The F-box protein ACRE189/ACIF1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato

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.     

Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp

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.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

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.     

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.     

CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence

To identify proteins involved in tomato Cf-9 resistance protein function, a yeast two-hybrid screen was undertaken using the cytoplasmic C-terminus of Cf-9 as bait. A thioredoxin-homologous clone, interacting specifically with Cf-9, was identified and called CITRX (Cf-9-interacting thioredoxin). Virus-induced gene silencing (VIGS) of CITRX resulted in an accelerated Cf-9/Avr9-triggered hypersensitive response in both tomato and Nicotiana benthamiana, accompanied by enhanced accumulation of reactive oxygen species, alteration of protein kinase activity and induction of defence-related genes. VIGS of CITRX also conferred increased resistance to the fungal pathogen Cladosporium fulvum in the otherwise susceptible Cf0 tomato. CITRX acts as a negative regulator of the cell death and defence responses induced through Cf-9, but not Cf-2. Recognition of the Cf-9 C-terminus by CITRX is necessary and sufficient for this negative regulation. This is the first study that implicates thioredoxin activity in the regulation of plant disease resistance.     

The Cf-9 disease resistance protein is present in an approximately 420-kilodalton heteromultimeric membrane-associated complex at one molecule per complex

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.     

Gene shuffling-generated and natural variants of the tomato resistance gene Cf-9 exhibit different auto-necrosis-inducing activities in Nicotiana species

Tomato Cf genes encode membrane-bound proteins with extracellular leucine-rich repeats, and confer resistance to the fungal tomato pathogen Cladosporium fulvum, and a hypersensitive response (HR) to C. fulvum-derived race-specific elicitors. Several Cf genes, including Cf-4 and Cf-9, are members of the highly homologous Hcr9 (homologues of C. fulvumresistance gene Cf-9) gene family. Hcr9s evolve mainly by sequence exchange between paralogues, by which novel Cf genes may be generated. To mimic this aspect of natural evolution, we generated chimeras between multiple Hcr9s in vitro by gene shuffling. The shufflants were tested for novel specificities by transient expression in Nicotiana benthamiana. Many shufflants induced an HR in the absence of fungal elicitors and were designated auto-activators. We also identified two natural Hcr9 auto-activators in the wild tomato species Lycopersicon peruvianum, which induced an HR upon expression in N. benthamiana. The Hcr9 auto-activators exhibit different auto-necrosis-inducing specificities in five selected species of the Nicotiana genus, and they were shown to function in the same signalling pathway as Cf-9. Auto-activating alleles of nucleotide binding site-leucine-rich repeat genes and the protein kinase Pto were previously described. The auto-activators described here, belonging to the Cf-like structural class of resistance genes, shed light on this important phenotype and may be used as tools to unravel the mechanisms by which this class of resistance proteins function.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

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.     

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis

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.     

K+ channels of Cf-9 transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9 elicitor-dependent signal transduction

The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (IK,in) and outward-rectifying (IK,out) K+ channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of IK,out and almost complete suppression of IK,in within 3-5 min. The K+ channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K+ channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 microM staurosporine and 100 microM H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K+ channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.     

Tomato mitogen-activated protein kinases LeMPK1, LeMPK2, and LeMPK3 are activated during the Cf-4/Avr4-induced hypersensitive response and have distinct phosphorylation specificities

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.     

Cladosporium fulvum overcomes Cf-2-mediated resistance by producing truncated AVR2 elicitor proteins

The Cf-2 gene of tomato confers resistance to strains of the biotrophic pathogenic fungus Cladosporium fulvum carrying avirulence gene Avr2. To allow dissection of the biochemical mechanism of perception of AVR2 by Cf-2, we set out to clone the Avr2 gene. Here, we report the functional cloning of Avr2 cDNA, based on the induction of a hypersensitive response (HR) by the encoded AVR2 protein in Cf2 tomato plants. Analysis of strains of C. fulvum that are virulent on Cf2 tomato lines revealed various independent frameshift mutations in the Avr2 open reading frame (ORF) and a point mutation resulting in a premature stop codon. All modifications result in the production of truncated AVR2 proteins. Interestingly, an additional modification involves the insertion of a LINE-like element, Cfl1, in the Avr2 ORF. Cfl1 is the first LINE-like element identified in C. fulvum and provides the first example of loss of avirulence of a plant pathogen caused by insertion of a retrotransposable element in an Avr gene. Rcr3 represents an additional plant protein that is specifically required for Cf-2-mediated resistance. Analysis of two different rcr3 mutant Cf2 tomato plants revealed that their ability to respond to AVR2 with a HR correlates with their degree of resistance to AVR2-producing strains of C. fulvum. These data support a role for Rcr3 in the perception of AVR2 by Cf-2.     

Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.     

CMPG1-dependent cell death follows perception of diverse pathogen elicitors at the host plasma membrane and is suppressed by Phytophthora infestans RXLR effector AVR3a

? Little is known about how effectors from filamentous eukaryotic plant pathogens manipulate host defences. Recently, Phytophthora infestans RXLR effector AVR3a has been shown to target and stabilize host E3 ligase CMPG1, which is required for programmed cell death (PCD) triggered by INF1. We investigated the involvement of CMPG1 in PCD elicited by perception of diverse pathogen proteins, and assessed whether AVR3a could suppress each. ? The role of CMPG1 in PCD events was investigated using virus-induced gene silencing, and the ability of AVR3a to suppress each was determined by transient expression of natural forms (AVR3a(KI) and AVR3a(EM)) and a mutated form, AVR3a(KI/Y147del) , which is unable to interact with or stabilize CMPG1. ? PCD triggered at the host plasma membrane by Cf-9/Avr9, Cf-4/Avr4, Pto/AvrPto or the oomycete pathogen-associated molecular pattern (PAMP), cellulose-binding elicitor lectin (CBEL), required CMPG1 and was suppressed by AVR3a, but not by the AVR3a(KI/Y147del) mutant. Conversely, PCD triggered by nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins R3a, R2 and Rx was independent of CMPG1 and unaffected by AVR3a. ? CMPG1-dependent PCD follows perception of diverse pathogen elicitors externally or in association with the inner surface of the host plasma membrane. We argue that AVR3a targets CMPG1 to block initial signal transduction/regulatory processes following pathogen perception at the plasma membrane.