bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ecdysone agonist inducible transcription in transgenic tobacco plants
Authors: Martinez Alberto , Sparks Caroline , Hart Cliff A. , Thompson John , Jepson Ian 
Published: March 2003

SummaryA novel chemical‐induced gene regulatory system for plants consisting of two molecular components is described. The first, or regulatory, cassette comprises a chimeric receptor composed of the hinge and ligand binding domains of the Heliothis virescens ecdysone receptor and the transactivation domain of the Herpes simplex VP16 protein fused to the DNA binding domain and transactivation of a mammalian glucocorticoid receptor. The second component, a reporter cassette, contains six copies of the glucocorticoid response element (GRE) fused to the minimal 35SCaMV promoter and β‐glucuronidase. The system uses a commercially available non‐steroidal ecdysone agonist, RH5992 (tebufenozide), as an inducer. Activation of gene expression is shown in both tobacco transient protoplasts and transgenic plants. The response is ligand dependent and is modulated by the change in minimal promoter context. The system is capable of inducing transgene activity up to 420‐fold corresponding to 150% of the activity observed with positive controls (35SCaMV:GUS).
The pre‐vacuolar t‐SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP
Authors: Bassham Diane C. , Raikhel Natasha V. 
Published: March 2003

SummaryMany proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans‐Golgi network are thought to fuse with a pre‐vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t‐SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to α‐SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with α‐SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre‐vacuolar compartment.
Downy mildew (Peronospora parasitica) resistance genes in Arabidopsis vary in functional requirements for NDR1, EDS1, NPR1 and salicylic acid accumulation
Authors: McDowell John M. , Cuzick Alayne , Can Canan , Beynon Jim , Dangl Jeffery L. , Holub Eric B. 
Published: March 2003

SummaryTo better understand the genetic requirements for R gene‐dependent defense activation in Arabidopsis, we tested the effect of several defense response mutants on resistance specified by eight RPP genes (for resistance to Peronospora parasitica) expressed in the Col‐0 background. In most cases, resistance was not suppressed by a mutation in the SAR regulatory gene NPR1 or by expression of the NahG transgene. Thus, salicylic acid accumulation and NPR1 function are not necessary for resistance mediated by these RPP genes. In addition, resistance conferred by two of these genes, RPP7 and RPP8, was not significantly suppressed by mutations in either EDS1 or NDR1. RPP7 resistance was also not compromised by mutations in EIN2, JAR1 or COI1 which affect ethylene or jasmonic acid signaling. Double mutants were therefore tested. RPP7 and RPP8 were weakly suppressed in an eds1‐2/ndr1‐1 background, suggesting that these RPP genes operate additively through EDS1, NDR1 and as‐yet‐undefined signaling components. RPP7 was not compromised in coi1/npr1 or coi1/NahG backgrounds. These observations suggest that RPP7 initiates resistance through a novel signaling pathway that functions independently of salicylic acid accumulation or jasmonic acid response components.
Conversion of compatible plant–pathogen interactions into incompatible interactions by expression of the Pseudomonas syringae pv. syringae 61 hrmA gene in transgenic tobacco plants
Authors: Shen Songhai , Li Qingshun , He Sheng‐Yang , Barker Kenneth R. , Li Debao , Hunt Arthur G. 
Published: March 2003

SummaryThe hrmA gene from Pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium P. syringae pv. tabaci in all examined tobacco cultivars. We expressed this gene in tobacco plants under the control of the tobacco Δ0.3 TobRB7 promoter, which is induced upon nematode infection in tobacco roots (
Opperman et al. 1994
, Science, 263, 221–223). A basal level of hrmA expression in leaves of transgenic plants activated the expression of pathogenesis‐related genes, and the transgenic plants exhibited high levels of resistance to multiple pathogens: tobacco vein mottling virus, tobacco etch virus, black shank fungus Phytophthora parasitica, and wild fire bacterium Pseudomonas syringae pv. tabaci. However, the hrmA transgenic plants were not significantly more resistant to root‐knot nematodes. Our results suggest a potential use of controlled low‐level expression of bacterial avr genes, such as hrmA, in plants to generate broad‐spectrum resistance to bacterial, fungal and viral pathogens.
Cell cycle function of a Medicago sativa A2‐type cyclin interacting with a PSTAIRE‐type cyclin‐dependent kinase and a retinoblastoma protein
Authors: Roudier François , Fedorova Elena , Györgyey Janos , Feher Attila , Brown Spencer , Kondorosi Adam , Kondorosi Eva 
Published: March 2003

SummaryIn plants multiple A‐type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1–A3), while in animal somatic cells a single type of cyclin A is required for cell‐cycle regulation from the S to M phases. We studied the function of an A2‐type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A‐type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti‐Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2‐M phase, the protein level after a rapid increase in S‐phase reached a plateau during the G2 phase. In the yeast two‐hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE‐motif‐containing cyclin‐dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2‐associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.
Development of clover yellow vein virus as an efficient, stable gene‐expression system for legume species
Authors: Masuta Chikara , Yamana Toshikazu , Tacahashi Yoko , Uyeda Ichiro , Sato Masanao , Ueda Shigenori , Matsumura Takeshi 
Published: March 2003

