Papers by Peter Palukaitis
Phytopathology, 2002
ABSTRACT Mixed infections of cucurbits by Cucumber mosaic virus (CMV) and potyviruses exhibit a s... more 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.
The plant pathology journal, 2013
The incidence and distribution of Tobacco mosaic virus (TMV) and related tobamoviruses was determ... more The incidence and distribution of Tobacco mosaic virus (TMV) and related tobamoviruses was determined using an enzyme-linked immunosorbent assay on 1,926 symptomatic horticultural crops and 107 asymptomatic weed samples collected from 78 highly infected fields in the major horticultural crop-producing areas in 17 provinces throughout Iran. The results were confirmed by host range studies and reverse transcription-polymerase chain reaction. The overall incidence of infection by these viruses in symptomatic plants was 11.3%. The coat protein (CP) gene sequences of a number of isolates were determined and disclosed to be a high identity (up to 100%) among the Iranian isolates. Phylogenetic analysis of all known TMV CP genes showed three clades on the basis of nucleotide sequences with all Iranian isolates distinctly clustered in clade II. Analysis using the complete CP amino acid sequence showed one clade with two subgroups, IA and IB, with Iranian isolates in both subgroups. The nucle...
Mobile genetic elements, 2014
Functional genomics in plants has been facilitated greatly by the use of plant viruses to carry s... more Functional genomics in plants has been facilitated greatly by the use of plant viruses to carry segments of host genes that can then promote the silencing of the RNAs expressed from the corresponding host genes; a process called virus-induced gene silencing (VIGS). The silencing of genes in filamentous fungi is either technically more problematic or labor-intensive, especially if transgenic plants need to be generated first. However, a recent paper from our team demonstrated that a plant virus could infect three related fungal species, as well as express a reporter gene ectopically, and also silence the correspondingly expressed reporter transgene. The gene expression and RNA silencing of the reporter gene was maintained for six passages in culture and also persisted in plants infected by the virus-infected fungus. Here, we consider how the virus can enter and migrate within the fungus, whether the virus can move back and forth between the fungus and the plant and the ramifications ...
Advances in virus research, 2003
Research on the molecular biology of cucumoviruses and their plant-virus interactions has been ve... more Research on the molecular biology of cucumoviruses and their plant-virus interactions has been very extensive in the last decade. Cucumovirus genome structures have been analyzed, giving new insights into their genetic variability, evolution, and taxonomy. A new viral gene has been discovered, and its role in promoting virus infection has been delineated. The localization and various functions of each viral-encoded gene product have been established. The particle structures of Cucumber mosaic virus (CMV) and Tomato aspermy virus have been determined. Pathogenicity domains have been mapped, and barriers to virus infection have been localized. The movement pathways of the viruses in some hosts have been discerned, and viral mutants affecting the movement processes have been identified. Host responses to viral infection have been characterized, both temporally and spatially. Progress has been made in determining the mechanisms of replication, gene expression, and transmission of CMV. T...
Methods in molecular biology (Clifton, N.J.), 2008
A variety of techniques have been used to examine plant viral genomes, the functions of virus-enc... more A variety of techniques have been used to examine plant viral genomes, the functions of virus-encoded proteins, plant responses induced by virus infection and plant-virus interactions. This overview considers these technologies and how they have been used to identify novel viral and plant proteins or genes involved in disease and resistance responses, as well as defense signaling. These approaches include analysis of spatial and temporal responses by plants to infection, and techniques that allow the expression of viral genes transiently or transgenically in planta, the expression of plant and foreign genes from virus vectors, the silencing of plants genes, imaging of live, infected cells, and the detection of interactions between viral proteins and plant gene products, both in planta and in various in vitro or in vivo systems. These methods and some of the discoveries made using these approaches are discussed.
Advances in Virus Research, 2003
Research on the molecular biology of cucumoviruses and their plant-virus interactions has been ve... more Research on the molecular biology of cucumoviruses and their plant-virus interactions has been very extensive in the last decade. Cucumovirus genome structures have been analyzed, giving new insights into their genetic variability, evolution, and taxonomy. A new viral gene has been discovered, and its role in promoting virus infection has been delineated. The localization and various functions of each viral-encoded gene product have been established. The particle structures of Cucumber mosaic virus (CMV) and Tomato aspermy virus have been determined. Pathogenicity domains have been mapped, and barriers to virus infection have been localized. The movement pathways of the viruses in some hosts have been discerned, and viral mutants affecting the movement processes have been identified. Host responses to viral infection have been characterized, both temporally and spatially. Progress has been made in determining the mechanisms of replication, gene expression, and transmission of CMV. The pathogenicity determinants of various satellite RNAs have been characterized, and the importance of secondary structure in satellite RNA-mediated interactions has been recognized. Novel plant genes specifying resistance to infection by CMV have been identified. In some cases, these genes have been mapped, and one resistance gene to CMV has been isolated and characterized. Pathogen-derived resistance has been demonstrated against CMV using various segments of the CMV genome, and the mechanisms of some of these forms of resistances have been analyzed. Finally, the nature of synergistic interactions between CMV and other viruses has been characterized. This review highlights these various achievements in the context of the previous work on the biology of cucumoviruses and their interactions with plants.
