William O. Dawson

Tobamovirus-plant interactions

It is clear that the genetic information responsible for the phenomenon we think of as TMV not only consists of the genes carried in the viral genome, but that numerous plant genes are equally important in viral gene functions. These gene products not only allow the virus to replicate, but may effect functions of evolution that determine what the virus is. Even the processes of pathogenesis and resistance appear to involve similarly precise plant interactions. The challenge of the future is to identify the plant genes involved in these precise interactions and to understand both components of genetic information that comprise plant viruses.

Tobacco mosaic virus coat protein: An elicitor of the hypersensitive reaction but not required for the development of mosaic symptoms in Nicotiana sylvestris

Specific nucleotide changes in the coat protein gene of tobacco mosaic virus (TMV) have been identified as responsible for the induction of the hypersensitive reaction (HR) in Nicotiana sylvestris. Each of these nucleotide changes resulted in amino acid substitutions in the coat protein. To determine if the altered viral RNA or the altered protein acted directly to elicit the HR, the coat protein translational starts were removed from full-length cDNA clones of the HR-inducing mutant TMV 25 and the systemically infecting TMV U1 strain. Infectious transcripts of these altered genomes failed to induce HR in inoculated leaves of N. sylvestris. These free-RNA mutants moved poorly out of inoculated leaves and produced a systemic mosaic symptom 9 to 12 weeks after inoculation. Infectious viral RNA, from both mutants, was recovered from inoculated and systemic mosaic leaves. Western blot analysis of both inoculated and noninoculated leaves revealed the presence of TMV-encoded 126-kDa protein and the absence of coat protein for both mutants. This study demonstrates that the coat protein of TMV 25 is an elicitor molecule responsible for the induction of HR in N. sylvestris and that the TMV coat protein is not required for the development of systemic mosaic symptoms.

Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement

The regulation of the internal open reading frame (ORF) of tobacco mosaic virus (TMV) that encodes the 30K movement protein was examined by constructing mutants in vitro with the putative coat protein subgenomic RNA promoter and leader sequences inserted upstream of the 30K ORF. A mutant with a 49-nucleotide fragment of the promoter region inserted replicated only transiently before being overtaken by a progeny wild-type virus with the insert deleted. A mutant with a 253-nucleotide promoter region fragment inserted replicated stably, and the inserted promoter was active in its new location. The production of 30K protein was not enhanced by this promoter/leader insertion to a level similar to that of coat protein. However, the accumulation of 30K protein was delayed, suggesting that different promoters/leader sequences determine the time of expression of the genes. This mutant was deficient in movement. A similar mutant, but with increased production of 30K protein, overcame the movement deficiency, suggesting that 30K protein is needed during the early stages of infection for efficient cell-to-cell movement of the virus.

Time course of TMV 30K protein accumulation in intact leaves

The kinetics of accumulation of the 30K and coat proteins of tobacco mosaic virus (TMV) which are expressed via subgenomic mRNAs were determined in near-synchronously infected cells of intact tobacco leaves. The 30K protein accumulated to maximal levels during the early stages of the infection as previously reported in tobacco protoplasts. However, in intact leaves, 30K protein accumulated for longer periods and attained higher concentrations than in protoplasts, remaining detectable in all later time samples. Detection of the coat protein, and also the 126K and 183K proteins, began at approximately the same time as the 30K protein (9-12 hr after initiation of replication), but their accumulation continued throughout the 72-hr sampling period. Most of the 30K protein accumulated before 24 hr, whereas most of the coat protein accumulated after 24 hr. This confirms that these two genes are regulated differently, both temporally and quantitatively.

Construction of tobacco mosaic virus subgenomic replicons that are replicated and spread systemically in tobacco plants

Abstract Two tobacco mosaic virus (TMV)derived replicons, created by deletion of most of the 126/183-kDa open reading frame (ORF), replicated and systemically invaded tobacco plants when supported by wild type TMV. One RNA replicon contained an internal direct repeat of 476 nucleotides from the 3′ end of the 30-kDa ORF. Although this RNA was replicated, most of the progeny were heterogeneous in size and smaller than the original transcript. A second TMV-derived RNA replicon, without any internally repeated sequences and containing a deletion of the 5′ portion of the 30-kDa ORF as well as most of the 126/183-kDa ORF, was created and coinoculated with wild type TMV as helper. This RNA also was replicated efficiently and systemically invaded tobacco plants. An examination of the sequences of cDNA clones obtained after PCR amplification of the progeny population of this RNA replicon demonstrated that the observed size heterogeneity was due to deletions and insertions adjacent to the artificially created deletion junction. These data demonstrate that a TMV infection is capable of supporting an artificially created RNA replicon, similar to defective interfering RNAs or satellites. However, these dependent RNAs were replicated without noticeably interfering with wild type TMV symptoms or replication.

