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The F-box protein ACRE189/ACIF1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato 总被引:2,自引:0,他引:2
van den Burg HA Tsitsigiannis DI Rowland O Lo J Rallapalli G Maclean D Takken FL Jones JD 《The Plant cell》2008,20(3):697-719
Virus-induced gene silencing identified the Avr9/Cf-9 RAPIDLY ELICITED gene ACRE189 as essential for the Cf-9- and Cf-4-mediated hypersensitive response (HR) in Nicotiana benthamiana. We report a role for ACRE189 in disease resistance in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). ACRE189 (herein renamed Avr9/Cf-9-INDUCED F-BOX1 [ACIF1]) encodes an F-box protein with a Leu-rich-repeat domain. ACIF1 is widely conserved and is closely related to F-box proteins regulating plant hormone signaling. Silencing of tobacco ACIF1 suppressed the HR triggered by various elicitors (Avr9, Avr4, AvrPto, Inf1, and the P50 helicase of Tobacco mosaic virus [TMV]). ACIF1 is recruited to SCF complexes (a class of ubiquitin E3 ligases), and the expression of ACIF1 F-box mutants in tobacco compromises the HR similarly to ACIF1 silencing. ACIF1 affects N gene-mediated responses to TMV infection, including lesion formation and salicylic acid accumulation. Loss of ACIF1 function also reduced confluent cell death induced by Pseudomonas syringae pv tabaci. ACIF1 silencing in Cf9 tomato attenuated the Cf-9-dependent HR but not Cf-9 resistance to Cladosporium fulvum. Resistance conferred by the Cf-9 homolog Cf-9B, however, was compromised in ACIF1-silenced tomato. Analysis of public expression profiling data suggests that Arabidopsis thaliana homologs of ACIF1 (VFBs) regulate defense responses via methyl jasmonate- and abscisic acid-responsive genes. Together, these findings support a role of ACIF1/VFBs in plant defense responses. 相似文献
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Functional analysis of Avr9/Cf-9 rapidly elicited genes identifies a protein kinase, ACIK1, that is essential for full Cf-9-dependent disease resistance in tomato 总被引:1,自引:0,他引:1
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Rowland O Ludwig AA Merrick CJ Baillieul F Tracy FE Durrant WE Fritz-Laylin L Nekrasov V Sjölander K Yoshioka H Jones JD 《The Plant cell》2005,17(1):295-310
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The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9 总被引:12,自引:0,他引:12
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The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed. 相似文献
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Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp
Thomas CM Tang S Hammond-Kosack K Jones JD 《Molecular plant-microbe interactions : MPMI》2000,13(4):465-469
We have previously shown that tomato Cf-9 induces an Avr9-dependent hypersensitive response (HR) in Nicotiana tabacum and potato. We show here that Cf-4 also induces an Avr4-dependent HR in two tobacco species (N. tabacum and N. benthamiana). The HR induced by Cf-4 and Cf-9 was compared in stable tobacco transgenics by a seedling lethal assay and resistance to recombinant Potato virus X expressing Avr4 or Avr9. We also compared HR induction with Agrobacterium-mediated transient expression. The Cf-4/Avr4 combination induced a more rapid HR than Cf-9/Avr9. Sensitive assays for Cf-9 and Cf-4 function should prove useful for structure/function analyses of these resistance proteins in tobacco. 相似文献
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Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis 总被引:13,自引:0,他引:13
Van der Hoorn RA Laurent F Roth R De Wit PJ 《Molecular plant-microbe interactions : MPMI》2000,13(4):439-446
The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family. 相似文献
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Kruijt M Kip DJ Joosten MH Brandwagt BF de Wit PJ 《Molecular plant-microbe interactions : MPMI》2005,18(9):1011-1021