Virology, 1988
RNAs from 13 strains of cucumber mosaic virus (CMV) were divided into two groups on the basis of ... more 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.
Virus Genes, 2000
Tomato aspermy virus RNAs derived from infectious cDNA clones exhibited a number of sequence alte... more Tomato aspermy virus RNAs derived from infectious cDNA clones exhibited a number of sequence alterations in the 5' non-translated region (NTR). These included a deletion of the first four residues in both RNAs 1 and 2, transversion of residue 5 from a G to a U in RNA 1, and transversion of A to C at position of 50 of RNA 1. These alterations were not stable in the infected plants while the insertion of a U residue between nucleotides 1 and 5 of RNA 1 was stable in the infected plants. Generation of these sequence alternations was not dependent upon either the host species or the concentration of the inoculum. The sequence alterations also did not occur on passage of wildtype virus. Rather, the sequence alterations related to transcription from the cauliflower mosaic virus 35S RNA promoter-driving infectious cDNAs. The alternations observed had no impact on symptoms or infectivity, but did affect the accumulation of specific viral RNAs. The data also demonstrated the existence of some plasticity in the sequence of the 5' NTR.
Virology, 1984
Two new satellite RNAs of cucumber mosaic virus (CMV) which did not induce necrosis on tomato in ... more Two new satellite RNAs of cucumber mosaic virus (CMV) which did not induce necrosis on tomato in the presence of CMV, B-sat RNA, and WL-sat RNA, were shown to be related by sequence to two well-characterized satellite RNAs of CMV: G-sat RNA (non-necrotic on tomato) and n-CARNA 5 (necrotic on tomato). Using the techniques of molecular hybridization analysis, RNA fingerprinting and partial RNA sequencing, B-sat RNA and WL-sat RNA were shown to be more closely related to each other (probably differing by only a small number of nucleotides) than to the other two satellite RNAs. Furthermore, B-sat RNA and WL-sat RNA showed greater sequence homology with G-sat RNA than with n-CARNA 5. WL-sat RNA, which induces a "white-leaf" disease on tomato in the presence of CMV [Gonsalves et al. (1982)., exhibited heterogeneity of sequence in at least one nucleotide position.
Virology, 1979
A viroid has been purified from avocado leaves infected by sunblotch disease and designated the a... more A viroid has been purified from avocado leaves infected by sunblotch disease and designated the avocado sunblotch viroid. It is a covalently closed circular RNA molecule with a molecular weight lower than that of chrysanthemum stunt viroid and citrus exocortis viroid while hybridization analysis with 32P-labeled complementary DNA indicated that it is a single RNA species. It could be detected as a stainable RNA band on polyacrylamide tube gel electrophoresis of partially purified extracts of only two of four avocado isolates with positive symptoms of sunblotch disease. However, the viroid was detected in all four isolates by hybridization analysis with 32P-complementary DNA; this procedure has potential use for the rapid indexing of sunblotch disease since the viroid was not present in an isolate of healthy avocado. It has yet to be shown that the viroid is the causative agent of sunblotch disease.
The Plant Journal, 2007
The cucumber mosaic virus (CMV) 2b protein suppresses RNA silencing and determines viral symptoms... more The cucumber mosaic virus (CMV) 2b protein suppresses RNA silencing and determines viral symptoms. Among Arabidopsis thaliana lines expressing 2b proteins from mild (LS and Q CMV) or severe (Fny CMV) strains, only Fny 2b-transgenic plants displayed strong symptom-like phenotypes in leaves, stems and flowers, together with stunting of main root growth and increased emergence of lateral roots. However, LS and Fny 2b proteins both enhanced lateral root length. Micro (mi)RNA-mediated cellular mRNA turnover was inhibited in Fny 2b-transgenic plants, but there was no evidence for this in LS 2b-transgenic plants. Both 2b proteins efficiently suppressed small interfering (si)RNA-mediated RNA silencing, suggesting that 2b proteins can target the siRNA pathway without disrupting miRNA-regulated RNA turnover. Thus, symptom induction is not an inevitable consequence of RNA silencing suppression. For CMV, strain-specific differences between the 2b silencing proteins determine whether only one or both small RNA-guided RNA destruction pathways are disrupted.
