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.     

Molecular evolution of virulence in natural field strains of Xanthomonas campestris pv. vesicatoria

The avrBs2 avirulence gene of the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria triggers disease resistance in pepper plants containing the Bs2 resistance gene and contributes to bacterial virulence on susceptible host plants. We studied the effects of the pepper Bs2 gene on the evolution of avrBs2 by characterizing the molecular basis for virulence of 20 X. campestris pv. vesicatoria field strains that were isolated from disease spots on previously resistant Bs2 pepper plants. All field strains tested were complemented by a wild-type copy of avrBs2 in their ability to trigger disease resistance on Bs2 plants. DNA sequencing revealed four mutant alleles of avrBs2, two of which consisted of insertions or deletions of 5 nucleotides in a repetitive region of avrBs2. The other two avrBs2 alleles were characterized by point mutations with resulting single amino acid changes (R403P or A410D). We generated isogenic X. campestris pv. vesicatoria strains by chromosomal avrBs2 gene exchange to study the effects of these mutations on the dual functions of avrBs2 in enhancing bacterial virulence and inducing plant resistance by in planta bacterial growth experiments. The deletion of 5 nucleotides led to loss of avrBs2-induced resistance on Bs2 pepper plants and abolition of avrBs2-mediated enhancement of fitness on susceptible plants. Significantly, the point mutations led to minimal reduction in virulence function of avrBs2 on susceptible pepper plants, with either minimal (R403P allele) or an intermediate level of (A410D allele) triggering of resistance on Bs2 plants. Consistent with the divergent selection pressures on avrBs2 exerted by the Bs2 resistance gene, our results show that avrBs2 is evolving to decrease detection by the Bs2 gene while at the same time maintaining its virulence function.     

An hrcU-homologous gene mutant of Xanthomonas campestris pv. glycines 8ra that lost pathogenicity on the host plant but was able to elicit the hypersensitive response on nonhosts.

Transposon mutagenesis was used to isolate nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra, which causes bacterial pustule disease in soybean. A 6.1-kb DNA region in which a mutation gave loss of pathogenicity was isolated and found to carry six open reading frames (ORFs). Four ORFs had homology with hrcU, hrcV, hrcR, and hrcS genes of Ralstonia solanacearum and X. campestris pv. vesicatoria. One nonpathogenic mutant, X. campestris pv. glycines H80, lost pathogenicity on soybean but was able to elicit the hypersensitive response (HR) on nonhost pepper and tomato plants. This mutant still multiplied as well as the wild type in the leaves or cotyledons of soybean. Although the DNA and amino acid sequences showed high homology with known hrp genes, the hrcU-homolog ORF is not required for HR induction on nonhost plants, pepper and tomato, or for the multiplication of bacteria in the host plant. This gene was only required for the pathogenic symptoms of X. campestris pv. glycines 8ra on soybean.     

A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3.

Strains of Xanthomonas campestris pv. vesicatoria that were avirulent in tomato leaves but virulent in pepper leaves were identified. A cloned gene, avrBsP, from one of the strains, Xv 87-7, converted a virulent strain in tomato to avirulent in tomato. A 1.7-kb subclone containing the avirulence gene cross-hybridized with the avirulence gene, which determines race 1 within the pepper group of strains (avrBs3). However, the two avirulence genes differ in their biological activity. The base sequences of the two avirulence genes were almost identical through the 1.7-kb segment of avrBsP, with significant differences only in some bases in the repeat region.     

Identification and expression profiling of tomato genes differentially regulated during a resistance response to Xanthomonas campestris pv. vesicatoria

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. Plants of the tomato line Hawaii 7981 are resistant to race T3 of X. campestris pv. vesicatoria expressing the type III effector protein AvrXv3 and develop a typical hypersensitive response upon bacterial challenge. A combination of suppression subtractive hybridization and microarray analysis identified a large set of cDNAs that are induced or repressed during the resistance response of Hawaii 7981 plants to X. campestris pv. vesicatoria T3 bacteria. Sequence analysis of the isolated cDNAs revealed that they correspond to 426 nonredundant genes, which were designated as XRE (Xanthomonas-regulated) genes and were classified into more than 20 functional classes. The largest functional groups contain genes involved in defense, stress responses, protein synthesis, signaling, and photosynthesis. Analysis of XRE expression kinetics during the tomato resistance response to X. campestris pv. vesicatoria T3 revealed six clusters of genes with coordinate expression. In addition, by using isogenic X. campestris pv. vesicatoria T2 strains differing only by the avrXv3 avirulence gene, we found that 77% of the identified XRE genes were directly modulated by expression of the AvrXv3 effector protein. Interestingly, 64% of the XRE genes were also induced in tomato during an incompatible interaction with an avirulent strain of Pseudomonas syringae pv. tomato. The identification and expression analysis of X. campestris pv. vesicatoria T3-modulated genes, which may be involved in the control or in the execution of plant defense responses, set the stage for the dissection of signaling and cellular responses activated in tomato plants during the onset of spot disease resistance.     

