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P Palukaitis

Publications and source records attributed to P Palukaitis.

At least 19 recordsLinked to original sources
PubMed2003

Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (-) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

Capsid Proteins↗
PubMed2001

Cucumber mosaic virus-plant interactions: identification of 3a protein sequences affecting infectivity, cell-to-cell movement, and long-distance movement.

Mutants of the Cucumber mosaic virus (CMV) movement protein (MP) were generated and analyzed for their effects on virus movement and pathogenicity in vivo. Similar to the wild-type MP, mutants M1, M2, and M3, promoted virus movement in eight plant species. Mutant M3 showed some differences in pathogenicity in one host species. Mutant M8 showed some host-specific alterations in movement in two hypersensitive hosts of CMV. Mutant M9 showed altered pathogenicity on three hosts and was temperature sensitive for long-distance movement, demonstrating that cell-to-cell and long-distance movement are distinct movement functions for CMV. Four mutants (M4, M5, M6, and M7) were debilitated from movement in all hosts tested. Mutants M4, M5, and M6 could be complemented in trans by the wild-type MP expressed transgenically, although not by each other or by mutant M9 (at the restrictive temperature). Mutant M7 showed an inability to be complemented in trans. From these mutants, different aspects of the CMV movement process could be defined and specific roles for particular sequence domains assigned. The broader implications of these functions are discussed.

Alanine↗
PubMed2001

A cucumber mosaic virus (CMV) RNA 1 transgene mediates suppression of the homologous viral RNA 1 constitutively and prevents CMV entry into the phloem.

Resistance to Cucumber mosaic virus (CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of Tobacco rattle virus vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism.

Cucumis sativus↗
PubMed2000

Characterisation of genetically modified cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins.

Biological active cDNA clones of cucumber mosaic virus (CMV) RNAs 1 and 2 were modified by addition of sequences that encode hexahistidine (His-tag) at the amino- (N-) or carboxy- (C-) terminus of the 1a and 2a proteins. These proteins are essential components of the viral RNA-dependent RNA polymerase (RdRp). In all but one case, addition of the His-tag did not significantly affect the yields of the corresponding viruses and the His-tag-encoding sequences were maintained after mechanical passages. No differences were observed among the in vitro activities of the modified vs. wild-type viral RdRps. Subcellular fractionation showed that 2a protein was found both membrane-associated and in the 30,000 x g soluble fraction. Both termini of the native His-tag 2a protein could bind to a resin containing nickel-nitrilotriacetic acid (Ni(2+)-NTA). Detergent-treated RdRp containing C-terminal His-tagged 1a and 2a proteins was chromatographed on Ni(2+)-NTA resin. The activity of the eluted RdRp was template- dependent, in contrast to pre-chromatography fractions. However, only a small proportion of the viral RdRp as well as numerous host proteins bound to and eluted from the resin under non-denaturing conditions.

Cell Membrane↗
PubMed2000

Subcellular distribution analysis of the cucumber mosaic virus 2b protein.

The cucumoviral 2b protein is a viral counterdefence factor that interferes with the establishment of virus-induced gene silencing in plants. Synthetic peptides were used to generate an antibody to the 2b protein encoded by the Fny strain of cucumber mosaic virus (Fny-CMV). This polyclonal antibody was able to recognize the Fny-CMV 2b protein in a 10000 g pellet fraction of infected tobacco. No protein of equivalent size was detected in mock-inoculated or tobacco mosaic virus-infected samples. This represents the first demonstration of 2b protein expression by a subgroup I strain of CMV. Subcellular fractionation experiments on CMV-infected tobacco leaf tissue showed that the Fny-CMV 2b protein accumulated within a fraction that sedimented at forces of less than 5000 g and that the 2b protein was solubilized only by treatment with urea or SDS. These results suggested that the 2b protein associates either with the nucleus or cytoskeleton of the host cell. Further analysis showed that the 2b protein was enriched in a fraction that sedimented through a 2.2 M sucrose cushion. This fraction was also enriched in histones, suggesting that the CMV 2b protein associates preferentially with the host cell nucleus.

