention of long-distance movement. As CTV infections often contain a population of multiple strains, we investigated whether the members of a population were capable of interaction, and what effect this would have on the infection process. We found that the tissue-tropism limitations of strain T36 in selective hosts could be overcome through interaction with a second strain, VT, increasing titer of, and number of cells infected by, T36. This interaction was strain-specific: other strains, T30 and T68, did not complement T36, indicating a requirement for compatibility between gene-products of the strains involved. This interaction was also host-specific, suggesting a second requirement of compatibility between the provided gene-product and host. These findings provide insight into the 'rules' that govern interaction between strains, and suggest an important mechanism by which viruses survive in a changing environment.

Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene.

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Logistic and Poisson models for infection by multicomponent plant viruses.

A model for the relationship between virus concentration and infectivity of multicomponent plant viruses is based on a combination of logistic and Poisson equations. Two separate equations are derived from the Poisson distribution assuming, (i) that infections occur only when a set of components containing the complete multicomponent genome is established at an infection site, but that any excess of components present does not reduce the probability of infection (no interference postulate); and (ii) that infection can occur only if a set of components containing the full genome reaches an infection site before it can be preempted by an incomplete set (competitive interference postulate). Postulate (i) affects the form of a dilution series without affecting N, the maximum possible number of infections (lesions), and postulate (ii) changes the value of N but not the form of the dilution series. There is a close correlation between the logit slope of a logistic dilution series and the form of the corresponding multiple Poisson dilution series for viruses with 2, 3 or 4 components. Calibrated by Poisson equations, the logit slope may thus suggest whether or not the virus components have invaded independently and infected similar infection sites. The methods of fitting the combined logistic-Poisson model are demonstrated by applying it to data for cowpea chlorotic mottle virus.

Transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA of tobacco mosaic virus.

We engineered cDNA of tobacco mosaic tobamovirus (TMV) into Agrobacterium tumefaciens for inoculation of plant cells. The resulting bacterial strains were used to transfect tobacco (Nicotiana tabacum cv. Xanthi and Xanthi/nc) with wild type and a defective virus. Lesion formation on Xanthi/nc tobacco was used to measure the timing and efficiency of transfection. Infections mediated by Agrobacterium produced lesions an average of two days later than infections produced by inoculation with virions. The addition of approximately 80 bp of non-viral sequences to the 5'-end of TMV transcripts abolished transfection. Transcripts with non-viral sequences at the 3'-end initiated infections, while precise transcript termination with a synthetic ribozyme sequence increased transfection frequencies two-fold. Culture conditions reported to induce genes of the vir region of the Agrobacterium Ti plasmid also increased the transfection frequency approximately two-fold. Therefore, in addition to the pararetroviruses and geminiviruses previously described, 'agroinoculation' may be used to infect plants with plus-sense RNA viruses.

Two paths of sequence divergence in the citrus tristeza virus complex.

ABSTRACT Comparison of a sampling of complementary DNA (cDNA) sequences from the Florida citrus tristeza virus (CTV) isolates T3 and T30 to the sequence of the genome of the Israeli isolate VT showed a relatively consistent or symmetrical distribution of nucleotide sequence identity in both the 5' and 3' regions of the 19.2-kb genome. In contrast, comparison of these sequences to the sequence of isolate T36 showed a dramatic decrease in sequence identity in the 5' proximal 11 kb of the genome. A cDNA probe derived from this region of the T36 genome hybridized to double-stranded RNA (dsRNA) of only 3 of 10 different Florida CTV isolates. In contrast, analogous probes from T3 and T30 hybridized differentially to the seven isolates not selected by the T36 probe. Primers designed from cDNA sequence for polymerase chain reaction (PCR) selectively amplified these 10 isolates, allowing them to be classified as similar to T3, T30, or T36. In contrast, individual cDNA probes derived from the 3' terminal open reading frames of the T3, T30, and T36 genomes all hybridized to dsRNA from all Florida CTV isolates tested, and PCR primers designed from the T36 capsid protein gene sequence amplified successfully from all isolates. Based on these data, we propose the creation of two groups of CTV, exemplified by the VT and T36 isolates, respectively. Isolates in the VT group, which include isolates VT, T3, and T30, have genomic sequence divergence that is relatively constant in proportion and distribution throughout the genome, and candidate isolates for that group could be considered strains of the same virus. The T36 group is differentiated from the VT group by the highly divergent 5' genomic sequence. This 5' region of the CTV genome, thus, can serve as a measure of the extent of sequence divergence and can be used to define new groups and group members in the CTV complex.

Characterization of the beet yellow stunt virus coat protein gene.

