[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent protein kinases (CDPKs) comprise a large family of serine/threonine kinases in plants and protozoans. We isolated two related CDPK cDNAs (NtCDPK2 and NtCDPK3) from Nicotiana tabacum. These CDPK transcripts are elevated after race-specific defence elicitation and hypo-osmotic stress. Transiently expressed myc-epitope-tagged NtCDPK2 in Nicotiana benthamiana and N.tabacum leaves showed a rapid transient interconversion to an activated form after elicitation and hypo-osmotic stress. The Avr9 race-specific elicitor caused a more pronounced and sustained response. This transition is due to phosphorylation of the CDPK. Immuno complex kinase assays with epitope-tagged NtCDPK2 showed that stress-induced phosphorylation and interconversion of NtCDPK2 correlates with an increase in enzymatic activity. The function of NtCDPK2 in plant defence was investigated by employing virus-induced gene silencing (VIGS) in N.benthamiana. CDPK-silenced plants showed a reduced and delayed hypersensitive response after race-specific elicitation in a gene-for-gene interaction, and lacked an accompanying wilting phenotype. Silencing correlated with loss of CDPK mRNA, whereas mRNA accumulation of mitogen-activated protein kinase WIPK remained unaltered.
The EMBO Journal 11/2001; 20(20):5556-67. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA microarrays are being used to comprehensively examine gene expression networks during the plant defense response that is triggered when a plant encounters a pathogen or an elicitor molecule. In addition to identifying new genes induced during defense, these studies are providing new insights into the complex pathways governing defense gene regulation.
[Show abstract][Hide abstract] ABSTRACT: To identify genes from the obligatory biotrophic oomycete Peronospora parasitica that are expressed during infection in Arabidopsis thaliana we employed cDNA-amplified fragment length polymorphism (AFLP) display. cDNA-AFLP fragments from infected and non-infected leaves were separated in parallel by gel electrophoresis and displayed by autoradiography. Most differential gene fragments were derived from P. parasitica.
[Show abstract][Hide abstract] ABSTRACT: The tomato Cf-9 gene confers resistance to races of the fungal pathogen Cladosporium fulvum expressing the Avr9 gene. cDNA amplified fragment length polymorphism analysis was used to display transcripts whose expression is rapidly altered during the Avr9- and Cf-9-mediated defense response in tobacco cell cultures. Diphenyleneiodonium was used to abolish the production of active oxygen species during gene induction. Of 30,000 fragments inspected, 290 showed altered abundance, of which 263 were induced independently of active oxygen species. cDNA clones were obtained for 13 ACRE (for Avr9/Cf-9 rapidly elicited) genes. ACRE gene induction occurred in the presence of cycloheximide. Avr9 induced ACRE gene expression in leaves. Surprisingly, ACRE genes were also rapidly but transiently induced in leaves in response to other stresses. The amino acid sequences of some ACRE proteins are homologous to sequences of known proteins such as ethylene response element binding protein transcription factors, the N resistance protein, a calcium binding protein, 13-lipoxygenase, and a RING-H2 zinc finger protein. Rapid induction of ACRE genes suggests that they play a pivotal role during plant defense responses.
The Plant Cell 07/2000; 12(6):963-77. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9-dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca(2)+ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9-dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.
The Plant Cell 06/2000; 12(5):803-16. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Resistance of tomato to the leaf mould fungus Cladosporium fulvum is controlled by the interaction between a plant-encoded resistance gene (Cf-9) and pathogen-encoded avirulence (Avr9) gene. Our objective is to understand the underlying molecular mechanisms that transmit the Cf-9/Avr9-dependent pathogen perception event and activate the plant defence response. Our approach toward the understanding of Cf-function is based on the analysis of early Cf-9/Avr9-mediated responses and signalling events. Because Cf-9 transgenically expressed in tobacco retains its specificity and activity to the Avr9 elicitor, signalling experiments were conducted in the heterologous system using these transgenic lines or derived Cf9 tobacco cell cultures. Among the earliest responses to the Avr9/Cf-9 elicitation event were rapid changes in ion-fluxes, the synthesis of active oxygen species (AOS), probably catalysed by a plant NADPH-oxidase, and the transient activation of two MAP kinases. These kinases were identified as WIPK (wounding-induced protein kinase) and SIPK (salicylic-acid induced kinase) from tobacco. Studies with pharmacological inhibitors suggested that the MAP kinases are located in an independent signalling pathway from the Avr9/Cf-9-dependent synthesis of AOS. SIPK and WIPK were involved in pathogen-related elicitation processes as well as in abiotic stress responses. This indicates that the plant defence is triggered via a signalling network that shares components with pathways originating from abiotic environmental stress stimuli.
[Show abstract][Hide abstract] ABSTRACT: Abstract Molecular genetic approaches were adopted in the model crucifer, Arabidopsis thaliana, to unravel components of RPP5- and RPP1-mediated disease resistance to the oomycete pathogen, Peronospora parasitica. The products of RPP5 and three genes comprising the RPP1 complex locus belong to a major subclass of nucleotide-binding/leucine-rich repeat (NB-LRR) resistance (R) protein that has amino-terminal homology to the cytoplasmic domains of Drosophila and mammalian Toll and interleukin-1 family receptors (the so called 'TIR' domain). Similarities in the domain architecture of these proteins and animal regulators of programmed cell death have also been observed. Mutational screens revealed a number of genes that are required for RPP5-conditioned resistance. Among these are EDS1 and PAD4. Both EDS1 and PAD4 precede the function of salicylic acid-mediated plant responses. The EDS1 and PAD4 genes were cloned and found to encode proteins with similarity to the catalytic site of eukaryotic lipases, suggesting that they may function by hydrolysing a lipid-based substrate.