Amino Acid Sequence↗
PubMed1999

The hypersensitive response to cucumber mosaic virus in Chenopodium amaranticolor requires virus movement outside the initially infected cell.

Cucumber mosaic virus (CMV) expressing the green fluorescent protein (GFP), and lacking either the 3a movement protein or the coat protein (CP), failed to induce a hypersensitive response producing local lesions in inoculated leaves of Chenopodium amaranticolor. Cytological analysis showed that both viral-encoded proteins are required for cell-to-cell movement of the virus and the simultaneous appearance of cellular necrosis. In the absence of either or both proteins, infection was confined to single, non-necrotized, epidermal cells. CMV with a mutation in the 3a protein (M8 CMV) could infect tobacco systemically but did not induce necrotic lesions in C. amaranticolor. In this host, the mutated 3a protein was unable to promote viral movement out of the initially infected epidermal cell. Movement-deficient CMV expressing wild-type (WT) 3a protein as a fusion to the GFP, as well as WT CP, also failed to induce necrosis. Finally, single epidermal cells infected with a movement-deficient CMV expressing WT 3a protein, WT CP, and free GFP did not show necrosis. These data indicate that viral movement out of the initially infected epidermal cell, and not the simultaneous expression in this cell of the 3a protein and the CP, is required for the induction of cell death.

Blotting, Western↗
PubMed1999

Host-specific cell-to-cell and long-distance movements of cucumber mosaic virus are facilitated by the movement protein of groundnut rosette virus.

The cucumovirus, cucumber mosaic virus (CMV), requires both the 3a movement protein (MP) and the capsid protein (CP) for cell-to-cell movement. Replacement of the MP of CMV with the MP of the umbravirus, groundnut rosette virus (GRV), which does not encode a CP, resulted in a hybrid virus, CMV(ORF4), which could move cell to cell in Nicotiana tabacum and long distance in N. benthamiana. After replacement of the CMV CP in CMV(ORF4) with the gene encoding the green fluorescent protein (GFP), the hybrid virus, CMV(ORF4.GFP), expressing both the GRV MP and the GFP, could move cell to cell but not systemically in either Nicotiana species. Immunoelectron microscopic analysis of cells infected by the hybrid viruses showed different cellular barriers in the vasculature preventing long-distance movement of CMV(ORF4) in N. tabacum and CMV(ORF4.GFP) in N. benthamiana. Thus the GRV MP, which shows limited sequence similarity to the CMV MP, was able to support CP-independent cell-to-cell movement of the hybrid virus, but CP was still required for long-distance movement and entry of particular vascular cells required functions encoded by different proteins.

Capsid↗
PubMed1998

Characterization of cucumber mosaic virus. VI. Generation of deletions in defective RNA 3s during passage in transgenic tobacco expressing the 3a gene.

Defective mutants of cucumber mosaic virus (CMV) RNA 3, containing deletions in the 3a gene were passaged in transgenic tobacco plants expressing the CMV 3a gene. After six passages, the various progeny RNA 3 were characterized. In all but one case, the size of the deletion increased. For the NheI-fs RNA 3 mutant of the Fny-strain of CMV (with an 8 nucleotide deletion), the deletion increased in the progeny viral RNA 3 to 570 nucleotides. For a similar frameshift mutant in RNA 3 of the M strain of CMV, either single RNA 3 species (with deletions of 579 or 627 nucleotides) or mixtures of RNA 3 deletion variants were observed in different plants. The DeltaE-H mutant (with a deletion of 202 nucleotides) of Fny-CMV RNA 3 underwent further deletion resulting in the loss of the entire 3a gene and flanking sequences. The DeltaKpnI mutant (deletion of 501 nucleotides) of Fny-CMV RNA 3 underwent a further deletion of 30 nucleotides. Except for the deletion progeny of the DeltaE-H RNA 3 mutant, the other defective RNA 3s all contained inframe deletions. It is proposed that the various deletions were created by different types of recombination and that packaging may be an important factor in the selection of particular defective RNA 3 variants.

