Evolution of a guarded decoy protease and its receptor in solanaceous plants.

Rcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.

Biological and Molecular Properties of Wild potato mosaic virus Isolates from Pepino (Solanum muricatum).

In 1976, a virus with flexuous, filamentous virions typical of the family Potyviridae was isolated from symptomatic pepino (Solanum muricatum) plants growing in two valleys in Peru's coastal desert region. In 2014, a virus with similar-shaped virions was isolated from asymptomatic fruits obtained from pepino plants growing in six coastal valleys and a valley in Peru's Andean highlands. Both were identified subsequently as Wild potato mosaic virus (WPMV) by serology or high-throughput sequencing (HTS). The symptoms caused by two old and seven new isolates from pepino were examined in indicator plants. Infected solanaceous hosts varied considerably in their sensitivities to infection and individual isolates varied greatly in virulence. All seven new isolates caused quick death of infected Nicotiana benthamiana plants and more than half of them killed infected plants of Physalis floridana and S. chancayense. These three species were the most sensitive to infection. The most virulent isolate was found to be BA because it killed five of eight solanaceous host species whereas CA was the least severe because it only killed N. benthamiana. Using HTS, complete genomic sequences of six isolates were obtained, with one isolate (FE) showing evidence of recombination. The distances between individual WPMV isolates in phylogenetic trees and the geographical distances between their collection sites were found to be unrelated. The individual WPMV isolates displayed nucleotide sequence identities of 80.9-99.8%, whereas the most closely related virus, Potato virus V (PVV), was around 75% identical to WPMV. WPMV, PVV, and Peru tomato virus formed clusters of similar phylogenetic diversity, and were found to be distinct but related viruses within the overall Potato virus Y lineage. WPMV infection seems widespread and of likely economic significance to pepino producers in Peru's coastal valleys. Because it constitutes the fifth virus found infecting pepino and this crop is entirely vegetatively propagated, development of healthy pepino stock programs is advocated.

Antibacterial Activities of Crude Secondary Metabolite Extracts from Pantoea Species Obtained from the Stem of Solanum mauritianum and Their Effects on Two Cancer Cell Lines.

Endophytes are microorganisms that are perceived as non-pathogenic symbionts found inside plants since they cause no symptoms of disease on the host plant. Soil conditions and geography among other factors contribute to the type(s) of endophytes isolated from plants. Our research interest is the antibacterial activity of secondary metabolite crude extracts from the medicinal plant Solanum mauritianum and its bacterial endophytes. Fresh, healthy stems of S. mauritianum were collected, washed, surface sterilized, macerated in PBS, inoculated in the nutrient agar plates, and incubated for 5 days at 30 °C. Amplification and sequencing of the 16S rRNA gene was applied to identify the isolated bacterial endophytes. These endophytes were then grown in nutrient broth for 7⁻14 days, after which sterilized Amberlite® XAD7HP 20⁻60 mesh (Merck KGaA, Darmstadt, Germany) resin was added to each culture to adsorb the secondary metabolites, which were later extracted using ethyl acetate. Concentrated crude extracts from each bacterial endophyte were tested for antibacterial activity against 11 pathogenic bacteria and two human cancer cell lines. In this study, a total of three bacterial endophytes of the Pantoea genus were identified from the stem of S. mauritianum. The antibacterial test showed that crude secondary metabolites of the endophytes and stem of S. mauritianum possessed antibacterial properties against pathogenic microbes such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with concentrations showing inhibition ranging from 0.0625 to 8.0000 mg/mL. The anticancer analysis showed an increase in cell proliferation when A549 lung carcinoma and UMG87 glioblastoma cell lines were treated with both the plant and endophytes' crude extracts. As far as we know, this is the first study of its kind on Solanum mauritianum in South Africa showing S. mauritianum endophytes having activity against some of the common human pathogenic organisms.

The ELR-SOBIR1 Complex Functions as a Two-Component Receptor-Like Kinase to Mount Defense Against Phytophthora infestans.

