Rene van Wezel - Academia.edu (original) (raw)

Papers by Rene van Wezel

Research paper thumbnail of Reinvestment Alert

This article cites 46 articles, 17 of which can be accessed free

Research paper thumbnail of Contribution of the Zinc Finger to Zinc and DNA Binding by a Suppressor of Posttranscriptional Gene Silencing

Journal of Virology, 2003

The zinc finger C 36 -X1-C 38 -X7-C 46 -X6-H 53 of the nuclearly localized C2 protein of Tomato y... more The zinc finger C 36 -X1-C 38 -X7-C 46 -X6-H 53 of the nuclearly localized C2 protein of Tomato yellow leaf curl virus China is involved in pathogenicity and suppression of posttranscriptional gene silencing (PTGS). Here, we demonstrate that the zinc finger is indispensable for the C2 protein to bind zinc and DNA. Mutation of cysteine residue C 36 , C 38 , or C 46 reduced the zinc and DNA binding capacity of C2 protein. When expressed from potato virus X, all three mutants, C2-C 36 R, C2-C 38 N, and C2-C 46 I, tagged with a green fluorescent protein (GFP) were still capable of transporting GFP into but aggregated abnormally in nuclei. Our data establish that zinc- and DNA-binding activity correlates with C2-mediated pathogenesis and PTGS suppression.

Research paper thumbnail of Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Planta, 1999

Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of s... more Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the¯avonol-de®cient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of¯avonols in this process, wild-type and¯avonol-de®cient pollen tubes were subjected to cytological and ultrastructural analyses and screened for dierences. The results showed that before disruption of the¯avonol-de®cient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural dierences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any signi®cant dierence. However, for the ®rst time, obvious morphological dierences were observed in the wall of the¯avonol-de®cient pollen tubes. We conclude that¯avonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins.

Research paper thumbnail of Fine structure of silicoflagellate double skeletons

Marine Micropaleontology, 2014

Research paper thumbnail of Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Planta, 1999

Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of s... more Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the¯avonol-de®cient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of¯avonols in this process, wild-type and¯avonol-de®cient pollen tubes were subjected to cytological and ultrastructural analyses and screened for dierences. The results showed that before disruption of the¯avonol-de®cient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural dierences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any signi®cant dierence. However, for the ®rst time, obvious morphological dierences were observed in the wall of the¯avonol-de®cient pollen tubes. We conclude that¯avonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins.

Research paper thumbnail of Suppression of local RNA silencing is not sufficient to promote cell-to-cell movement of Turnip crinkle virus in Nicotiana benthamiana

Plant Signaling & Behavior, 2009

The biological relationship between suppression of RNA silencing and virus movement poses an intr... more The biological relationship between suppression of RNA silencing and virus movement poses an intriguing question in virus-plant interactions. Here, we have used a local RNA silencing assay, based on a movement-deficient Turnip crinkle virus TCV/ GFPΔCP, to investigate the influence of silencing suppression by three different viral suppressors: the TCV 38K coat protein (CP), the 126K protein of Tobacco mosaic virus (TMV), and P19 of Tomato bushy stunt virus (TBSV) on cell-to-cell movement and long-distance spread of TCV/GFPΔCP. First, we found that TCV CP blocked the induction of local RNA silencing, but failed to support virus trafficking in silencing-suppressed transgenic plants, although it acted as a functional movement protein in nontransformed plants. Second, we demonstrated that the TMV 126K suppressor inhibited TCV/GFPΔCP-mediated RNA silencing, but did not facilitate intercellular spread of the chimaeric carmovirus. However, TMV and TMVΔCP prevented the initiation of RNA silencing by TCV/GFPΔCP and caused TCV/GFPΔCP to move between cells, although only TMV supported its long-distance spread. Third, TBSV P19 functioned as a movement protein for TCV/GFPΔCP and as a silencing suppressor in non-transformed and silencing-suppressed transgenic plants. We further identified three types of mutant P19 proteins that possessed no or varied functionality in silencing suppression and in the facilitation of carmovirus movement. These results suggest that, although suppression of local RNA silencing is essential for the maintenance of viral RNA, recovery of cell-to-cell movement and long-distance spread of movement-deficient carmoviruses is not a direct consequence of such silencing suppression.