Molecular Plant-Microbe Interactions, 2002
The approximately 12-kDa 2b protein, encoded by all cucumoviruses, had been shown to play an impo... more The approximately 12-kDa 2b protein, encoded by all cucumoviruses, had been shown to play an important role in viral long-distance movement, hypervirulence, and suppression of post-transcriptional gene silencing. The role of the 2b gene in the hypervirulence of Cucumber mosaic virus (CMV) and whether hypervirulence was linked to movement were analyzed using a hybrid virus (CMV-qw), generated by replacing the 2b gene in a subgroup II strain, Q-CMV, with the 2b gene from a subgroup IA strain, WAII-CMV. CMV-qw was more virulent than Q-CMV or WAII-CMV on most of the host plant species tested. Northern blot and nucleotide sequence analyses demonstrated that CMV-qw was stably maintained during the course of infection and upon passage. Kinetic studies revealed that the hypervirulence induced by the hybrid virus was associated with neither increased viral RNA accumulation nor more rapid viral movement per se, suggesting that other functions of the 2b protein are important in determining the hypervirulence.
MGG Molecular & General Genetics, 1990
The organization of the intergenic spacer of a 9.04 kb tomato ribosomal RNA gene (rDNA) was deter... more The organization of the intergenic spacer of a 9.04 kb tomato ribosomal RNA gene (rDNA) was determined. The 3258 bp spacer contains two major repeat elements enclosing a region which includes 351 bp of an 81.8% A -T rich sequence. A block of nine 53 bp repeats begins 388 bp downstream from the 3' end of the 25S rRNA. The A -T rich domain is followed by a block of six 141 bp repeats terminating 818 bp upstream from the 5 r end of the 18S rRNA. Major pre-rRNAs of 7.6 and 6.5 kb were observed by Northern hybridization analysis. The 5' termini of these RNAs were identified through combined S1 nuclease and primer extension analyses. The 7.6 kb RNA is likely to be the primary transcript; its 5' terminus lies within a sequence motif, TATA(R)TA(N)GGG, conserved at the termini of transcripts mapped in three other plant species. The 6.5 kb RNA is interpreted as a 5' end processed transcript derived from the 7.6 kb RNA. Comparative analysis of transcribed sequences revealed a 25 bp domain of the intergenic spacer which is relatively conserved among five plant species. The conservation of spacer sequences in plants is in contrast to the extensive sequence divergence of the intergenic spacer in other non-plant systems and suggests a conserved function directed by these sequences.
Molecular plant-microbe interactions : MPMI, 2010
The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been v... more The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO prot...
Journal of Molecular Biology, 2004
RNA-protein interactions are fundamental for different aspects of molecular biology such as gene ... more RNA-protein interactions are fundamental for different aspects of molecular biology such as gene expression, assembly of biomolecular complexes or macromolecular transport. The 3a movement protein (MP) of a plant virus, Cucumber mosaic virus (CMV), forms ribonucleoprotein (RNP) complexes with viral RNA, capable of trafficking from cell-to-cell throughout the infected plant only in the presence of the CMV capsid protein (CP). However, deletion of the C-terminal 33 amino acid residues of the CMV MP (in the mutant designated 3aDeltaC33 MP) resulted in CP-independent cell-to-cell movement. The biological differences in the behaviour of CMV wild type (wt) 3a MP and 3aDeltaC33 MP could have been a consequence of differences in the RNA-binding properties of the two MPs detected previously using biochemical assays on ensembles of molecules. To investigate the physical mechanisms of MP-RNA interactions at a single molecule level, we applied atomic force microscopy to measure for the first time unbinding forces between these individual binding partners. Minimal unbinding forces determined for individual interaction of the CMV RNA molecule with the CMV wt or truncated MPs were estimated to be approximately 45 pN and approximately 90 pN, respectively, suggesting that the distinct differences in the strength of MP-RNA interactions for the wt MP and truncated MP are attributable to the molecular binding mechanism. We also demonstrated that molecules of both CMV 3a MP and 3aDeltaC33 MP were capable of self-interaction with minimal unbinding forces of approximately 50 pN and approximately 70 pN, respectively, providing a physical basis for the cooperative mechanism of the RNA binding. The significance of intermolecular force measurements for understanding the structural and functional aspects of viral RNP formation and trafficking is discussed.
