Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper.

The AvrBs3 protein of the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria is targeted to host-plant cells by the bacterial Hrp type III secretion system. In pepper plants containing the Bs3 resistance gene, AvrBs3 induces the hypersensitive response (HR). AvrBs3 recognition is thought to occur in the plant cell nucleus as HR induction is dependent on nuclear localization signals (NLSs) and an acidic transcription activation domain (AAD). In a search for AvrBs3-interacting pepper proteins using the yeast two-hybrid system, we have isolated eight different classes of cDNA inserts including two genes for importin alpha proteins. Importin alpha is part of the nuclear import machinery and interacts with AvrBs3 through an NLS in the carboxy-terminus of the protein, both in yeast and in vitro. The mechanism of AvrBs3 recognition was further studied by analysis of the C-terminal AAD. This putative transcription-activation domain was shown to be required for AvrBs3 HR-inducing activity, and could be functionally replaced with the VP16 AAD from the Herpes simplex virus. Our data support the model in which the AvrBs3 effector localizes to the nucleus, where the Bs3-mediated surveillance system of resistant plants detects AvrBs3 through its interference with host gene transcription.

Genetic mapping and functional analysis of the tomato Bs4 locus governing recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 protein.

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.

Type III secretion and in planta recognition of the Xanthomonas avirulence proteins AvrBs1 and AvrBsT.

summary The hrp gene cluster of the plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) encodes a type III secretion system required for the delivery of virulence and avirulence proteins into the plant. Some of these effector proteins, e.g. AvrBs1 and AvrBsT, are recognized by pepper plants carrying corresponding resistance genes, triggering the hypersensitive reaction (HR). In this study, epitope tagged AvrBs1 and AvrBsT proteins were detected in culture supernatants only in the presence of a functional type III apparatus and not in a hrcV mutant, showing that both proteins are secreted by Xcv in an hrp-dependent manner. Expression of both avirulence genes is constitutive and independent of the hrp gene regulators, hrpG and hrpX. Transient expression of avrBs1 and avrBsT in resistant host plants using Agrobacterium tumefaciens-mediated gene transfer resulted in the induction of a specific HR. This indicates that recognition occurs intracellularly, and suggests that during the Xcv infection, AvrBs1 and AvrBsT are translocated from Xcv into the plant cell. We describe a conserved protein motif which is present in the N-terminal region of all known Xcv avirulence proteins and discuss its potential role in translocation into plant cells.

HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant.

The interaction between the plant pathogen Xanthomonas campestris pv. vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system. Among type III-secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions. Using non-polar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X. c. pv. vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis. Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non-conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion. Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell. In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp-dependent manner. As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process. We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell.

How the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria conquers the host.

Abstract Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on pepper and tomato. Pathogenicity on susceptible plants and the induction of the hypersensitive reaction (HR) on resistant plants requires a number of genes, designated hrp, most of which are clustered in a 23-kb chromosomal region. Nine hrp genes encode components of a type III protein secretion apparatus that is conserved in Gram-negative plant and animal pathogenic bacteria. We have recently demonstrated that Xcv secretes proteins into the culture medium in a hrp-dependent manner. Substrates of the Hrp secretion machinery are pathogenicity factors and avirulence proteins, e.g. AvrBs3. The AvrBs3 protein governs recognition, i.e. HR induction, when bacteria infect pepper plants carrying the corresponding resistance gene Bs3. Intriguingly, the AvrBs3 protein contains eukaryotic signatures such as nuclear localization signals (NLS), and has been shown to act inside the plant cell. We postulate that AvrBs3 is transferred into the plant cell via the Hrp type III pathway and that recognition of AvrBs3 takes place in the plant cell nucleus.

Gene-for-gene interactions: bacterial avirulence proteins specify plant disease resistance.

Resistance of plants to bacterial pathogens is often controlled by corresponding genes for resistance and avirulence in host and pathogen, respectively. Fifty years after discovery of the genetic basis of gene-for-gene interactions, several avirulence and plant resistance genes have been isolated and are being studied on the molecular level. Tremendous progress has been made due to a better understanding of type III secretion systems that are required for bacterial pathogenicity. We are beginning to grasp how the plant actually recognizes bacterial avirulence determinants. The current view is that the bacterium translocates avirulence proteins into the host cell by the Hrp type III secretion system and that recognition occurs in the plant cell.

Bacterial avirulence proteins as triggers of plant disease resistance.

Disease resistance in plants is often characterized by matching resistance and avirulence genes in host and pathogen, respectively. It has recently been shown that expression of bacterial avirulence genes in plants induces resistance gene-dependent defense reactions. The finding that avirulence proteins act inside plant cells represents a major advance in our understanding of host-pathogen specificity.

Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell.

The molecular mechanism by which bacterial avirulence genes mediate recognition by resistant host plants has been enigmatic for more than a decade. In this paper we provide evidence that the Xanthomonas campestris pv. vesicatoria avirulence protein AvrBs3 is recognized inside the plant cell. Transient expression of avrBs3 in pepper leaves, using Agrobacterium tumefaciens for gene delivery, results in hypersensitive cell death, specifically on plants carrying the resistance gene Bs3. In addition, for its intracellular recognition, AvrBs3 requires nuclear localization signals that are present in the C-terminal region of the protein. We propose that AvrBs3 is translocated into plant cells via the Xanthomonas Hrp type III secretion system and that nuclear factors are involved in AvrBs3 perception.

HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two-component response regulators.

