Detection, Distribution, and Genetic Variability of 'Candidatus Liberibacter' Species Associated with Zebra Complex Disease of Potato in North America.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for 'Candidatus Liberibacter solanacearum' and 'Candidatus Liberibacter psyllaurous' were evaluated in conventional and real-time PCR assays. All PCR primers were specific for 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. 'Ca. Liberibacter' species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using 'Ca. L. solanacearum'-specific primers and primers specific for the ß-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for 'Ca. Liberibacter' detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The 'Ca. Liberibacter' species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' "strains" investigated in this study. 'Ca. L. solanacearum' and 'Ca. L. psyllaurous' were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of 'Ca. L. solanacearum' and reverse primer of 'Ca. L. psyllaurous' in ZC-affected potato samples. This finding clarifies the current taxonomic status of 'Ca. L. solanacearum' and 'Ca. L. psyllaurous'. The detection of 'Ca. L. solanacearum' from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

Colony establishment and maintenance of the eriophyid wheat curl mite Aceria tosichella for controlled transmission studies on a new virus-like pathogen.

High plains disease (HPD) is of serious economic concern for wheat and corn production, but little is known about the virus-like causal agent. In the field, HPD is often associated with Wheat streak mosaic virus (WSMV) and both pathogens are transmitted by the same eriophyid wheat curl mite, Aceria tosichella Keifer. The objective of this study was to develop methods for establishing and maintaining HPD-transmitting wheat curl mite colonies for their use in studies on HPD. Towards this goal, mite colonies from a mixed infection source were separated into colonies either (i). not viruliferous; (ii). only transmitting WSMV; or (iii). only transmitting HPD. Maintenance of these colonies required strictly separated incubator facilities and adaptation of mite-suitable transfer techniques to permit frequent passages of mites to healthy plants. The established colonies provided reliable sources of infective material to study the progression of HPD and/or WSMV in plants using sensitive immuno-detection assays. In conclusion, we have developed reliable methods with a poorly studied arthropod vector to examine the biology and properties of a new virus-like disease.

Retention of a small replicase gene segment in tomato bushy stunt virus defective RNAs inhibits their helper-mediated trans-accumulation.

Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.

Genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion.

The plus-sense single-stranded RNA of tomato bushy stunt virus (TBSV) encodes a 19-kDa protein, which is translated from a 3' proximal open reading frame (p19) that is entirely nested within the cell-to-cell movement gene (p22). Expression of the cytosolic p19-protein induces either a systemic lethal collapse in Nicotiana benthamiana and N. clevelandii, or necrotic local lesions on resistant N. tabacum. In spinach, the p19-protein is required at high abundance for efficient systemic invasion. This study aimed to determine whether these seemingly different host-dependent biological activities are governed by the same or separate regions on the 172 amino acid p19-protein. For this purpose, codons for charged amino acids predicted to be exposed on the surface of the polypeptide and presumably available for host-specific interactions, were targeted for mutagenesis. A total of 12 mutants were generated, which had no deficiencies in replication or cell-to-cell movement, and substitution of amino acids at the extreme N-terminal end or within the carboxyl 70 amino acids failed to cause a noticeable biological effect on plants. However, mutations dispersed between positions 43 and 85 on the N-terminal half prevented the onset of a systemic lethal necrosis on N. benthamiana and N. clevelandii. With one exception, the same mutants elicited mostly chlorotic, rather than necrotic, local lesions on N. tabacum. Mutations in the central region, which substituted Arg with Gly at positions 72 or 75-78, impaired the ability of TBSV to systemically invade spinach plants. However, substitution with Ala instead of Gly at position 72 had minimal effects on systemic spread in spinach, suggesting the possible influence of protein structure effects. The implications are that regions on the N-terminal portion of the p19-protein mediate interactions in a host-dependent manner and that a central region is required for all activities either by a direct effect of the amino acids or through maintenance of structural integrity.

Rapid delivery of foreign genes into plants by direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector.

Tomato bushy stunt virus (TBSV) cDNA, positioned between a modified cauliflower mosaic virus 35S promoter and the hepatitis delta virus antigenomic ribozyme with a downstream nopaline synthase gene polyadenylation signal, established infections upon rub-inoculation of plants with intact plasmids. Application of this methodology produced a TBSV DNA-based gene vector which yielded readily detectable levels of localized foreign gene expression in inoculated leaves. This is the first demonstration of an infectious DNA from a member of the Tombusviridae which permits rapid TBSV-mediated foreign-gene expression upon direct rub-inoculation of miniprep DNA onto a variety of plant species.