The ELICITIN RESPONSE protein (ELR) from Solanum microdontum can recognize INF1 elicitin of Phytophthora infestans and trigger defense responses. ELR is a receptor-like protein (RLP) that lacks a cytoplasmic signaling domain and is anticipated to require interaction with a signaling-competent receptor-like kinase. SUPPRESSOR OF BIR1-1 (SOBIR1) has been proposed as a general interactor for RLPs involved in immunity and, as such, is a potential interactor for ELR. Here, we investigate whether SOBIR1 is required for response to INF1 and resistance to P. infestans and whether it associates with ELR. Our results show that virus-induced gene silencing of SOBIR1 in Nicotiana benthamiana leads to loss of INF1-triggered cell death and increased susceptibility to P. infestans. Using genetic complementation, we found that the kinase activity of SOBIR1 is required for INF1-triggered cell death. Coimmunoprecipitation experiments showed that ELR constitutively associates with potato SOBIR1 in planta, forming a bipartite receptor complex. Upon INF1 elicitation, this ELR-SOBIR1 complex recruits SERK3 (SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3) leading to downstream signaling activation. Overall, our study shows that SOBIR1 is required for basal resistance to P. infestans and for INF1-triggered cell death and functions as an adaptor kinase for ELR.

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor.

Plant-pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface-localized receptor Ve1 confers race-specific resistance against race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.

The plant genetic background affects the efficiency of the pepper major nematode resistance genes Me1 and Me3.

KEY MESSAGE: The plant genetic background influences the efficiency of major resistance genes to root-knot nematodes in pepper and has to be considered in breeding strategies. Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous plant parasites worldwide. Since the use of most chemical nematicides is being prohibited, genetic resistance is an efficient alternative way to protect crops against these pests. However, nematode populations proved able to breakdown plant resistance, and genetic resources in terms of resistance genes (R-genes) are limited. Sustainable management of these valuable resources is thus a key point of R-gene durability. In pepper, Me1 and Me3 are two dominant major R-genes, currently used in breeding programs to control M. arenaria, M. incognita and M. javanica, the three main RKN species. These two genes differ in the hypersensitive response induced by nematode infection. In this study, they were introgressed in either a susceptible or a partially resistant genetic background, in either homozygous or heterozygous allelic status. Challenging these genotypes with an avirulent M. incognita isolate demonstrated that (1) the efficiency of the R-genes in reducing the reproductive potential of RKNs is strongly affected by the plant genetic background, (2) the allelic status of the R-genes has no effect on nematode reproduction. These results highlight the primary importance of the choice of both the R-gene and the genetic background into which it is introgressed during the selection of new elite cultivars by plant breeders.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

Improvement in antioxidant activity, angiotensin-converting enzyme inhibitory activity and in vitro cellular properties of fermented pepino milk by Lactobacillus strains containing the glutamate decarboxylase gene.

BACKGROUND: The purpose of this study was to evaluate the functional potential of fermented pepino extract (PE) milk by Lactobacillus strains containing the glutamate decarboxylase (GAD) gene. Three Lactobacillus strains were selected, including L. brevis BCRC 12310, L. casei BCRC 14082 and L. salivarius subsp. salivarius BCRC 14759. The contents of free amino acids, total phenolics content, total carotenoids and the associated functional and antioxidant abilities were analyzed, including angiotensin-converting enzyme (ACE) inhibition activity, 1,1-diphenyl-2-picylhydrazyl (DPPH) radical-scavenging ability and oxygen radical absorbance capacity (ORAC). Cell proliferation of fermented PE milk was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: Compared to the unfermented PE, fermented PE milk from Lactobacillus strains with the GAD gene showed higher levels of total phenolics, γ-aminobutyric acid, ACE inhibitory activity, DPPH, and ORAC. The viability of human promyelocytic leukemia cells (HL-60) determined by the MTT method decreased significantly when the cells were incubated with the PE and the fermented PE milk extracts. CONCLUSION: The consumption of fermented PE milk from Lactobacillus strains with the GAD gene is expected to benefit health. Further application as a health food is worthy of investigation. © 2012 Society of Chemical Industry.

Inbreeding increases susceptibility to powdery mildew (Oidium neolycopersici) infestation in horsenettle (Solanum carolinense L).

Inbreeding is common in flowering plants, but relatively few studies have examined its effects on interactions between plants and other organisms, such as herbivores and pathogens. In a recent paper, we documented effects of inbreeding depression on plant volatile signaling phenotypes, including elevated constitutive volatile emissions (and consequently greater herbivore recruitment to inbred plants) but reduced emission of key herbivore-induced volatiles that attract predatory and parasitic insects to damaged plants. While the effects of inbreeding on plant-insect interactions have been explored in only a few systems, even less is known about its effects on plant-pathogen interactions. Here we report the effects of inbreeding on horsenettle susceptibility to powdery mildew (Oidium neolycopersici), including more rapid onset of infection in inbred plants, particularly when plants were not previously damaged. These data suggest that inbreeding may increase plant susceptibility to pathogen infection and, therefore, may potentially facilitate pathogen establishment in natural populations.