Research paper thumbnail of Protoperidinium tricingulatum sp. nov. (Dinophyceae), a new motile form of a round, brown, and spiny dinoflagellate cyst

Phycological Research, 2000

A small, broadly ovoidal and heterotrophic dinoflagellate containing round, brownish, and spiny c... more A small, broadly ovoidal and heterotrophic dinoflagellate containing round, brownish, and spiny cyst was found in the water column of Huibertsplaat in the Wadden Sea off the coast of the Netherlands. This dinoflagellate had these conspicuous morphological characters: a five-sided first apical plate (1′), only three cingular plates, and an extremely small first antapical plate. Based on these morphological features, Protoperidinium tricingulatum Kawami, vanWezel, Koeman et Matsuoka is described as a new species. The flagellar pore of P. tricingulatum is covered with a small fin, which rises from the left side of the right sulcal plate to the large V-shaped posterior sulcal plate. This feature suggests that P. tricingulatum is assigned to the Abé's Monovela Group. The cyst stage of P. tricingulatum was positively linked to the vegetative stage by comparison of the ribosomal 5.8S rDNA, internal transcribed spacers (ITS1 and ITS2). Living cysts of P. tricingulatum are round, brownish, and covered with many slender spines bearing capitate or cauliforate distal ends. The cyst also possesses a theropylic archeopyle formed by a slit corresponding to parasutures between three apical and two apical intercaraly plates. These morphological characters indicate that this species is morphologically related to two dinoflagellate cystgenera Islandinium and Echinidinium.

Research paper thumbnail of Mutation of Three Cysteine Residues in Tomato yellow leaf curl virus-China C2 Protein Causes Dysfunction in Pathogenesis and Posttranscriptional Gene—Silencing Suppression

Molecular Plant-Microbe Interactions, 2002

The nuclear localized C2 protein of the monopartite bego-movirus Tomato yellow leaf curl virus-Ch... more The nuclear localized C2 protein of the monopartite bego-movirus Tomato yellow leaf curl virus-China (TYLCV-C) contributes to viral pathogenicity. Here, we have investigated TYLCV-C C2 protein domains that play a role in the phenotype. Alignment of the C2 protein with 67 homologues from monopartite and bipartite begomoviruses re-vealed that a putative zinc-finger motif C36-X1-C38-X7-C46-X6-H53-X4-H58C59 and four potential phosphorylation sites(T52, S61, Y68, and S74) are highly conserved. When ex-pressed from a Potato virus X (PVX) vector, TYLCV-C C2 protein mutants C2-T52M, C2-H58S, C2-C59S, C2-S61R, and C2-S74D, like the wild-type C2 protein, induced local necrotic ringspots and systemic necrosis in Nicotiana ben-thamiana plants. Mutants C2-H53P and C2-Y68D produced irregular necrotic lesions on inoculated leaves that were distinct from the wild-type phenotype. In contrast, mutants C2-C36R, C2-C38N, and C2-C46I induced chlorosis and mosaic symptoms rather than necrosis. We demonst...

Research paper thumbnail of Gene C2 of the Monopartite Geminivirus Tomato yellow leaf curl virus -China Encodes a Pathogenicity Determinant That Is Localized in the Nucleus

Molecular Plant-Microbe Interactions, 2001

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent ... more Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.

Research paper thumbnail of Differential roles of geminivirus Rep and AC4 (C4) in the induction of necrosis in Nicotiana benthamiana

Molecular Plant Pathology, 2002

The replication-associated protein (Rep) of two distinct begomoviruses, the bipartite African cas... more The replication-associated protein (Rep) of two distinct begomoviruses, the bipartite African cassava mosaic virus (ACMV) and the monopartite Tomato yellow leaf curl virus-China (TYLCV-C), elicits a reaction resembling a hypersensitive response (HR), associated with the induction of local necrosis and a systemic burst of hydrogen peroxide production, when expressed from a potato virus X vector in Nicotiana benthamiana. Transient expression of the ACMV Rep after Agrobacterium infiltration of N. benthamiana also triggered an HR-like response. We have identified a region of the ACMV Rep, referred to as the HR-like determinant domain (HRD, amino acids 119-179) that is essential for induction of the phenotype. Two additional regions have been identified (amino acids 1-85 and 86-118) that have various effects on the Rep-mediated phenotype, suggesting that structural constraints are imposed on the functional HRD. The co-expression of Rep with either AC4 or C4, expressed from overlapping open reading frames, triggers systemic necrosis in infected-tissues, but AC4 or C4 alone is neither an inducer nor enhancer of the HR-like phenotype. We propose that ACMV AC4 and TYLCV-C C4 may counter the plant defence mechanism that is initiated by the Rep-mediated local HR-like phenotype.