The Cf-4 and Cf-9 genes originate from the wild tomato species Lycopersicon hirsutum and L. pimpinellifolium and confer resistance to strains of the leaf mold fungus Cladosporium fulvum that secrete the Avr4 and Avr9 elicitor proteins, respectively. Homologs of Cf-4 and Cf-9 (Hcr9s) are located in several clusters and evolve mainly through sequence exchange between homologs. To study the evolution of Cf genes, we set out to identify functional Hcr9s that mediate recognition of Avr4 and Avr9 (designated Hcr9-Avr4s and Hcr9-Avr9s) in all wild tomato species. Plants responsive to the Avr4 and Avr9 elicitor proteins were identified throughout the genus Lycopersicon. Open reading frames of Hcr9s from Avr4- and Avr9-responsive tomato plants were polymerase chain reaction-amplified. Several Hcr9s that mediate Avr4 or Avr9 recognition were identified in diverged tomato species by agroinfiltration assays. These Hcr9-Avr4s and Hcr9-Avr9s are highly identical to Cf-4 and Cf-9, respectively. Therefore, we conclude that both Cf-4 and Cf-9 predate Lycopersicon speciation. These results further suggest that C. fulvum is an ancient pathogen of the genus Lycopersicon, in which Cf-4 and Cf-9 have been maintained by selection pressure imposed by C. fulvum. 相似文献
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Tomato mitogen-activated protein kinases LeMPK1, LeMPK2, and LeMPK3 are activated during the Cf-4/Avr4-induced hypersensitive response and have distinct phosphorylation specificities 总被引:3,自引:0,他引:3
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Tomato (Solanum lycopersicum) plants with the Cf-4 resistance gene recognize strains of the pathogenic fungus Cladosporium fulvum that secrete the avirulence protein Avr4. Transgenic tomato seedlings coexpressing Cf-4 and Avr4 mount a hypersensitive response (HR) at 20 degrees C, which is suppressed at 33 degrees C. Within 120 min after a shift from 33 degrees C to 20 degrees C, tomato mitogen-activated protein (MAP) kinase (LeMPK) activity increases in Cf-4/Avr4 seedlings. Searching tomato genome databases revealed at least 16 LeMPK sequences, including the sequence of LeMPK1, LeMPK2, and LeMPK3 that cluster with biotic stress-related MAP kinase orthologs from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). LeMPK1, LeMPK2, and LeMPK3 are simultaneously activated in Cf-4/Avr4 seedlings, and, to reveal whether they are functionally redundant or not, recombinant LeMPKs were incubated on PepChip Kinomics slides carrying peptides with potential phosphorylation sites. Phosphorylated peptides and motifs present in them discriminated between the phosphorylation specificities of LeMPK1, LeMPK2, and LeMPK3. LeMPK1, LeMPK2, or LeMPK3 activity was specifically suppressed in Cf-4-tomato by virus-induced gene silencing and leaflets were either injected with Avr4 or challenged with C. fulvum-secreting Avr4. The results of these experiments suggested that the LeMPKs have different but also overlapping roles with regard to HR and full resistance in tomato. 相似文献
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Li W Ahn IP Ning Y Park CH Zeng L Whitehill JG Lu H Zhao Q Ding B Xie Q Zhou JM Dai L Wang GL 《Plant physiology》2012,159(1):239-250
The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis. 相似文献
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The Cf-9 disease resistance protein is present in an approximately 420-kilodalton heteromultimeric membrane-associated complex at one molecule per complex
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The tomato Cf-9 gene confers race-specific resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In tobacco, Cf-9 confers a hypersensitive response to the Avr9 peptide. To investigate Cf-9 protein function in initiating defense signaling, we engineered a functional C-terminal fusion of the Cf-9 gene with the TAP (Tandem Affinity Purification) tag. In addition, we established a transient expression assay in Nicotiana benthamiana leaves for the production of functional Cf-9:myc and Cf-9:TAP. Transiently expressed Cf-9:myc and Cf-9:TAP proteins induced an Avr9-dependent hypersensitive response, consistent with previous results with stably transformed tobacco plants and derived cell suspension cultures expressing c-myc-tagged Cf-9. Gel filtration of microsomal fractions solubilized with octylglucoside revealed that the Cf-9 protein, either as c-myc or TAP fusions, migrated at a molecular mass of 350 to 475 kD. By using blue native gel electrophoresis, the molecular size was confirmed to be approximately 