Base Sequence↗
PubMed1998

Transgenically expressed cucumber mosaic virus RNA 1 simultaneously complements replication of cucumber mosaic virus RNAs 2 and 3 and confers resistance to systemic infection.

Tobacco plants transformed with a cDNA copy of RNA 1 of the Fny strain of cucumber mosaic virus (CMV) promoted the asymptomatic accumulation of inoculated viral RNAs 2 and 3, which could be detected in noninoculated leaves, suggesting that the transgene also permitted viral long-distance movement. Typical symptoms of infection appeared later and correlated with the appearance of viral RNA 1 regenerated from the transgenic mRNA. Although all R0-generation plants were susceptible to Fny-CMV, one line displaying variable susceptibility to the virus in R1-and R2-generations led to selected R3-generation lines with systemic resistance to Fny-CMV. In the inoculated leaves of resistant plants, a dramatic decrease in the accumulation of viral RNA 1 was observed, relative to susceptible plants. No viral RNAs were detected in noninoculated leaves of the resistant plants, but such leaves were susceptible to infection. Furthermore, these leaves could sustain replication of inoculated CMV RNAs 2 and 3, indicating that a complete transgene-silencing had not been induced. Although a transgene-mediated, CMV RNA 1-suppression occurred in the inoculated leaf of resistant plants, the absence of a complete systemically acquired silencing suggests the existence of additional interferences with viral infection that prevented systemic infection by viral RNAs 2 and 3.

Cucumovirus↗
PubMed1998

Characterization of cucumber mosaic virus. V. Cell-to-cell movement requires capsid protein but not virions.

To ascertain the importance of amino-terminal proximal capsid protein (CP) sequences in cel-to-cell movement, virion formation, and stabilization, two CP mutants of cucumber mosaic virus (CMV) were generated by deletion of sequences encoding CP amino acids 15-40 (delta Sal-Nru) or 26-40 (delta Sac-Nru). Wildtype CMV and CMV containing delta Sac-Nru could infect systemically four host species, although symptoms induced by the two viruses usually were different CMV containing delta Sal-Nru could only infect Nicotiana benthamiana and N. clevelandii systemically, but only slowly, suggesting phloem-independent long-distance movement. A variant mutant designated delta Sal-Nru* could systemically infect N. tabacum as well as the above two Nicotiana species, rapidly, but could not systemically infect Cucurbita pepo. Virus particles could not be detected in plants infected by delta Sal-Nru, while delta Sal-Nru* and delta Sac-Nru formed particles of lower stabilities than for wildtype virus. The CPs of delta Sal-Nru and delta Sal-Nru* could bind RNA in vitro, although less strongly than delta Sac-Nru or wildtype CMV. These data indicate that amino-terminal proximal sequences of the CMV CP interact with viral RNA and are required for the formation of stable virions. Moreover, while the CP is necessary for cell-to-cell movement, the ability to form virions is not a prerequisite for cell-to-cell movement.

Amino Acid Sequence↗
PubMed1998

Amino acid changes in the coat protein of cucumber mosaic virus differentially affect transmission by the aphids Myzus persicae and Aphis gossypii.

Cucumber mosaic virus (CMV) transmission by two aphid species was investigated using strains of the virus with modified coat proteins. Chimeric viral RNA 3 cDNA constructs were designed with coat protein-encoding sequences from the efficiently aphid-transmitted Fny-CMV and the very poorly aphid-transmitted M-CMV. Different regions of the viral coat protein variably influenced transmission; the magnitude of effects depended on the aphid species used as vector. Five amino acid changes in the coat protein of M-CMV (positions 25, 129, 162, 168, and 214) were required to restore efficient transmission by Myzus persicae. This contrasts with previously demonstrated, more limited changes required for restoration of transmission by Aphis gossypii. A construct with modified amino acids 129, 162, and 168 was efficiently transmitted by A. gossypii, while remaining poorly transmissible by M. persicae. Transmission of Fny-CMV by single aphids of A. gossypii and M. persicae was 54.0% and 27.9%, respectively. Single coat protein amino acid changes in Fny-CMV dramatically reduced transmission by M. persicae, while showing little effect on transmission by A. gossypii.