Research paper thumbnail of Novel System for the Simultaneous Analysis of Geminivirus DNA Replication and Plant Interactions in Nicotiana benthamiana

Journal of Virology, 2003

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector bas... more The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replicationassociated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R 2 , R 5 , R 7 and K 11 or H 56 , L 57 and H 58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.

Research paper thumbnail of Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells

Journal of Virology, 2004

A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic l... more A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFP⌬CP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFP⌬CP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFP⌬CP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFP⌬CP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.

Research paper thumbnail of Functional Characterization of the Nuclear Localization Signal for a Suppressor of Posttranscriptional Gene Silencing

Journal of Virology, 2003

The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active su... more The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17KVQHRIAKKTTRRRR31. When expressed from potato virus X, C2-RRRR31DVGG (in which the four consecutive arginine residues 28RRRR31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K17D-GFP, C2-HR21DV-GFP, and C2-KK25DI-GFP localized to nuclei and produced necrosis, but only C2-K17D-GFP suppressed PTGS. The N-terminal portions C21-31 and C217-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establ...

Research paper thumbnail of Virus survival of RNA silencing without deploying protein-mediated suppression in Nicotiana benthamiana

FEBS Letters, 2004

RNA silencing represents an evolutionarily conserved defence mechanism that plays a key antiviral... more RNA silencing represents an evolutionarily conserved defence mechanism that plays a key antiviral role in protecting plants and animals against virus infection. To counterattack, plant, animal and fungal viruses produce proteins capable of suppressing RNA silencing. Here, we report an unprecedented phenomenon that Potato virus X, a single-stranded positive RNA virus, is able to survive RNA silencing without deploying protein-mediated anti-silencing by revealing an unexpected symptom re-emergence and re-accumulation of viral RNAs and proteins in plants maintaining strong RNA silencing. Our results provide evidence that a plant virus may have developed a getaway strategy to survive RNA silencing.

Research paper thumbnail of Hypovirus Papain-Like Protease p29 Suppresses RNA Silencing in the Natural Fungal Host and in a Heterologous Plant System

Eukaryotic Cell, 2006

Virulence-attenuating hypoviruses of the speciesCryphonectriahypovirus 1 (CHV1) encode a papain-l... more Virulence-attenuating hypoviruses of the speciesCryphonectriahypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungusCryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed inC. parasiticastrains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenicNicotiana benthamianaline 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral de...

Research paper thumbnail of Fine structure of silicoflagellate double skeletons

Research paper thumbnail of Reinvestment Alert

This article cites 46 articles, 17 of which can be accessed free

Research paper thumbnail of Contribution of the Zinc Finger to Zinc and DNA Binding by a Suppressor of Posttranscriptional Gene Silencing

Journal of Virology, 2003

The zinc finger C 36 -X1-C 38 -X7-C 46 -X6-H 53 of the nuclearly localized C2 protein of Tomato y... more The zinc finger C 36 -X1-C 38 -X7-C 46 -X6-H 53 of the nuclearly localized C2 protein of Tomato yellow leaf curl virus China is involved in pathogenicity and suppression of posttranscriptional gene silencing (PTGS). Here, we demonstrate that the zinc finger is indispensable for the C2 protein to bind zinc and DNA. Mutation of cysteine residue C 36 , C 38 , or C 46 reduced the zinc and DNA binding capacity of C2 protein. When expressed from potato virus X, all three mutants, C2-C 36 R, C2-C 38 N, and C2-C 46 I, tagged with a green fluorescent protein (GFP) were still capable of transporting GFP into but aggregated abnormally in nuclei. Our data establish that zinc- and DNA-binding activity correlates with C2-mediated pathogenesis and PTGS suppression.

Research paper thumbnail of Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Planta, 1999

Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of s... more Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the¯avonol-de®cient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of¯avonols in this process, wild-type and¯avonol-de®cient pollen tubes were subjected to cytological and ultrastructural analyses and screened for dierences. The results showed that before disruption of the¯avonol-de®cient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural dierences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any signi®cant dierence. However, for the ®rst time, obvious morphological dierences were observed in the wall of the¯avonol-de®cient pollen tubes. We conclude that¯avonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins.

Research paper thumbnail of Fine structure of silicoflagellate double skeletons

Marine Micropaleontology, 2014

Research paper thumbnail of Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Planta, 1999

Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of s... more Despite the vital role that¯avonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the¯avonol-de®cient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of¯avonols in this process, wild-type and¯avonol-de®cient pollen tubes were subjected to cytological and ultrastructural analyses and screened for dierences. The results showed that before disruption of the¯avonol-de®cient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural dierences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any signi®cant dierence. However, for the ®rst time, obvious morphological dierences were observed in the wall of the¯avonol-de®cient pollen tubes. We conclude that¯avonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins.