Broad-spectrum protection against tombusviruses elicited by defective interfering RNAs in transgenic plants.

We have designed a DNA cassette to transcribe defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenic Nicotiana benthamiana plants from tombusvirus infections. To produce RNAs with authentic 5' and 3' termini identical to those of the native B10 DI RNA, the DI RNA sequences were flanked by ribozymes (RzDI). When RzDI RNAs transcribed in vitro were mixed with parental TBSV transcripts and inoculated into protoplasts or plants, they became amplified, reduced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F1 and F2 RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformed N. benthamiana plants and transformed negative controls died. Although infection of transgenic RzDI plants initially induced moderate to severe symptoms, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tombus-like virus and to unrelated viruses.

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene.

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.

The enigma of pX: A host-dependent cis-acting element with variable effects on tombusvirus RNA accumulation.

Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.

The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells.

We analyzed the distribution of the cauliflower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF- and the derivative mutant-microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunofluorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.

Plant virus gene vectors for transient expression of foreign proteins in plants.

The development of plant virus gene vectors for expression of foreign genes in plants provides attractive biotechnological tools to complement conventional breeding and transgenic methodology. The benefits of virus-based transient RNA and DNA replicons versus transgenic gene expression include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. These characteristics are especially advantageous when very high levels of gene expression are desired within a short time, although instability of the foreign gene in the viral genome can present some problems. The strategies that have been tested for foreign gene expression in various virus-based vectors include gene replacement, gene insertion, epitope presentation, use of virus controlled gene expression cassettes, and complementation. Recent reports of the utilization of virus vectors for foreign gene expression in fundamental research and biotechnology applications are discussed.

Tomato bushy stunt virus spread is regulated by two nested genes that function in cell-to-cell movement and host-dependent systemic invasion.

We have investigated the importance of two small nested genes (p19 and p22) located near the 3' end of the genome of tomato bushy stunt virus (TBSV) for infectivity in several hosts. Our results show that both genes are dispensable for replication and transcription and that the p19 gene encodes a soluble protein, whereas the p22 gene specifies a membrane-associated protein. Assays using TBSV derivatives that have the beta-glucuronidase gene substituted for the capsid protein gene demonstrate that p22 is required for cell-to-cell movement in all plants tested. Mutations inactivating p19 ameliorate the severe necrotic systemic symptoms elicited by wild-type TBSV in Nicotiana benthamiana and Nicotiana clevelandii, but p19 does not obviously affect movement in these hosts. However, in some local lesion hosts p19 influences the lesion diameter, which suggests that it has an auxiliary host-dependent role in movement. This notion is supported by the observation that p19 is required for long-distance spread of TBSV in spinach and for systemic infection of pepper plants. Thus, movement of TBSV is regulated by two nested genes; p22 governs cell-to-cell movement and p19 has a host-specific role in systemic invasion.

Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.

The effect of defective interfering RNAs on the accumulation of tomato bushy stunt virus proteins and implications for disease attenuation.

Tombusviruses, of which tomato bushy stunt virus (TBSV) is the type member, spontaneously generate defective interfering RNAs (DIs) that are known to interfere with viral accumulation and symptom development. We show that co-infection with TBSV and DIs causes a dramatic reduction in accumulation of TBSV subgenomic RNAs and corresponding TBSV proteins with a less dramatic reduced accumulation of the genomic RNA and the replicase proteins. Associated with this differentially regulated suppression was a greatly reduced expression of both the p19 protein, which is responsible for severe symptoms, and the p22 protein, which is associated with cell-to-cell movement of the virus. Therefore, the results suggest that the protective effect of DIs may be due to selective inhibition of p19 and p22 expression in addition to reduced replication of genomic RNA.

Host effects and sequences essential for accumulation of defective interfering RNAs of cucumber necrosis and tomato bushy stunt tombusviruses.