Gene-for-gene interactions between Pseudomonas syringae pv. phaseolicola and Phaseolus.

The gene for cultivar-specific avirulence to Phaseolus vulgaris cv. Tendergreen in races 3 and 4 of Pseudomonas syringae pv. phaseolicola was isolated and sequenced. Genomic clones from libraries of race 3 in pLAFR1 and race 4 in pLAFR3, which altered the phenotype of race 5 from virulent to avirulent in Tendergreen, were found to possess a common approximately 15-kb region of DNA that contained the determinant of avirulence. Subcloning and insertion mutagenesis with Tn1000 located an avirulence gene within a 1.4-kb BglII/HindIII DNA fragment in races 3 and 4. Comparison of the nucleotide sequences of regions of DNA that confer avirulence confirmed that both races have an identical gene for avirulence (designated avrPph3) comprising 801 base pairs (bp) and predicted to encode a cytoplasmic protein of 28,703 Da. A sequence, TGCAACCGAAT, 91% homologous to the motif found in promoter regions of avrB and avrD from P. s. pv. glycinea was located 89-99 bp upstream of the start of the open-reading frame 1. Hybridization experiments showed that avrPph3 was not plasmid-borne and was absent from isolates of P. s. pv. phaseolicola races 1, 2, 5, 6, 7, and 8, P. cichorii, P. s. pvs. coronafaciens, glycinea, maculicola, pisi, syringae, and tabaci. Cosegregation studies of crosses between cultivars resistant (Tendergreen) and susceptible (Canadian Wonder) to races 3 and 4 and transconjugants of race 5 confirmed that a gene-for-gene relationship controls specificity in the interaction between Tendergreen and races 3 and 4 of P. s. pv. phaseolicola.     

Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.

A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrB0 and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants containing avrC induced a light brown necrotic HR within 48 h on the soybean cultivars Acme, Peking, Norchief, and Flambeau. An additional avirulence gene, avrB1, cloned from race 1, appeared to be identical to avrB0 from race 0. The avrB0 and avrC genes from race 0 were characterized by restriction enzyme mapping, Southern blot analysis, Tn5 transposon mutagenesis, and site-directed gene replacements. The effects of these three genes on the in planta bacterial growth of race 4 transconjugants have also been examined. The identification and cloning of avrB1 provides genetic evidence for a gene-for-gene interaction in the bacterial blight disease of soybean, as avrB1 from race 1 interacts with the soybean disease resistance locus, Rpg1.     

Characterization of a unique chromosomal copper resistance gene cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.     

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and wild tomato relative Lycopersicon pennellii

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.     

Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae.

Races of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, interact with cultivars of rice in a gene-for-gene specific manner. Multiple DNA fragments of various sizes from all strains of X. o. pv. oryzae hybridized with avrBs3, an avirulence gene from Xanthomonas campestris pv. vesicatoria, in Southern blots; this suggests the presence of several homologs and possibly a gene family. A genomic library of a race 2 strain of X. o. pv. oryzae, which is avirulent on rice cultivars carrying resistance genes xa-5, Xa-7, and Xa-10, was constructed. Six library clones, which hybridized to avrBs3, altered the interaction phenotype with rice cultivars carrying either xa-5, Xa-7, or Xa-10 when present in a virulent race 6 strain. Two avirulence genes, avrXa7 and avrXa10, which correspond to resistance genes Xa-7 and Xa-10, respectively, were identified and partially characterized from the hybridizing clones. On the basis of transposon insertion mutagenesis, sequence homology, restriction mapping, and the presence of a repeated sequence, both genes are homologs of avirulence genes from dicot xanthomonad pathogens. Two BamHI fragments that are homologous to avrBs3 and correspond to avrXa7 and avrXa10 contain a different number of copies of a 102-bp direct repeat. The DNA sequence of avrXa10 is nearly identical to avrBs3. We suggest that avrXa7 and avrXa10 are members of an avirulence gene family from xanthomonads that control the elicitation of resistance in mono- and dicotyledonous plants.