Peter Palukaitis

Virus movement in infected plants

Abstract The movement of plant viruses from cell to cell is controlled by the action of virus‐encoded proteins. With the realization that understanding the mechanism involved will lead to a broad comprehension of virus disease and may open new ways to engineer virus resistance, this phenomenon has recently received much attention. Sequence homologies between different movement proteins indicates that they may share a common origin and some functional similarities. However, groupings based upon protein structure, function as determined by complementation in double infections or host range, are not correlated. The proteins influence plasmodesmatal structure to allow the passage of viral genomes or virus particles, and one protein has been shown to have single‐stranded nucleic acid binding properties. Transgenic plants, heterologous protein‐expression systems, and cell biological approaches will determine how these proteins interact with host components to elicit complex structural changes which result in sy...

Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: generation of infectious RNA transcripts.

SummaryFull-length cDNA copies of cucumber mosaic virus (CMV) RNAs 1 and 2 of the Fny strain were constructed from partial cDNA clones and were cloned downstream of bacteriophage T7 promoters. In one pair of clones, transcription proceeded from an unaltered T7 promoter such that in vitro transcripts representing RNAs 1 and 2 contained an additional 17 nucleotides at their 5′ termini. In a second pair of clones, the T7 promoter/cDNA junction was altered by oligonucleotide-directed mutagenesis such that the in vitro transcripts contained only an additional G residue at their 5′ ends. In addition, a full-length cDNA copy of Fny-CMV RNA 3 was constructed from two overlapping cDNA clones and was cloned downstream of an altered T7 promoter such that the resultant in vitro transcripts also contained only an additional G residue at their 5′ ends. In vitro transcripts derived from all clones contained an additional C residue at their 3′ ends. In vitro transcripts representing RNAs 1, 2 and 3 which contained an additional residue at each terminus were shown to be infectious together in several hosts of CMV.

The Movement Protein of Cucumber Mosaic Virus Traffics into Sieve Elements in Minor Veins of Nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a–green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a–GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a–GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.

Replicase-Mediated Resistance to Plant Virus Disease

Publisher Summary This chapter discusses resistance induced by replicase and polymerase genes of RNA viruses—a phenomenon termed replicase-mediated resistance. Pathogen-mediated resistance is not a manifestation of classical cross-protection, although both may have common features in their mechanisms. Two characteristics of replicase-mediated resistance affect the potential utility of the technique for control of plant virus disease. Foremost among these is the near immunity induced in plants transformed with replicase genes. Replicase-mediated resistance is particularly useful for vegetatively propagated plants in which a delay in disease would be an unacceptable means of control. Trees and vines would fall into this category. As this concept is relatively new, its commercial application is just being developed. Foremost is the work with PLRV, in their effort several lines of Russet Burbank potatoes transformed with a full-length gene of ORF2b of PLRV showed a high degree of field immunity over several years and in several locations. Potential environment concerns include recombination among the transgene and other viruses, viral synergism, and seed transmissibility.

Virulence and Differential Local and Systemic Spread of Cucumber mosaic virus in Tobacco are Affected by the CMV 2b Protein

A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVdelta2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVdelta2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVdelta2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movement profile of Fny-CMVdelta2b results in the absence of disease symptoms in tobacco.

The plant defense response to cucumber mosaic virus in cowpea is elicited by the viral polymerase gene and affects virus accumulation in single cells.

Resistance to infection in cowpea by strains of cucumber mosaic virus (CMV) involves a local, hypersensitive response (HR) and a localization of infection. These responses can be separated by mutation at two sites (nucleotides 1978 and 2007, in codons 631 and 641) in the CMV 2a polymerase gene. Changes to both sites of a restricted strain allow systemic infection without an HR and increase the accumulation of both the 2a protein and viral RNA in protoplasts, while changing position 1978 alone results in a systemic infection, a systemic HR, and an increase in viral RNA accumulation in protoplasts. It is suggested that the inhibition response observed in protoplasts, where an HR does not occur, leads to localization of infection in whole plants and that different plant genes are involved in eliciting the HR and the localization response.