SummaryA highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green fluorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC‐Pro on pClYVV to create three recombinant plasmids: pClYVV‐GFP, pClYVV‐GFP‐GS, and pClYVV‐GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green fluorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was confirmed by RT–PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and flowered early. As the GS gene, one of the nodulin genes for nitrogen fixation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.
A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid‐potentiated oxidative burst
Authors: Mur Luis A. J. , Brown Ian R. , Darby Robert M. , Bestwick Charles S. , Bi Yong‐Mei , Mansfield John W. , Draper John 
Published: March 2003

SummaryThe role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild‐type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S‐SH‐L). Challenge of 35S‐SH‐L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild‐type tobacco, both the onset of plant cell death and the induction of an active oxygen species‐responsive promoter (AoPR1‐GUS) were delayed following challenge of 35S‐SH‐L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild‐type tobacco, whereas in 35S‐SH‐L plants the onset of H2O2 accumulation was delayed by 2–3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1–2 h following inoculation in wild‐type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence‐gene induction, factors that together may determine the outcome of plant–pathogen interactions.
The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface
Authors: Chiou Tzyy‐Jen , Liu Henry , Harrison Maria J. 
Published: March 2003

SummaryThe movement of phosphate from the soil into plant root cells is the first of many crucial transport events required to supply phosphorous (P) to cells throughout the plant. In addition to the ability to acquire phosphate from the soil, the majority of the vascular plants are able to form arbuscular mycorrhizal associations in which phosphate may be delivered to the cortex via a fungus. Previously, we cloned two phosphate transporter genes, MtPT1 and MtPT2 from Medicago truncatula roots. Complementation of a yeast phosphate transport mutant revealed that MtPT1 is a functional phosphate transporter and Northern analyses revealed that MtPT1 is expressed exclusively in roots (Liu et al., 1998, Mol. Plant‐Microbe Interact. 11, 14–22). Utilising an antibody specific for MtPT1, we have analysed the accumulation and spatial expression patterns of the MtPT1 transporter. MtPT1 transcript and protein levels show close correlation and increase dramatically in the roots in response to phosphate starvation. MtPT1 protein levels decrease in roots during development of a symbiosis with arbuscular mycorrhizal (AM) fungi, indicating that this transporter is not involved in symbiotic phosphate transport. Membrane fractionation and analysis of a MtPT1/GFP fusion protein revealed that MtPT1 is located in the plasma membrane, while in situ hybridisation and immunolocalisation demonstrate the presence of MtPT1 transcripts and protein in the epidermal cells and root hairs of M. truncatula roots. MtPT1 shows expression patterns consistent with a role specifically in the acquisition of phosphate from the soil and is distinct from the other phosphate transporter of this class described to date.
Different modes of de novo telomere formation by plant telomerases
Authors: Fitzgerald Matthew S. , Shakirov Eugene V. , Hood Elizabeth E. , McKnight Thomas D. , Shippen Dorothy E. 
Published: March 2003

SummaryThe telomerase reverse transcriptase can recognize broken chromosome ends and add new telomeres de novo in a reaction termed ‘chromosome healing’. Here we investigate new telomere formation in vitro by telomerases from a variety of flowering plant species. Comparing the electrophoretic mobilities and nucleotide sequences of the products, we uncovered three different modes of new telomere formation. The soybean telomerase, designated a Class I enzyme, only elongated DNA primers ending in telomeric nucleotides. Arabidopsis and maize telomerases, designated Class II enzymes, efficiently extended completely non‐telomeric sequences by positioning the 3′ terminus at a preferred site on the RNA template. Silene latifolia and sorghum telomerases constituted class III enzymes that elongated non‐telomeric DNA primers by annealing them at alternative sites on the RNA template. For all enzymes, errors were prevalent during synthesis of the first two repeats, likely reflecting lateral instability of the primer 3′ terminus on the template during the initial rounds of elongation. Class III telomerases, however, were five‐ to 13‐fold more error prone than class II, generating more mistakes in distal repeats added to the primers. This remarkable variability in enzyme–DNA interactions among plant telomerases does not reflect phylogenetic relationships, and therefore implies that the telomerase active site can evolve rapidly.
Dissection of RNA‐mediated ryegrass mosaic virus resistance in fertile transgenic perennial ryegrass (Lolium perenne L.)
Authors: Xu Jianping , Schubert Jörg , Altpeter Fredy 
Published: March 2003