Research paper thumbnail of Suppression of local RNA silencing is not sufficient to promote cell-to-cell movement of Turnip crinkle virus in Nicotiana benthamiana

Plant Signaling & Behavior, 2009

The biological relationship between suppression of RNA silencing and virus movement poses an intr... more The biological relationship between suppression of RNA silencing and virus movement poses an intriguing question in virus-plant interactions. Here, we have used a local RNA silencing assay, based on a movement-deficient Turnip crinkle virus TCV/ GFPΔCP, to investigate the influence of silencing suppression by three different viral suppressors: the TCV 38K coat protein (CP), the 126K protein of Tobacco mosaic virus (TMV), and P19 of Tomato bushy stunt virus (TBSV) on cell-to-cell movement and long-distance spread of TCV/GFPΔCP. First, we found that TCV CP blocked the induction of local RNA silencing, but failed to support virus trafficking in silencing-suppressed transgenic plants, although it acted as a functional movement protein in nontransformed plants. Second, we demonstrated that the TMV 126K suppressor inhibited TCV/GFPΔCP-mediated RNA silencing, but did not facilitate intercellular spread of the chimaeric carmovirus. However, TMV and TMVΔCP prevented the initiation of RNA silencing by TCV/GFPΔCP and caused TCV/GFPΔCP to move between cells, although only TMV supported its long-distance spread. Third, TBSV P19 functioned as a movement protein for TCV/GFPΔCP and as a silencing suppressor in non-transformed and silencing-suppressed transgenic plants. We further identified three types of mutant P19 proteins that possessed no or varied functionality in silencing suppression and in the facilitation of carmovirus movement. These results suggest that, although suppression of local RNA silencing is essential for the maintenance of viral RNA, recovery of cell-to-cell movement and long-distance spread of movement-deficient carmoviruses is not a direct consequence of such silencing suppression.

Research paper thumbnail of Protoperidinium tricingulatum sp. nov. (Dinophyceae), a new motile form of a round, brown, and spiny dinoflagellate cyst

Phycological Research, 2000

A small, broadly ovoidal and heterotrophic dinoflagellate containing round, brownish, and spiny c... more A small, broadly ovoidal and heterotrophic dinoflagellate containing round, brownish, and spiny cyst was found in the water column of Huibertsplaat in the Wadden Sea off the coast of the Netherlands. This dinoflagellate had these conspicuous morphological characters: a five-sided first apical plate (1′), only three cingular plates, and an extremely small first antapical plate. Based on these morphological features, Protoperidinium tricingulatum Kawami, vanWezel, Koeman et Matsuoka is described as a new species. The flagellar pore of P. tricingulatum is covered with a small fin, which rises from the left side of the right sulcal plate to the large V-shaped posterior sulcal plate. This feature suggests that P. tricingulatum is assigned to the Abé's Monovela Group. The cyst stage of P. tricingulatum was positively linked to the vegetative stage by comparison of the ribosomal 5.8S rDNA, internal transcribed spacers (ITS1 and ITS2). Living cysts of P. tricingulatum are round, brownish, and covered with many slender spines bearing capitate or cauliforate distal ends. The cyst also possesses a theropylic archeopyle formed by a slit corresponding to parasutures between three apical and two apical intercaraly plates. These morphological characters indicate that this species is morphologically related to two dinoflagellate cystgenera Islandinium and Echinidinium.

Research paper thumbnail of Mutation of Three Cysteine Residues in Tomato yellow leaf curl virus-China C2 Protein Causes Dysfunction in Pathogenesis and Posttranscriptional Gene—Silencing Suppression

Molecular Plant-Microbe Interactions, 2002

The nuclear localized C2 protein of the monopartite bego-movirus Tomato yellow leaf curl virus-Ch... more The nuclear localized C2 protein of the monopartite bego-movirus Tomato yellow leaf curl virus-China (TYLCV-C) contributes to viral pathogenicity. Here, we have investigated TYLCV-C C2 protein domains that play a role in the phenotype. Alignment of the C2 protein with 67 homologues from monopartite and bipartite begomoviruses re-vealed that a putative zinc-finger motif C36-X1-C38-X7-C46-X6-H53-X4-H58C59 and four potential phosphorylation sites(T52, S61, Y68, and S74) are highly conserved. When ex-pressed from a Potato virus X (PVX) vector, TYLCV-C C2 protein mutants C2-T52M, C2-H58S, C2-C59S, C2-S61R, and C2-S74D, like the wild-type C2 protein, induced local necrotic ringspots and systemic necrosis in Nicotiana ben-thamiana plants. Mutants C2-H53P and C2-Y68D produced irregular necrotic lesions on inoculated leaves that were distinct from the wild-type phenotype. In contrast, mutants C2-C36R, C2-C38N, and C2-C46I induced chlorosis and mosaic symptoms rather than necrosis. We demonst...