An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts.

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.

Diversity, distribution, and evolution of Solanum bulbocastanum late blight resistance genes.

Knowledge on the evolution and distribution of late blight resistance genes is important for a better understanding of the dynamics of these genes in nature. We analyzed the presence and allelic diversity of the late blight resistance genes Rpi-blb1, Rpi-blb2, and Rpi-blb3, originating from Solanum bulbocastanum, in a set of tuber-bearing Solanum species comprising 196 different taxa. The three genes were only present in some Mexican diploid as well as polyploid species closely related to S. bulbocastanum. Sequence analysis of the fragments obtained from the Rpi-blb1 and Rpi-blb3 genes suggests an evolution through recombinations and point mutations. For Rpi-blb2, only sequences identical to the cloned gene were found in S. bulbocastanum accessions, suggesting that it has emerged recently. The three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their spread is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2, and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2, and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach, some accessions could be identified that contain novel, as yet unknown, late blight resistance factors in addition to the Rpi-blb1, Rpi-blb2, and Rpi-blb3 genes.

Mapping and cloning of late blight resistance genes from Solanum venturii using an interspecific candidate gene approach.

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late-blight-resistant cultivars but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized further. Here, we report on the cloning of dominant Rpi genes from S. venturii. Rpi-vnt1.1 and Rpi-vnt1.3 were mapped to chromosome 9 using nucleotide binding site (NBS) profiling. Subsequently, a Tm-2(2)-based allele mining strategy was used to clone both genes. Rpi-vnt1.1 and Rpi-vnt1.3 belong to the coiled-coil NBS leucine-rich repeat (LRR) class of plant R genes and encode predicted peptides of 891 and 905 amino acids (aa), respectively, which share 75% amino acid identity with the Tomato mosaic virus resistance protein Tm-2(2) from tomato. Compared with Rpi-vnt1.1, Rpi-vnt1.3 harbors a 14-aa insertion in the N-terminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum.

Evidence of parietal amine oxidase activity in Solanum torvum Sw. stem calli after Ralstonia solanacearum inoculation.

Calli induced from Solanum torvum stem explants were inoculated with Ralstonia solanacearum under partial vacuum. All calli showed a hypersensitive response after infiltration. Furthermore, amine oxidase activity with aldehyde and H(2)O(2) production was detected in semi-purified cell walls of calli infiltrated by the bacteria. Due to its preferential affinity for monoamines, this enzyme is supposed to have monoamine oxidase-like (MAO-like) activity. Moreover, the presence of hydroxyl radicals in the aromatic cycle alters the oxidative deamination kinetics of potential substrates. Indeed, the oxidation of dopamine (+2, OH) was shown to be faster than that of tyramine (+1, OH), which in turn was faster than that of phenylethylamine (0, OH). The MAO-like catalytic activity was significantly inhibited by some reducing agents such as sodium bisulphite and cysteine, and also by tryptamine under anaerobiosis. This latter result suggested that the prosthetic group of the MAO-like enzyme could be a tyrosine-derived 6-hydroxytopaquinone structure. Finally, the sigmoid kinetics of the MAO-like enzyme in semi-purified cell walls did not correspond to that expected for a purified MAO, suggesting that the kinetics were affected by some factors present in cell walls.

Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments.