SummaryRyegrass mosaic virus (RgMV) frequently reduces yield and persistence of perennial ryegrass. We introduced an untranslatable RgMV coat protein (RgMV‐CP) gene into perennial ryegrass using particle bombardment to explore the potential of RNA‐mediated virus resistance. Nine months after inoculation with the RgMV‐Bulgaria strain, the most resistant transgenic line showed no immuno‐detectable RgMV‐CP in all replications of the primary transformant and a sexual progeny. A significant influence of the transgenic line, virus strain and the period after inoculation on the RgMV level was observed. Typical examples of recovery resistance from initially susceptible plants were not identified. Molecular analysis revealed RgMV resistance operates by targeted RNA degradation resulting in post‐transcriptional transgene silencing (PTGS) along with inhibition of virus RNA replication and was not associated with a threshold of transgene expression. Suppression of PTGS by RgMV differed significantly in two progenies with identical RgMV‐CP transgene integration and a similar RgMV‐CP transgene methylation pattern, suggesting the presence of quantitative components controlling the stability of PTGS in perennial ryegrass. This study extends RNA‐mediated virus resistance, post transcriptional gene silencing and crop improvement by genetic engineering to an agronomically important perennial monocot.
Gene‐associated CpG islands in plants as revealed by analyses of genomic sequences
Authors: Ashikawa Ikuo 
Published: March 2003

SummaryWe screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG‐rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG‐rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG‐rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5′‐end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5′‐end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue‐specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5′‐end of the genes.
Light regulation of circadian clock‐controlled gene expression in rice
Authors: Sugiyama Nobuko , Izawa Takeshi , Oikawa Tetsuo , Shimamoto Ko 
Published: March 2003

SummaryUsing transgenic rice seedlings expressing a firefly luciferase (luc) gene under the control of a rice CAB (chlorophyll a/b binding protein) promoter, we demonstrated how light affects circadian clocks in the transcription of the CAB gene. Rhythmic luc expression was observed for more than 5 d under constant light and dark (DD) conditions after light/dark entrainment. After a light pulse was applied at different time points in DD various temporal patterns of CAB gene expression were individually observed. We first examined two distinct properties related to the entrainment mechanism of the circadian clock: fluence‐rate dependence of free‐running periods (FRPs) and phase resetting by a light pulse. Although fluence‐rate dependent shortening of FRP was demonstrated, the FRP in DD was almost equal to that in constant light of a middle fluence‐rate, indicating that this fluence‐rate dependence may not fully describe the entrainment of the circadian clock in rice. Typical phase responses of the circadian clock by a single light pulse were also observed at the transcriptional level in rice seedlings. Thus, the phase resettings upon the light/dark transitions of daily cycles may be sufficient to explain the entrainment mechanisms of rice. We have further demonstrated that, in addition to having a gating effect to acute response, a light pulse can activate the circadian clock‐controlled CAB1R gene expression at the first circadian peak in a phase‐dependent manner. This suggests that light activates circadian clock activity in the diurnal CAB gene expression under daily light/dark cycles.
Trans‐dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responses
Authors: Pontier Dominique , Miao Zhong‐He , Lam Eric 
Published: March 2003

SummarySalicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic‐leucine‐zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans‐dominant mutant that specifically eliminates DNA‐binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA‐binding activities is correlated with suppression of two xenobiotic‐responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis‐related (PR) genes by SA. In addition, these TGA‐suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.
Characterization of Arabidopsis enhanced disease susceptibility mutants that are affected in systemically induced resistance
Authors: Ton Jurriaan , De Vos Martin , Robben Charlotte , Buchala Anthony , Métraux Jean‐Pierre , Van Loon L. C. , Pieterse Corné M. J. 
Published: March 2003

SummaryIn Arabidopsis, the rhizobacterial strain Pseudomonas fluorescens WCS417r triggers jasmonate (JA)‐ and ethylene (ET)‐dependent induced systemic resistance (ISR) that is effective against different pathogens. Arabidopsis genotypes unable to express rhizobacteria‐mediated ISR against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) exhibit enhanced disease susceptibility towards this pathogen. To identify novel components controlling induced resistance, we tested 11 Arabidopsis mutants with enhanced disease susceptibility (eds) to pathogenic P. syringae bacteria for WCS417r‐mediated ISR and pathogen‐induced systemic acquired resistance (SAR). Mutants eds4‐1, eds8‐1 and eds10‐1 failed to develop WCS417r‐mediated ISR, while mutants eds5‐1 and eds12‐1 failed to express pathogen‐induced SAR. Whereas eds5‐1 is known to be blocked in salicylic acid (SA) biosynthesis, analysis of eds12‐1 revealed that its impaired SAR response is caused by reduced sensitivity to this molecule. Analysis of the ISR‐impaired eds mutants revealed that they are non‐responsive to induction of resistance by methyl jasmonate (MeJA) (eds4‐1, eds8‐1 and eds10‐1), or the ET precursor 1‐aminocyclopropane‐1‐carboxylate (ACC) (eds4‐1 and eds10‐1). Moreover, eds4‐1 and eds8‐1 showed reduced expression of the plant defensin gene PDF1.2 after MeJA and ACC treatment, which was associated with reduced sensitivity to either ET (eds4‐1) or MeJA (eds8‐1). Although blocked in WCS417r‐, MeJA‐ and ACC‐induced ISR, eds10‐1 behaved normally for several other responses to MeJA or ACC. The results indicate that EDS12 is required for SAR and acts downstream of SA, whereas EDS4, EDS8 and EDS10 are required for ISR acting either in JA signalling (EDS8), ET signalling (EDS4), or downstream JA and ET signalling (EDS10) in the ISR pathway.