Research paper thumbnail of Gene C2 of the Monopartite Geminivirus Tomato yellow leaf curl virus -China Encodes a Pathogenicity Determinant That Is Localized in the Nucleus

Molecular Plant-Microbe Interactions, 2001

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent ... more Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.

Research paper thumbnail of Differential roles of geminivirus Rep and AC4 (C4) in the induction of necrosis in Nicotiana benthamiana

Molecular Plant Pathology, 2002

The replication-associated protein (Rep) of two distinct begomoviruses, the bipartite African cas... more The replication-associated protein (Rep) of two distinct begomoviruses, the bipartite African cassava mosaic virus (ACMV) and the monopartite Tomato yellow leaf curl virus-China (TYLCV-C), elicits a reaction resembling a hypersensitive response (HR), associated with the induction of local necrosis and a systemic burst of hydrogen peroxide production, when expressed from a potato virus X vector in Nicotiana benthamiana. Transient expression of the ACMV Rep after Agrobacterium infiltration of N. benthamiana also triggered an HR-like response. We have identified a region of the ACMV Rep, referred to as the HR-like determinant domain (HRD, amino acids 119-179) that is essential for induction of the phenotype. Two additional regions have been identified (amino acids 1-85 and 86-118) that have various effects on the Rep-mediated phenotype, suggesting that structural constraints are imposed on the functional HRD. The co-expression of Rep with either AC4 or C4, expressed from overlapping open reading frames, triggers systemic necrosis in infected-tissues, but AC4 or C4 alone is neither an inducer nor enhancer of the HR-like phenotype. We propose that ACMV AC4 and TYLCV-C C4 may counter the plant defence mechanism that is initiated by the Rep-mediated local HR-like phenotype.

Research paper thumbnail of Novel System for the Simultaneous Analysis of Geminivirus DNA Replication and Plant Interactions in Nicotiana benthamiana

Journal of Virology, 2003

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector bas... more The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replicationassociated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R 2 , R 5 , R 7 and K 11 or H 56 , L 57 and H 58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.

Research paper thumbnail of Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells

Journal of Virology, 2004

A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic l... more A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFP⌬CP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFP⌬CP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFP⌬CP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFP⌬CP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.

Research paper thumbnail of Functional Characterization of the Nuclear Localization Signal for a Suppressor of Posttranscriptional Gene Silencing

Journal of Virology, 2003

The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active su... more The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17KVQHRIAKKTTRRRR31. When expressed from potato virus X, C2-RRRR31DVGG (in which the four consecutive arginine residues 28RRRR31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K17D-GFP, C2-HR21DV-GFP, and C2-KK25DI-GFP localized to nuclei and produced necrosis, but only C2-K17D-GFP suppressed PTGS. The N-terminal portions C21-31 and C217-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establ...

Research paper thumbnail of Virus survival of RNA silencing without deploying protein-mediated suppression in Nicotiana benthamiana

FEBS Letters, 2004

RNA silencing represents an evolutionarily conserved defence mechanism that plays a key antiviral... more RNA silencing represents an evolutionarily conserved defence mechanism that plays a key antiviral role in protecting plants and animals against virus infection. To counterattack, plant, animal and fungal viruses produce proteins capable of suppressing RNA silencing. Here, we report an unprecedented phenomenon that Potato virus X, a single-stranded positive RNA virus, is able to survive RNA silencing without deploying protein-mediated anti-silencing by revealing an unexpected symptom re-emergence and re-accumulation of viral RNAs and proteins in plants maintaining strong RNA silencing. Our results provide evidence that a plant virus may have developed a getaway strategy to survive RNA silencing.

Research paper thumbnail of Hypovirus Papain-Like Protease p29 Suppresses RNA Silencing in the Natural Fungal Host and in a Heterologous Plant System

Eukaryotic Cell, 2006

Virulence-attenuating hypoviruses of the speciesCryphonectriahypovirus 1 (CHV1) encode a papain-l... more Virulence-attenuating hypoviruses of the speciesCryphonectriahypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungusCryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed inC. parasiticastrains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenicNicotiana benthamianaline 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral de...

Research paper thumbnail of Fine structure of silicoflagellate double skeletons