BACKGROUND: Quantitative phenotypic variation of agronomic characters in crop plants is controlled by environmental and genetic factors (quantitative trait loci = QTL). To understand the molecular basis of such QTL, the identification of the underlying genes is of primary interest and DNA sequence analysis of the genomic regions harboring QTL is a prerequisite for that. QTL mapping in potato (Solanum tuberosum) has identified a region on chromosome V tagged by DNA markers GP21 and GP179, which contains a number of important QTL, among others QTL for resistance to late blight caused by the oomycete Phytophthora infestans and to root cyst nematodes. RESULTS: To obtain genomic sequence for the targeted region on chromosome V, two local BAC (bacterial artificial chromosome) contigs were constructed and sequenced, which corresponded to parts of the homologous chromosomes of the diploid, heterozygous genotype P6/210. Two contiguous sequences of 417,445 and 202,781 base pairs were assembled and annotated. Gene-by-gene co-linearity was disrupted by non-allelic insertions of retrotransposon elements, stretches of diverged intergenic sequences, differences in gene content and gene order. The latter was caused by inversion of a 70 kbp genomic fragment. These features were also found in comparison to orthologous sequence contigs from three homeologous chromosomes of Solanum demissum, a wild tuber bearing species. Functional annotation of the sequence identified 48 putative open reading frames (ORF) in one contig and 22 in the other, with an average of one ORF every 9 kbp. Ten ORFs were classified as resistance-gene-like, 11 as F-box-containing genes, 13 as transposable elements and three as transcription factors. Comparing potato to Arabidopsis thaliana annotated proteins revealed five micro-syntenic blocks of three to seven ORFs with A. thaliana chromosomes 1, 3 and 5. CONCLUSION: Comparative sequence analysis revealed highly conserved collinear regions that flank regions showing high variability and tandem duplicated genes. Sequence annotation revealed that the majority of the ORFs were members of multiple gene families. Comparing potato to Arabidopsis thaliana annotated proteins suggested fragmented structural conservation between these distantly related plant species.

Changes in the antioxidant status in leaves of Solanum species in response to elicitor from Phytophthora infestans.

Three Solanum genotypes with various polygenic resistance levels to the oomycete pathogen Phytophthora infestans (Mont.) De Bary were studied for their antioxidant response to the pathogen culture filtrate (CF). Detached plant leaves were treated with CF for 6, 18 and 30 h, and assayed for changes in hydrogen peroxide content, total ascorbate and glutathione pools and redox ratios (reduced form to total pool), as well as for changes in activities of ascorbate peroxidase, glutathione reductase and glutathione-S-transferase. In CF treated leaves of non-host resistant S. nigrum var. gigantea and field resistant S. tuberosum cv Bzura, the H(2)O(2) content did not change in comparison to water treated control leaves, whereas in the susceptible S. tuberosum clone H-8105 it decreased below the control level. In CF treated leaves of all genotypes, the total ascorbate pools were relatively unaltered and their redox ratio changed only transiently. In Bzura leaves the total glutathione content increased earlier than in the two other genotypes. The glutathione redox ratio remained rather stable, except for the susceptible clone H-8105, where it decreased transiently by about 42%. The relative increases in activity of all the studied enzymes were the highest in the susceptible clone H-8105. The results are discussed in the light of oxidative processes occurring in CF treated leaves. We conclude that stringent control of pro- and anti-oxidant reactions bringing the H(2)O(2) and/or cellular redox state to the threshold level is decisive for deployment of an effective defense strategy.

Expression levels of avrBs3-like genes affect recognition specificity in tomato Bs4- but not in pepper Bs3-mediated perception.

The tomato Bs4 disease resistance gene mediates recognition of avrBs4-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria to give a hypersensitive response (HR). Here, we present the characterization of the Bs4 promoter and its application for low-level expression of bacterial type III effector proteins in planta. Real-time polymerase chain reaction showed that Bs4 is constitutively expressed at low levels and that transcript abundance does not change significantly upon infection with avrBs4-containing xanthomonads. A 302-bp promoter fragment was found to be sufficient to promote Bs4 gene function. Previous studies have shown that high, constitutive in planta expression of avrBs3 (AvrBs3 and AvrBs4 proteins are 96.6% identical) via the Cauliflower mosaic virus 35S (35S) promoter triggers a Bs4-dependent HR whereas X. campestris pv. vesicatoria-mediated delivery of AvrBs3 into the plant cytoplasm does not. Here, we demonstrate that, when expressed under control of the weak Bs4 promoter, avrBs3 does not trigger a Bs4-dependent HR whereas avrBs4 does. In contrast, the pepper Bs3 gene, which mediates recognition of AvrBs3- but not AvrBs4-delivering xanthomonads, retains its recognition specificity even if avrBs4 was expressed in planta from the strong 35S promoter. Importantly, Bs4 promoter-driven expression of hax3, hax4 (two recently isolated avrBs3-like genes), avrBs3, and avrBs4 resulted in identical reactions as observed upon infection with X. campestris pv. vesicatoria strains that express the respective avr gene, suggesting that the protein levels expressed under control of the Bs4 promoter are similar to those that are translocated by the bacterial type III secretion system.