Rapid, Random Evolution of the Genetic Structure of Replicating Tobacco Mosaic Virus Populations

The effects of temperature and type of host on the evolution of variants in replicating populations of tobacco mosaic virus (TMV) originating from an in vitro RNA transcript of a cDNA clone were studied. The phenotypic markers measured were temperature-sensitive (ts) replication and induction of necrotic local lesions (nl) on Nicotiana sylvestris. The proportions of ts variants were about the same under all conditions tested, but changes in temperature or host had strong effects on proportions of nl variants. During replication at elevated temperatures in tobacco or tomato, percentages of nl variants were reduced rapidly to about one tenth their initial values. In contrast, when virus replicated at 25 degrees in tobacco, little change in the average proportion of nl variants was observed, and in tomato, only a slight average reduction occurred. Virus replicating at 25 degrees in Solanum nigrum or Petunia hybrida suffered effects similar to those observed in tobacco or tomato, respectively. However, at 25 degrees in Physalis floridana, there was strong selection against nl variants, which eventually reduced them to essentially undetectable levels. In all hosts tested at 25 degrees, surprisingly large, apparently random changes in the proportion of nl variants occurred in individual plants. These experiments showed that virus populations can evolve rapidly on a time scale of days, and that an element of randomness is an important component in the initiation of change.

Examination of the mode of action of ribavirin against tobacco mosaic virus.

Ribavirin inhibited a specific step of tobacco mosaic virus (TMV) replication. Time-courses of sensitivity of TMV multiplication in asynchronous and synchronous infections demonstrated that ribavirin inhibited an early function that occurs prior to the period of viral RNA and protein synthesis. In the synchronous infection, ribavirin inhibited the synthesis of both single-stranded genomic RNA and double-stranded replicative form RNA when treatment began during the first 4 h, but there was no inhibition when treatment began at 12 h or later, even though most RNA synthesis occurred after 12 h. Viral protein synthesis also was inhibited when treatment began during the first 4 h and not when treatment began later. Unlike the mechanisms proposed against animal viruses, ribavirin appears to inhibit TMV replication by preventing an early function that is necessary to initiate viral RNA synthesis.

Pathogen Avirulence Genes and Elicitors of Plant Defense

The hypersensitive response (HR) is an active, inducible defense reaction employed by plants against pathogens. Its main superficial feature is the rapid necrosis of plant cells surrounding the infection site of an invading pathogen. Of more functional importance, 30 or more plant genes are specifically derepressed following recognition of a pathogen, some of which account for the inhibition of subsequent pathogen invasion. These ‘defense response genes’ control a wide range of expressive mechanisms associated with the HR, including the production of chitinases, antiviral proteins, protease inhibitors, s-1,3 glucanases and hydroxyproline-rich glycoproteins as well as encoding enzymes involved in the production of lignin, callose and phytoalexins at the infection site.

Time-course of actinomycin D inhibition of tobacco mosaic virus multiplication relative to the rate of spread of the infection.

The time-course of inhibition of tobacco mosaic virus synthesis in mechanically inoculated leaves by actinomycin D was not correlated with cell-to-cell spread of the infection, as previously shown to be the case with 2-thiouracil and guanidine inhibition. The amount of inhibition by actinomycin D declined after inoculation much more rapidly than the infection spread. An actinomycin-D-sensitive step or its product occurred in cells before they became infected.

Characterization of RNA-dependent RNA polymerases in uninfected and cowpea chlorotic mottle virus-infected cowpea leaves: Selective removal of host RNA polymerase from membranes containing CCMV RNA replicase

Abstract Extracts from Cowpea chlorotic mottle virus (CCMV)-infected Cowpea leaves contained membrane-bound (31,000 g pellet) and soluble (31,000 g supernatant) RNA-dependent RNA polymerase activities. The membrane-bound RNA-dependent RNA polymerase (CCMV RNA replicase) increased 12-fold 4 days after inoculation. The viral RNA synthesis in vitro proceeded linearly for 20 min and required the four nucleoside triphosphates and Mg 2+ ions for activity. Manganese ion was a poor substitute for Mg 2+ . Optimal enzymatic activity in vitro was unaffected by exogenous RNA or KCI. The CCMV RNA replicase product was predominantly heterodisperse single-stranded RNAs, some of which comigrated with CCMV virion RNA. Small amounts of large double-stranded RNAs were also products of the replicase reaction. The soluble RNA-dependent RNA polymerase from CCMV-infected or healthy Cowpea leaves required the four nucleoside triphosphates and Mg 2+ ions for activity. Its activity in the in vitro assay was stimulated by adding exogenous RNAs but was inhibited by KCI. The product of the soluble RNA-dependent RNA polymerase was predominantly double-stranded RNA of approximately 4 to 6 S. RNA-dependent RNA polymerase activity, similar to that detected in the soluble fraction, was detected in the membrane pellet. This activity, which complicates the analysis of viral replicase assay, was removed without affecting CCMV RNA replicase activity by washing the 31,000 g pellets with buffer containing 0.5 M KCI. The KCI treatment aids in preparation of membrane-bound fractions devoid of host RNA-dependent RNA polymerase activity and high in viral replicase activity.