Passage of cucumber necrosis virus (CNV) containing defective interfering (DI) RNAs through cucumber plants decreased the accumulation of DI RNAs to undetectable levels. Subsequent passages in two Nicotiana species (Nicotiana benthamiana or N. clevelandii) resulted in the appearance of DI RNA species that were larger than the DI RNAs observed during exclusive serial passages of CNV through the Nicotiana species. Sequence analysis of cloned cDNAs corresponding to the two DI RNA populations indicated that the smaller CNV-DI RNAs contained the four conserved regions (I through IV) of the genome typical of tombusvirus DI RNAs, whereas the larger DI RNAs were of similar organization but had a direct repeat of the middle portion of the molecule. This result suggests that the host has an influence on the type of DI RNA that accumulates during consecutive high multiplicity of infection passages. A comparative analysis of deletions targeting the individual conserved regions in both CNV and tomato bushy stunt virus (TBSV) DI RNAs revealed that only region III was completely dispensable for accumulation of either DI RNA species. More refined deletion analyses in regions I and II indicated that smaller segments of 75 and 35 nucleotides (nt), respectively, could be deleted without abolishing infectivity. The dispensable sequences in region II of both TBSV and CNV DI RNAs mapped to the top portion of a putative stem-loop structure. These studies indicate that both essential and nonessential sequences are conserved in DI RNAs. The essential sequences in regions I, II, and IV likely contain important cis-acting elements, whereas nonessential regions such as region III may play secondary roles such as optimally spacing cis-acting elements or maintaining the DI RNA at an overall size that is stable.

The tomato bushy stunt virus replicase proteins are coordinately expressed and membrane associated.

Two open reading frames at the 5'-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92-kDa (p92) readthrough product. From amino acid sequence comparisons with other small single-stranded RNA viruses, these proteins resemble viral components of the replicase-transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the carboxy-terminal "half" of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 readthrough protein, but the anti-p92 antibody was specific for p92. Immunoblot analyses revealed that at an early stage of infection both proteins were associated with the membrane fractions isolated from virus-infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic fraction. At all time points in plants and protoplasts p33 was about 20-fold more abundant than p92. A series of mutations derived from an infectious cDNA clone demonstrated that both the p33 and the p92 proteins were required for replication in protoplasts and the ratio of the two proteins was maintained in the replication-competent mutants. The wild-type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants (UAC or UAU) that were predicted to express only p92 were not viable in protoplasts.

The putative zinc finger of a caulimovirus is essential for infectivity but does not influence gene expression.

Plant pararetroviruses, such as caulimoviruses, and animal retroviruses have in common the presence of a highly conserved arrangement of cysteines and a histidine in the precursor of the capsid protein. The composition of these amino acids resembles a zinc finger element, a structure that is common to a class of eukaryotic proteins that regulate gene expression. The role of the putative zinc finger in the life-cycle of caulimoviruses was investigated by introducing specific mutations in the coat protein coding region of a cloned and infectious form of figwort mosaic virus, a caulimovirus. This mutated viral genome, which no longer encoded the conserved cysteine and histidine residues, was not infectious in plants. Transient expression assays in protoplasts showed that expression of a reporter gene inserted at different places in the genome was not detectably influenced by the coat protein or its putative zinc finger. It appears that the zinc finger-like element of caulimoviruses is not involved in the regulation of gene expression. These observations support a model which predicts a function of the zinc finger in specific recognition and packaging of viral RNA into virions prior to reverse transcription.

Regulation of caulimovirus gene expression and the involvement of cis-acting elements on both viral transcripts.

In a further analysis of gene regulation of figwort mosaic virus (FMV), a caulimovirus, we studied transient gene expression with modified viral genomes in Nicotiana edwardsonii cell suspension protoplasts. The results demonstrated that the presence of the promoter for the full-length RNA interferes with expression from the separate downstream promoter for gene VI. In addition, expression of gene VI was inhibited by cis-acting sequences within gene VI itself. Both inhibitory effects could be partially relieved by coelectroporation with a plasmid that produces gene VI protein, demonstrating that expression of gene VI is transactivated by its own product. Subsequent expression studies with partially redundant FMV plasmids containing a reporter gene in frame with gene IV showed that efficient transactivation of CAT expression relies on a cis-acting element inside the downstream gene VI. Insertions of a transcriptional terminator upstream of the cis-acting element for premature termination of transcription showed that the cis-acting region is not a DNA element but is active only as a feature of the RNA transcript. We conclude that the cis-acting element, together with the transacting gene VI product, enhances expression of all major genes, including gene VI, from the polycistronic mRNA and the separate mRNA for gene VI.