Robert A. Owens

Sensitive and Rapid Diagnosis of Potato Spindle Tuber Viroid Disease by Nucleic Acid Hybridization

A sensitive and reliable new method for the detection of potato spindle tuber viroid in potato tubers has been developed. The method is based on hybridization of highly radioactive recombinant DNA to viroid RNA that has been attached to a solid support. The method can be automated and permits the rapid testing of large numbers of tubers.

Movement of Potato Spindle Tuber Viroid Reveals Regulatory Points of Phloem-Mediated RNA Traffic

Increasing evidence indicates that the phloem mediates traffic of selective RNAs within a plant. How an RNA enters, moves in, and exits the phloem is poorly understood. Potato spindle tuber viroid (PSTVd) is a pathogenic RNA that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. The viroid RNA genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent probe to study mechanisms that regulate RNA traffic. Our analyses of PSTVd traffic in Nicotiana benthamianayielded evidence that PSTVd movement within sieve tubes does not simply follow mass flow from source to sink organs. Rather, this RNA is transported into selective sink organs. Furthermore, two PSTVd mutants can enter the phloem to spread systemically but cannot exit the phloem in systemic leaves of tobacco (Nicotiana tabacum). A viroid most likely has evolved structural motifs that mimic endogenous plant RNA motifs so that they are recognized by cellular factors for traffic. Thus, analysis of PSTVd traffic functions may provide insights about endogenous mechanisms that control phloem entry, transport, and exit of RNAs.

Site-specific mutagenesis of potato spindle tuber viroid cDNA: : Alterations within premelting region 2 that abolish infectivity.

SummaryThe infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C → U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered ‘wild type’ sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.

Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.

Redox-cycling and H2O2 generation by fabricated catecholic films in the absence of enzymes.

Phenolic matrices are ubiquitous in nature (e.g., lignin, melanin, and humics) but remain largely intractable to characterize. We examined an abiotic phenol-polysaccharide matrix fabricated by the anodic grafting of catechol to chitosan films. Previous studies have shown that catechol-modified chitosan films are redox-active and can be repeatedly interconverted between oxidized and reduced states. Here we developed quantitative electrochemical methods to characterize biorelevant redox properties of the catechol-modified chitosan films. Our analysis demonstrates that these films can (i) accept electrons from biological reductants (e.g., ascorbate and nicotinamide adenine dinucleotide phosphate, NADPH) and (ii) donate electrons in a model biological oxidation process. Furthermore, these films can donate electrons to O(2) to generate H(2)O(2). The demonstration that abiotic catechol-chitosan films possess catalytic activities in the absence of enzymes suggests the possibility that phenolic matrices may play an important role in redox cycling and reactive oxygen species (ROS) signaling in biology and the environment.

Viroid Pathogenicity: One Process, Many Faces

Despite the non-coding nature of their small RNA genomes, the visible symptoms of viroid infection resemble those associated with many plant virus diseases. Recent evidence indicates that viroid-derived small RNAs acting through host RNA silencing pathways play a key role in viroid pathogenicity. Host responses to viroid infection are complex, involving signaling cascades containing host-encoded protein kinases and crosstalk between hormonal and defense-signaling pathways. Studies of viroid-host interaction in the context of entire biochemical or developmental pathways are just beginning, and many working hypotheses have yet to be critically tested.

Potato spindle tuber viroid infections mediated by the Ti plasmid of Agrobacterium tumefaciens.

SummaryFull length copies of potato spindle tuber viroid (PSTV) were introduced into plant cells using an Agrobacterium tumefaciens vector. Crown galls containing the PSTV DNA were induced on tomato plants, and the plants analysed for systemic replication of the viroid. Two separately derived multimeric PSTV insertions in the T-DNA were infectious on every plant inoculated. However, monomeric PSTV gave rise to significant levels of infection only when an adjacent plant promoter could direct transcription of + strand PSTV RNA. Our results suggest that this experimental system will be useful for the analysis of viroid replication. A second application of the results may be the use of systemic viral infection as a sensitive assay for transient expression of transformed DNA. re]19850917 rv]19851203 ac]19851210

Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid.

A viroid has been isolated from tomato plants affected by Indian bunchy top disease of tomato (Lycopersicon esculentum Mill.). In dot blot hybridization assays with 32P-labelled cRNA probes specific for the detection of various viroids, the Indian viroid was shown to be most closely related to the citrus exocortis viroid (CEVd). Sequence determination showed that the viroid consists of 372 nucleotides and confirmed its close relationship with CEVd. The viroid, for which we propose the acronym CEVd-t, differs from the Australian CEVd strains A and B by 36 and 47 nucleotides, respectively, and from the Spanish grapevine isolate by 52 changes. A phylogenetic analysis confirmed the closet relationship with CEVd in all structural domains, except the pathogenicity and left-terminal domains, which are closely related to the corresponding domains of the potato spindle tuber and tomato apical stunt viroids, respectively.

Measurement of viroid sequence homology by hybridization with complementary DNA prepared in vitro

DNA complementary to potato spindle tuber viroid (YSTV cDNA) has been used in RNA DNA hybridization experiments to study PSTV replication in a variety of hosts and to measure the amount of sequence homology between YSTV and several independently isolated viroids. When PSTV cDNA was hybridized with low molecular weight RNA isolated from PSTV-infected tomato at different times after inoculation, PSTV . PSTV cDNA hybrids were detectable before symptoms began to appear. Low molecular weight RNA was prepared from PSTV-infected tomato, tobacco, Gynura, and chrysanthemum and was analyzed by polyacrylamide gel electrophoresis and hybridization to PSTV cDNA. The identical electrophoretic mobility of PSTV isolated from each host and identical thermal stability of the corresponding RNA DNA hybrids suggest that host-specific changes in viroid sequence do not occur. Previously unreported viroids have been isolated from Columnea erythrophae and from a random selection of Solanum phureja and S. stenotomum introductions. The electrophoretic mobility of the Solanum viroids is very similar to that of PSTV, whereas the Columnea viroid and the previously reported chrysanthemum stunt viroid migrate significantly faster than PSTV. Measurement of the degree of sequence homology between PSTV and the various viroid isolates suggests that whereas the Solunum viroids are closely related to PSTV, chrysanthemum stunt and Columnea viroids are not closely related to PSTV.

Both Point Mutation and RNA Recombination Contribute to the Sequence Diversity of Citrus Viroid III

Field-grown citrus trees often harbor complex mixtures of 4–5 different viroid species, and the presence of citrus viroid III (CVd-III) has been shown to reduce the rate of tree growth without inducing disease. To more fully define the structure of its quasi-species, we have examined nine citrus viroid complexes for the presence of previously undescribed sequence variants of CVd-III. Analysis of 86 full-length cDNAs generated from these nine viroid complexes by RT-PCR revealed the presence of 20 new CVd-III variants. Chain lengths ranged from 293–297 nucleotides, and sequence changes were confined largely to the lower portions of the central conserved region and variable domain. The previously described variants CVd-IIIa (297 nt) and CVd-IIIb (294 nt) were clearly predominant, but phylogenetic analysis indicated that certain isolates may contain representatives of two additional fitness peaks. At least one group of CVd-III variants appears to have arisen as a result of RNA recombination. Populations recovered from diseased/declining trees were the most diverse, but even dwarfing isolates originating from old line Shamouti trees showed considerable variability.