ABSTRACT The beet yellow stunt virus (BYSV) genome contains at least nine open reading frames (ORFs) that code for proteins ranging from 6 to 66 kDa. Based on amino acid sequence comparisons, the coat protein (CP) was previously identified as the product of ORF7. We expressed the product of ORF7 in bacteria and confirmed that ORF7 codes for the BYSV CP by immunoblotting. BYSV is a phloem-limited virus, and virus CP antigen of a quality sufficient for diagnostic antisera production has not been available. To produce BYSV antigen free of plant host contaminants, ORF7 was cloned into a pMAL bacterial expression vector. The resulting fusion protein was affinity-purified and used as an antigen to raise anti-BYSV CP antisera in rabbits and guinea pigs. Using these antisera, an indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA)-based diagnostic system was developed. This indirect DAS-ELISA format enabled reliable detection of BYSV in tissue extracts from virus-infected lettuce diluted up to 5,000 times. The diagnostic system developed may enable large-scale epidemiological studies of BYSV using simple serological techniques. The antisera raised had a titer exceeding 1 x 10(5) in immunoblots and easily detected the 23.7-kDa BYSV CP in virus-infected lettuce and sowthistle plants. In these two plant species, BYSV CP was detected as two closely migrating bands during electrophoresis, which may suggest posttranslational CP modifications. To further characterize the BYSV CP gene, the 5'-untranslated region (UTR) of the BYSV CP subgenomic RNA (sgRNA) was cloned and sequenced. The CP-encoding, approximately 1.9-kb sgRNA has an AT-rich, 66-nucleotide-long 5'-UTR colinear to the genomic sequence upstream of ORF7.

Differential tropism in roots and shoots infected by Citrus tristeza virus.

Virus tropism is a result of interactions between virus, host and vector species, and determines the fate of an infection. In this study, we examined the infection process of Citrus tristeza virus (CTV) in susceptible and resistant species, and found that the tropism of CTV is not simply phloem limited, but tissue specific. In resistant species, virus infection was not prevented, but mostly restricted to the roots. This phenomenon was also observed after partial replacement of genes of one CTV strain from another, despite both parental strains being capable of systemic infection. Finally, the roots remained susceptible in the absence of viral gene products needed for systemic infection of shoots. Our results suggest that all phloem cells within a plant are not equally susceptible and that changes in host or virus may produce a novel tropism: restriction by the host to a location where further virus spread is prevented.

Citrus tristeza virus-host interactions.

Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range. There is little understanding of how the virus causes severe disease in some citrus and none in others. Movement and distribution of CTV differs considerably from that of well-studied viruses of herbaceous plants where movement occurs largely through adjacent cells. In contrast, CTV systemically infects plants mainly by long-distance movement with only limited cell-to-cell movement. The virus is transported through sieve elements and occasionally enters an adjacent companion or phloem parenchyma cell where virus replication occurs. In some plants this is followed by cell-to-cell movement into only a small cluster of adjacent cells, while in others there is no cell-to-cell movement. Different proportions of cells adjacent to sieve elements become infected in different plant species. This appears to be related to how well viral gene products interact with specific hosts. CTV has three genes (p33, p18, and p13) that are not necessary for infection of most of its hosts, but are needed in different combinations for infection of certain citrus species. These genes apparently were acquired by the virus to extend its host range. Some specific viral gene products have been implicated in symptom induction. Remarkably, the deletion of these genes from the virus genome can induce large increases in stem pitting (SP) symptoms. The p23 gene, which is a suppressor of RNA silencing and a regulator of viral RNA synthesis, has been shown to be the cause of seedling yellows (SY) symptoms in sour orange. Most isolates of CTV in nature are populations of different strains of CTV. The next frontier of CTV biology is the understanding how the virus variants in those mixtures interact with each other and cause diseases.

Tobacco mosaic virus protein synthesis is correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis.

The synthesis rates of three proteins of tobacco mosaic virus (TMV), 160, 110, and 17.5 kDa, were monitored at intervals after interruption of synthesis of TMV RNA. Following inhibition of synthesis of both single-stranded and double-stranded RNAs by shifting wild type TMV to 40 degrees or ts mutant III2-35 to 35 degrees, the synthesis rates of viral proteins declined sequentially, with that of the larger proteins declining faster. When viral RNA synthesis was prevented with cordycepin, synthesis rates of the 110 and 160-kDa proteins declined rapidly, while the 17.5-kDa protein decreased more slowly. These data imply that the functional mRNA is transitory, probably nascent RNA, and that each protein is produced independently. The process of translation of viral mRNA was not temperature sensitive and occurred normally for brief periods after shift to restrictive temperatures. When single-stranded RNA synthesis was inhibited differentially from double-stranded RNA synthesis, protein synthesis was correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis. Following a shift of ts mutant IV-35 to 35 degrees, a shift that immediately stopped single-stranded RNA synthesis without inhibiting double-stranded RNA synthesis, all three viral proteins continued to be produced normally. Also, after return of wild type TMV to 25 degrees after a 1-hr incubation at 40 degrees, viral protein and double-stranded RNA synthesis recovered in parallel to the normal rate after 8 hr whereas single-stranded RNA synthesis, which had been reduced more drastically, recovered more slowly after 16 hr.

Guanidine inhibits tobacco mosaic virus RNA synthesis at two stages.

Guanidine (GD) inhibited tobacco mosaic virus (TMV) RNA synthesis at two different steps, depending on the concentration of GD used. The time-course of inhibition by 40 mM GD coincided with the time-course of RNA synthesis. However, 10 mM GD inhibited an earlier step that occurred prior to viral RNA synthesis.