Journal of General Virology, 2004
The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor... more The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber. In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species. Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV. Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function. Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species. Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization. The 2b protein interacted with an Arabidopsis thaliana karyopherin a protein (AtKAPa) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants. However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPa. These data suggest that the 2b protein localizes to the nucleus by using the karyopherin a-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts. Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement. 3Present address:
Journal of General Virology, 1989
The nucleotide sequence of RNA 1 of the Fny strain (Subgroup I) of cucumber mosaic virus (CMV) wa... more The nucleotide sequence of RNA 1 of the Fny strain (Subgroup I) of cucumber mosaic virus (CMV) was determined and compared at both the nucleic acid and protein levels with the corresponding sequence of RNA 1 of the Q strain (Subgroup II) of CMV. Fny-CMV RNA 1 consisted of 3357 nucleotides and contained a single long open reading frame (ORF) of 2979 nucleotides, whereas Q-CMV RNA 1 consists of 3389 nucleotides and contains a single ORF of 2973 nucleotides. The levels of sequence homology between the two RNAs were 76~ at the nucleotide level and 85~o at the protein level. These homologies were distributed widely over the molecules, with 45 of the non-conservative differences in amino acid sequence located between amino acids 503 and 705, and another 15 ~ of the differences located between amino acids 224 and 298. While the C-terminal 141 amino acids contain more basic than acidic amino acids, the region of greatest amino acid sequence heterogeneity, amino acids 503 to 600, contained a preponderance of acidic amino acids in the putative translation products of RNAs 1 of both Q-CMV and Fny-CMV. The last 180 nucleotides of the 3'-terminal non-coding region of Fny-CMV RNAs 1 and 2 were 96~ homologous, whereas the sequence homology between Fny-CMV RNA 1 and Q-CMV RNA 1 was 64~ in this region. Furthermore, the tRNA-like secondary structures formed by the 3'-terminal non-coding regions of Fny-CMV RNAs 1 and 2 were virtually identical. By contrast, there was only 84~ sequence homology between the 5"-terminal non-coding regions of these two RNAs and 81~ sequence homology between the 5'-terminal non-coding regions of Q-CMV RNA 1 and Fny-CMV RNA 1. The non-equivalent divergence in the non-coding regions of these RNAs, as well as possible functions for the translation product of RNA 1, are discussed.
Journal of General Virology, 2004
Defective (D) RNAs were generated in tobacco upon passage of two isolates of Cucumber mosaic viru... more Defective (D) RNAs were generated in tobacco upon passage of two isolates of Cucumber mosaic virus (CMV) initially derived from RNA transcripts of cDNA clones. In both cases, the D RNA was derived by a single in-frame deletion of either 339 or 411 nt within the 3a gene of Fny-CMV RNA 3 or M-CMV RNA 3, respectively. The generation of D RNAs was rare and occurred with two CMV isolates, the virions of which were known to differ in physico-chemical properties. The Fny-CMV D RNA 3, designated D RNA 3-1, was maintained by passage together with Fny-CMV in tobacco, but was lost by passage in squash. D RNA 3-1 accumulated in the inoculated squash cotyledons but not in upper, systemically infected leaves. Virions purified from infected squash cotyledons or leaf mesophyll protoplasts did not contain D RNA 3-1. Therefore, the failure of D RNA 3-1 to accumulate in squash leaves systemically infected by CMV was due to a lack of encapsidation of the D RNA 3-1 and movement out of the inoculated leaves.
Journal of General Virology, 1980
A new and rapid extraction procedure is described for the isolation and partial purification of l... more A new and rapid extraction procedure is described for the isolation and partial purification of low mol. wt. RNA as well as DNA from 5oo g batches of chrysanthemum stunt viroid-infected chrysanthemum. The chrysanthemum stunt viroid was then purified to homogeneity from this extract by one cycle of non-denaturing polyacrylamide slab gel electrophoresis followed by one cycle of denaturing slab gel electrophoresis. The covalently closed circular form and the linear form of the viroid co-migrated on the first gel but were well separated on the second. The yield of circular chrysanthemum stunt viroid was 200/,g/kg of infected chrysanthemum shoots and of the linear form was 35/*g/kg.
Uploads
Papers by Peter Palukaitis