420 kD. Our results suggest that only one Cf-9 protein molecule is present in the Cf-9 complex and that Cf-9 is part of a membrane complex consisting of an additional glycoprotein partner(s). The high structural similarity between Cf proteins and Clavata2 (CLV2) of Arabidopsis, together with the similarity of molecular mass between Cf-9 and CLV complexes (420 and 450 kD, respectively), led us to investigate whether Cf-9 is integrated into membrane-associated protein complexes like those formed by CLV1 and CLV2. Unlike CLV2, the Cf-9 protein did not form disulfide-linked heterodimers, no ligand (Avr9)-dependent shift in the molecular mass of the Cf-9 complex was detected, and no Rho-GTPase-related proteins were found associated with Cf-9 under the conditions tested. Thus, Cf-9-dependent defense signaling and CLV2-dependent regulation of meristem development seem to be accomplished via distinct mechanisms, despite the structural similarity of their key components Cf-9 and CLV2. 相似文献
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Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins 总被引:15,自引:0,他引:15
The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins. 相似文献
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Introgression of resistance trait Cf-4 from wild tomato species into tomato cultivar MoneyMaker (MM-Cf0) has resulted in the near-isogenic line MM-Cf4 that confers resistance to the fungal tomato pathogen Cladosporium fulvum. At the Cf-4 locus, five homologues of Cladosporium resistance gene Cf-9 (Hcr9s) are present. While Hcr9-4D represents the functional Cf-4 resistance gene matching Avr4, Hcr9-4E confers resistance towards C. fulvum by mediating recognition of the novel avirulence determinant Avr4E. Here, we report the isolation of the Avr4E gene, which encodes a cysteine-rich protein of 101 amino acids that is secreted by C. fulvum during colonization of the apoplastic space of tomato leaves. By complementation we show that Avr4E confers avirulence to strains of C. fulvum that are normally virulent on Hcr9-4E-transgenic plants, indicating that Avr4E is a genuine, race-specific avirulence determinant. Strains of C. fulvum evade Hcr9-4E-mediated resistance either by a deletion of the Avr4E gene or by production of a stable Avr4E mutant protein that carries two amino acid substitutions, Phe(82)Leu and Met(93)Thr. Moreover, we demonstrate by site-directed mutagenesis that the single amino acid substitution Phe(82)Leu in Avr4E is sufficient to evade Hcr9-4E-mediated resistance. 相似文献
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Brading PA Hammond-Kosack KE Parr A Jones JD 《The Plant journal : for cell and molecular biology》2000,23(3):305-318
Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants. 相似文献
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Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans 总被引:3,自引:0,他引:3
The tomato Cf9 resistance gene induces an Avr9-dependent hypersensitive response (HR) in tomato and transgenic Solanaceae spp. We studied whether the Cf9 gene product responded functionally to the corresponding Avr9 gene product when introduced in a heterologous plant species. We successfully expressed the Cf9 gene under control of its own promoter and the Avr9 or Avr9R8K genes under control of the p35S1 promoter in transgenic oilseed rape. We demonstrated that the transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as reported for Cladosporium fulvum. An Avr9-dependent HR was induced in Cf9 oilseed rape upon injection of intercellular fluid containing Avr9. We showed Avr9-specific induction of PR1, PR2, and Cxc750 defense genes in oilseed rape expressing CJ9. Cf9 x Avr9 oilseed rape did not result in seedling death of the F1 progeny, independent of the promoters used to express the genes. The F1 (Cf9 x Avr9) plants, however, were quantitatively more resistant to Leptosphaeria maculans. Phytopathological analyses revealed that disease development of L. maculans was delayed when the pathogen was applied on an Avr9-mediated HR site. We demonstrate that the CJ9 and Avr9 gene can be functionally expressed in a heterologous plant species and that the two components confer an increase in disease resistance. 相似文献