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.     

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.     

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.     

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

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.     

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.     

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.     

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.     

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.     

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.     

No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation 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.     

Specific recognition of AVR4 and AVR9 results in distinct patterns of hypersensitive cell death in tomato, but similar patterns of defence-related gene expression

Hypersensitive cell death occurs in tomato seedlings that are derived from a cross between plants that express a resistance (Cf) gene against the pathogenic fungus Cladosporium fulvum and plants that contain the matching avirulence (Avr) gene originating from this fungus. The pattern of Cf-9/Avr9- and Cf-4/Avr4-induced necrosis in these F₁ seedlings was found to differ significantly. Macroscopic observation revealed that in F₁ tomato seedlings containing both Cf-9 and Avr9, numerous necrotic spots developed that were scattered over the entire cotyledon, while the midvein and primary veins remained unaffected. In seedlings containing both Cf-4 and Avr4, however, initially only one or a few necrotic spots developed on each cotyledon, in most cases in the midvein and occasionally in primary veins. Subsequently, these spots turned rapidly into lesions that enlarged along the midvein and primary veins, eventually causing the cotyledons to wilt and abscise. These observations were confirmed by detailed histological studies. Production of the AVR proteins in adult tomato plants carrying the matching Cf gene, employing potato virus X, resulted in similar patterns of necrosis. RNA gel blot analysis demonstrated that both Avr4 and Avr9, controlled by the CaMV 35S promoter, were highly expressed in seedlings already at one day post-emergence, indicating that the distinct necrotic patterns are not due to differences in Avr expression levels. We have analysed the expression of many genes involved in defence signalling pathways and the defence response itself, during the onset of the Cf/Avr-initiated hypersensitive response (HR). Although most of the genes were expressed stronger and faster in Cf-4/Avr4 seedlings than in Cf-9/Avr9 seedlings at the onset of HR, no significant qualitative differences in the expression of genes involved in downstream signalling were observed when Cf-4/Avr4- and Cf-9/Avr9-induced defence responses were compared.     

Cladosporium fulvum circumvents the second functional resistance gene homologue at the Cf-4 locus (Hcr9-4E ) by secretion of a stable avr4E isoform

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.     

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.     

CITRX thioredoxin is a putative adaptor protein connecting Cf-9 and the ACIK1 protein kinase during the Cf-9/Avr9- induced defence response

Tomato Cf-9, a receptor-like protein (RLP), confers resistance to races of the fungal pathogen Cladosporium fulvum that express the Avr9 avirulence gene. CITRX (Cf-9-interacting thioredoxin) was previously identified in a yeast two-hybrid screen as a protein interacting with the cytoplasmic domain of Cf-9 and shown to be a negative regulator of the cell death induced after Cf-9/Avr9 interaction. ACIK1 is a Ser/Thr protein kinase that is specifically required for the Cf-9 and Cf-4 dependent defence response in tomato. In this paper we present data suggesting that CITRX may act as an adaptor recruiting the ACIK1 kinase to the cytoplasmic domain of Cf-9 upon elicitation with the Avr9 peptide. Interestingly, the catalytic activities of both CITRX and ACIK1 are not required for their interaction.