Characterization of Synergy Between Cucumber mosaic virus and Potyviruses in Cucurbit Hosts

ABSTRACT Mixed infections of cucurbits by Cucumber mosaic virus (CMV) and potyviruses exhibit a synergistic interaction. Zucchini squash and melon plants coinfected by the potyvirus Zucchini yellow mosaic virus (ZYMV) and either Fny-CMV (subgroup IA) or LS-CMV (subgroup II) displayed strong synergistic pathological responses, eventually progressing to vascular wilt and plant death. Accumulation of Fny- or LS-CMV RNAs in a mixed infection with ZYMV in zucchini squash was slightly higher than infection with CMV strains alone. There was an increase in CMV (+) strand RNA levels, but no increase in CMV (-) RNA3 levels during mixed infection with ZYMV. Moreover, only the level of capsid protein from LS-CMV increased in mixed infection. ZYMV accumulated to similar levels in singly and mixed infected zucchini squash and melon plants. Coinfection of squash with the potyvirus Watermelon mosaic virus (WMV) and CMV strains increased both the Fny-CMV RNA levels and the LS-CMV RNA levels. However, CMV (-) strand RNA3 levels were increased little or not at all for CMV on coinfection with WMV. Infection of CMV strains (LS and Fny) containing satellite RNAs (WL47-sat RNA and B5*-sat RNA) reduced the accumulation of the helper virus RNA, except when B5*-sat RNA was mixed with LS- CMV. However, mixed infection containing ZYMV and the CMV strains with satellites reversed the suppression effect of satellite RNAs on helper virus accumulation and increased satellite RNA accumulation. The synergistic interaction between CMV and potyviruses in cucurbits exhibited different features from that documented in tobacco, indicating there are differences in the mechanisms of potyvirus synergistic phenomena.

A single amino acid substitution in the coat protein of cucumber mosaic virus induces chlorosis in tobacco.

Some strains of cucumber mosaic virus (CMV) induce a bright yellow/white chlorosis in tobacco instead of the light green/dark green mosaic induced by most CMV strains. This property is controlled by RNA 3 of this tripartite virus. Recombination between cDNA clones of RNA 3 from a green mosaic strain, Fny-CMV, and a chlorotic strain, M-CMV, and inoculation of infectious transcripts of the chimeric RNAs 3, together with RNAs 1 and 2 of Fny-CMV, localized the chlorosis induction domain to a region of the coat protein gene containing two nucleotide differences. Site-directed mutagenesis of one nucleotide to change the codon for Leu129 in the M-CMV coat protein to Pro129 of Fny-CMV changed the phenotype from chlorotic to green mosaic, whereas the opposite change in phenotype was observed when the Pro129 in the Fny-CMV coat protein was altered to Ser129. Thus, the local secondary structure surrounding amino acid 129 rather than a particular amino acid per se is involved in chlorosis induction.

Characterization of cucumber mosaic virus. I. Molecular heterogeneity mapping of RNA 3 in eight CMV strains.

RNAs from 13 strains of cucumber mosaic virus (CMV) were divided into two groups on the basis of their ability to hybridize to cDNA of either Fny-CMV RNA or WL-CMV RNA. The extent of the cross-hybridization within one of these groups was analyzed by an RNA protection assay. A cDNA clone of RNA 3 of the Fny strain of CMV was placed in a transcription vector between bacterial promoters T3 and T7. Labeled, minus-sense RNA transcripts prepared from all or part of the cDNA to RNA 3 of Fny-CMV were annealed to the genomic RNA of each of a number of cucumoviruses and digested with RNases. The patterns of RNA fragments protected from digestion were specific for each CMV strain and revealed the extent and location of heterogeneity among the viruses as well as within the Fny-CMV natural population. This approach will allow the differences in host range and disease processes to be correlated with variations in genomic RNAs.

The role of the Cucumber mosaic virus 2b protein in viral movement and symptom induction.

The Cucumber mosaic virus (CMV) 2b protein is a counter-defense factor and symptom determinant. Conserved domains in the 2b protein sequence were mutated in the 2b gene of strain Fny-CMV. The effects of these mutations were assessed by infection of Nicotiana tabacum, N. benthamiana, and Arabidopsis thaliana (ecotype Col-0) with mutant viruses and by expression of mutant 2b transgenes in A. thaliana. We confirmed that two nuclear localization signals were required for symptom induction and found that the N-terminal domain was essential for symptom induction. The C-terminal domain and two serine residues within a putative phosphorylation domain modulated symptom severity. Further infection studies were conducted using Fny-CMVdelta2b, a mutant that cannot express the 2b protein and that induces no symptoms in N. tabacum, N. benthamiana, or A. thaliana ecotype Col-0. Surprisingly, in plants of A. thaliana ecotype C24, Fny-CMVdelta2b induced severe symptoms similar to those induced by the wild-type virus. However, C24 plants infected with the mutant virus recovered from disease while those infected with the wild-type virus did not. Expression of 2b transgenes from either Fny-CMV or from LS-CMV (a mild strain) in Col-0 plants enhanced systemic movement of Fny-CMVdelta2b and permitted symptom induction by Fny-CMVdelta2b. Taken together, the results indicate that the 2b protein itself is an important symptom determinant in certain hosts. However, they also suggest that the protein may somehow synergize symptom induction by other CMV-encoded factors.