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of pepper and tomato plants. Expression of its basic pathogenicity genes, the hrp genes, is induced in planta and in XVM2 medium and is dependent on the hrp regulatory gene hrpXv for five out of six loci in the 23-kb hrp cluster. Here we describe the isolation of a novel hrp gene, hrpG, that was identified after chemical mutagenesis and that is located next to the hrpXv gene. In a hrpG mutant induction of expression of the seven loci hrpA to hrpF, and hrpXv is abolished, suggesting that hrpG functions at the top of the hrp gene regulatory cascade. hrpG is the only gene in the locus and encodes a putative protein of 263 amino acids with a molecular mass of 28.9 kDa. The HrpG amino acid sequence shows similarity to response regulator proteins of the OmpR subclass of two-component systems, being mostly related to the ChvI proteins of Agrobacterium tumefaciens and Rhizobium spp., and TctD of Salmonella typhimurium. Expression of hrpG is low in complex medium, is increased in XVM2 by a factor of four, and is independent of other hrp loci. A model on hrp gene regulation in Xanthomonas campestris pv. vesicatoria is discussed.

The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity.

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato.

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum.

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter-beta-glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.

Molecular communication between host plant and the fungal tomato pathogen Cladosporium fulvum.

Host genotype specificity in interactions between biotrophic fungal pathogens and plants in most cases complies with the gene-for-gene model. Success or failure of infection is determined by absence or presence of complementary genes, avirulence and resistance genes, in the pathogen and the host plant, respectively. Resistance, expressed by the induction of a hypersensitive response followed by other defence responses in the host, is envisaged to be based on recognition of the pathogen, mediated through direct interaction between products of avirulence genes of the pathogen (the so-called race-specific elicitors) and receptors in the host plant, the putative products of resistance genes. The interaction between the biotrophic fungus Cladosporium fulvum and its only host tomato is a model system to study fungus-plant gene-for-gene relationships. Here we report on isolation, characterization and biological function of putative pathogenicity factors ECP1 and ECP2 and the race-specific elicitors AVR4 and AVR9 of C. fulvum and cloning and regulation of their encoding genes. Disruption of ecp1 and ecp2 genes has no clear effect on pathogenicity of C. fulvum. Disruption of the avr9 gene, which codes for the race-specific 28 amino acid AVR9 elicitor, in wild type avirulent races, leads to virulence on tomato genotypes carrying the complementary resistance gene Cf9. The avirulence gene avr4 encodes a 105 amino acid race-specific elicitor. A single basepair change in the avirulence gene avr4 leads to virulence on tomato genotypes carrying the Cf4 resistance gene.

The AVR9 race-specific elicitor of Cladosporium fulvum is processed by endogenous and plant proteases.

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces a hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is highly expressed when C. fulvum is growing in the plant and the elicitor accumulates in infected leaves as a 28-amino acid (aa) peptide. In C. fulvum grown in vitro, the peptide elicitor is not produced in detectable amounts. To produce significant amounts of the AVR9 elicitor in vitro, the coding and termination sequences of the avr9 gene were fused to the constitutive gpd-promoter (glyceraldehyde 3-phosphate dehydrogenase) of Aspergillus nidulans. Transformants of C. fulvum were obtained that highly expressed the avr9 gene in vitro and produced active AVR9 peptide elicitors. These peptides were partially sequenced from the N terminus and appeared to consist of 32, 33, and 34 aa's, respectively, and are the precursors of the mature 28-aa AVR9 peptide. We demonstrated that plant factors process the 34-aa peptide into the mature 28-aa peptide. We present a model for the processing of AVR9 involving cleavage of a signal peptide during excretion and further maturation by fungal and plant proteases into the stable 28-aa peptide elicitor.

Characterization of two putative pathogenicity genes of the fungal tomato pathogen Cladosporium fulvum.

The fungus Cladosporium fulvum is a biotrophic pathogen of tomato. On susceptible tomato plants, the fungus grows abundantly in the extracellular spaces between the mesophyll cells. The mechanism by which C. fulvum is able to establish and maintain basic compatibility on its one and only host species, the tomato, is unknown. The isolation and characterization of pathogenicity factors and the corresponding genes will provide insight into the mechanism by which C. fulvum colonizes its host. Two putative pathogenicity genes of C. fulvum encoding proteins, which occur abundantly in the extracellular space of infected tomato leaves, were isolated and characterized (ecp1 and ecp2). The DNA sequences of these ecp genes (encoding extracellular protein) do not share homology to any sequence present in the DNA databases. The ecp genes are highly expressed in planta but not in vitro, suggesting that they play a significant role in pathogenesis.

Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis.

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.

Cloning and characterization of cDNA of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum, causal agent of tomato leaf mold.

A race-specific peptide elicitor from Cladosporium fulvum induces a hypersensitive response on Cf9 tomato genotypes. We have hypothesized that the avirulence of fungal races on Cf9 genotypes is due to the production of this elicitor by an avirulence gene, avr9. To obtain cDNA clones of the avr9 gene, oligonucleotide probes were designed based on the amino acid sequence determined previously. In northern blot analysis, one oligonucleotide detected an mRNA of 600 nucleotides in tomato-C. fulvum interactions involving fungal races producing the elicitor. A primer extension experiment indicated that the probe hybridized to a region near position 270 of the mRNA. The probe was used to screen a cDNA library made from poly(A)+ RNA from an appropriate compatible tomato-C. fulvum interaction. One clone was obtained corresponding to the mRNA detected by the oligonucleotide probe. Sequence analysis revealed that this clone encoded the avr9 elicitor. By isolating longer clones and by RNA sequencing, the primary structure of the mRNA was determined. The mRNA contains an open reading frame of 63 amino acids, including the sequence of the elicitor at the carboxyterminus. A time course experiment showed that the avr9 mRNA accumulates in a compatible tomato-C. fulvum interaction in correlation with the increase of fungal biomass. The avr9 gene is a single-copy gene that is absent in fungal races which are virulent on tomato Cf9 genotypes. Possible functions of the avirulence gene are discussed.