[Show abstract][Hide abstract] ABSTRACT: In Arabidopsis ecotype Landsberg erecta (Ler), RPP5 confers resistance to the pathogen Peronospora parasitica. RPP5 is part of a clustered multigene family encoding nucleotide binding-leucine-rich repeat (LRR) proteins. We compared 95 kb of DNA sequence carrying the Ler RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the Ler and Col-0 RPP5 haplotypes exhibit remarkable intraspecific polymorphism. The RPP5 gene family probably evolved by extensive recombination between LRRs from an RPP5-like progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different numbers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes were, or are, functional. The RPP5 haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.
The Plant Cell 12/1999; 11(11):2099-112. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.
The Plant Cell 11/1999; 11(10):1841-52. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A major class of plant disease resistance (R) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana, EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs (PR1 and PDF1.2, respectively) for these two pathways in wild-type and eds1 mutant plants after different challenges. The results suggest that EDS1 functions upstream of salicylic acid-dependent PR1 mRNA accumulation and is not required for jasmonic acid-induced PDF1.2 mRNA expression.
Proceedings of the National Academy of Sciences 04/1999; 96(6):3292-7. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.
The Plant Cell 03/1999; 11(2):273-87. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.
Proceedings of the National Academy of Sciences 03/1999; 96(4):1761-6. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tomato Cf-4 and Cf-9 genes map at a genetically complex locus on the short arm of chromosome 1 and confer resistance against Cladosporium fulvum through recognition of different pathogen-encoded avirulence determinants. Cf-4 and Cf-9 are members of a large gene family (Hcr9s, Homologues of Cladosporium fulvum resistance gene Cf-9), some of which encode additional distinct recognition specificities. A genetic analysis of the majority of Hcr9s suggests that their distribution is spatially restricted to the short arm of chromosome 1. Two loci of clustered Hcr9 genes have been analyzed physically that mapped distal (Northern Lights) and proximal (Southern Cross) to the Cf-4/9 locus (Milky Way). Sequence homologies between intergenic regions at Southern Cross and Milky Way indicate local Hcr9 duplication preceded cluster multiplication. The multiplication of clusters involved DNA flanking Hcr9 sequences as indicated by conserved lipoxygenase sequences at Southern Cross and Milky Way. The similar spatial distribution of Hcr9 clusters in different Lycopersicon spp. suggests Hcr9 cluster multiplication preceded speciation.
[Show abstract][Hide abstract] ABSTRACT: A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.
The Plant Cell 02/1999; 11(1):87-99. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant resistance (R) genes have evolved specific recognition capabilities in defense against pathogens. The evolution of R gene function and maintenance of R gene diversity within a plant species are therefore of great interest. In the Arabidopsis accession Wassilewskija, the RPP1 region on chromosome 3 contains four genetically linked recognition specificities, conditioning resistance to different isolates of the biotrophic oomycete Peronospora parasitica (downy mildew). We show that three of four tightly linked genes in this region, designated RPP1-WsA, RPP1-WsB, and RPP1-WsC, encode functional products of the NBS-LRR (nucleotide binding site-leucine-rich repeat) R protein class. They possess a TIR (Toll, interleukin-1, resistance) domain that is characteristic of certain other NBS-LRR-type R proteins, but in addition, they have unique hydrophilic or hydrophobic N termini. Together, the three RPP1 genes account for the spectrum of resistance previously assigned to the RPP1 region and thus comprise a complex R locus. The distinct but partially overlapping resistance capabilities conferred by these genes are best explained by the hypothesis that each recognizes a different pathogen avirulence determinant. We present evidence suggesting that the RPP genes at this locus are subject to the same selective forces that have been demonstrated for structurally different LRR-type R genes.
The Plant Cell 12/1998; 10(11):1847-60. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In many plant-pathogen interactions resistance to disease is controlled by the interaction of plant-encoded resistance (R) genes and pathogen-encoded avirulence (Avr) genes. The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an ideal system to study the molecular basis of pathogen perception by plants. A total of four tomato genes for resistance to C. fulvum (Cf-2, Cf-4, Cf-5 and Cf-9) have been isolated from two genetically complex chromosomal loci. Their gene products recognize specific C. fulvum-encoded avirulence gene products (Avr2, Avr4, Avr5 and Avr9) by an unknown molecular mechanism. Cf genes encode extracellular membrane-anchored glycoproteins comprised predominantly of 24 amino acid leucine-rich repeats (LRRs). Cf genes from the same locus encode proteins which are more than 90% identical. Most of the amino-acid sequence differences correspond to the solvent-exposed residues within a beta-strand/beta-turn structural motif which is highly conserved in LRR proteins. Sequence variability within this motif is predicted to affect the specificity of ligand binding. Our analysis of Cf gene loci at the molecular level has shown they comprise tandemly duplicated homologous genes, and suggests a molecular mechanism for the generation of sequence diversity at these loci. Our analysis provides further insight into the molecular basis of pathogen perception by plants and the organization and evolution of R gene loci.
Philosophical Transactions of The Royal Society B Biological Sciences 10/1998; 353(1374):1413-24. · 6.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, "transit complex", and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.
Proceedings of the National Academy of Sciences 09/1998; 95(17):10312-6. · 9.81 Impact Factor