Animals↗
PubMed1998

Diversity among isolates of squash mosaic virus.

cDNA clones of RNA-2 of two isolates of squash mosaic virus (SqMV) were constructed and sequenced, revealing 87% sequence similarity. In Northern blot hybridization analyses, DNA probes made from these clones defined two SqMV hybridization subgroups. This grouping was verified by reciprocal hybridizations of purified RNA from five SqMV isolates, as probed with cDNA made from a member of each subgroup. Comparison of the RNA-2 sequence among the two SqMV isolates, and the reported sequence of other comoviruses, showed that SqMV constitutes one of four major branches in a phylogenetic tree of the genus. Analysis of the terminal noncoding sequences showed that although potentially similar folding patterns may form, neither nucleotide sequence nor secondary structural elements are highly conserved among comoviruses. In vitro translation products from purified RNA-1 of each subgroup (encoding the viral proteases) were found to process the polyprotein generated by in vitro translation of purified RNA-2 from either subgroup.

Amino Acid Sequence↗
PubMed1998

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.

Journal Article↗
PubMed1997

Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus.

cDNA clones of cucumber mosaic virus (CMV) RNA 3 were modified to express the jellyfish green fluorescent protein (GFP) in place of the 3a movement protein (MP) or coat protein (CP), as fusions to the N (GFP-3a) or C (3a-GFP) terminus of the MP or from a separate open reading frame as part of tricistronic RNAs 3. CMV RNA transcripts containing the individual modified RNAs 3 were unable to infect either Nicotiana tabacum or Nicotiana benthamiana systemically. Infection, as measured by confocal microscopy of GFP fluorescence, generally was limited to one to three epidermal cells at each inoculation site. Limited cell-to-cell movement, but not systemic movement, could be detected by complementation involving expression of MP and CP from two different RNA 3 constructs, each also expressing GFP. Infection involving RNA 3 expressing the GFP-3a fusion showed bright granules of variable size distributed predominantly and nonuniformly throughout the cytoplasm and, to a lesser extent, associated with the cell wall in single fluorescent cells, while infections expressing the 3a-GFP fusion showed bright, punctate fluorescence associated only with the cell wall. Infected cells expressing either 3a-GFP or free GFP showed a halo of less bright, fluorescent, neighboring cells, indicating limited movement of GFP. The initially infected cells also allowed movement of 10-kDa fluorescent dextran to the neighboring halo cells, while infection did not spread, suggesting different requirements for movement of either MP or dextran versus RNA.

Capsid↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

Characterization of cucumber mosaic virus. III. Localization of sequences in the movement protein controlling systemic infection in cucurbits.

Cucumber mosaic virus (CMV), generated from biologically active cDNA clones of Fny-CMV RNA 1 plus 2 and Sny-CMV RNA 3, derived from the Fny- and Sny-strains of CMV, was able to infect tobacco but not squash plants systemically. In squash, viral RNA, movement protein, and coat protein all accumulated in the inoculated cotyledons. The lack of systemic infection was associated with a reduced rate of cell-to-cell movement within the cotyledons. The restricted movement mapped to two sequence changes in the codons of amino acids 51 and 240 of the Sny-CMV 3a gene. These same sequence changes previously were shown to be associated with high levels of 3a protein accumulation and chronic vs acute, cyclic infection typical of Sny-CMV vs Fny-CMV [Gal-on et al. (1996). Virology 226, 354-361]. Fny-CMV, mutated in the codons of 3a gene amino acids 51 and 240, was still able to infect several solanaceous hosts (tobacco, tomato, and pepper) systemically, but did not elicit a typical CMV systemic infection on any of several cucurbit hosts (cucumber, melon, or squash). The significance of the location of amino acid positions 51 and 240 in the 3a movement protein is discussed.

Cucumis sativus↗