Gerald V. Minsavage

Gene-for-gene relationships specifying disease resistance in Xanthomonas campestris pv. vesicatoria - pepper interactions.

In this study, we describe the cloning and characterization of three avirulence genes from X.c. pv. vesicatoria. We present evidence that these avirulence genes restrict the host range of X.c. pv. vesicatoria strains

Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998.

The molecular and genetic control of the interaction between tomato races of Xanthomonas campestris pv. vesicatoria (XcvT) and tomato was studied. Based on inoculation phenotype and analysis of in planta bacterial growth, tomato line Hawaii 7998 is resistant to XcvT race 1 75-3 but not to XcvT race 2 89-1. Two cosmid clones from a genomic library of XcvT race 1 75-3 converted the normally virulent race 2 89-1 to avirulence on Hawaii 7998. The two clones contained the previously isolated, nonhost avirulence gene avrRxv, and their activity was localized to a 2.1-kbp subclone of avrRxv. avrRxv inhibits growth of race 2 89-1 in the resistant line Hawaii 7998 and an insertional mutation in avrRxv prevents this inhibition. In addition, a dramatic increase in electrolyte leakage of leaves of Hawaii 7998 occurred after 12-hr postinfiltration with race 2 89-1 carrying avrRxv. The nucleotide sequence of avrRxv revealed one major open reading frame (ORF) that accords well with activity analysis of nested deletions. ORF 2-2 encodes a putative protein of 374 amino acids with a molecular weight of 42.1 kDa and a pI of 10.7. Inheritance of the avrRxv-specific resistance in Hawaii 7998 was studied in a total of 587 F2 individuals from crosses between Hawaii 7998 and susceptible lines. The inheritance of avrRxv-specific resistance in Hawaii 7998 appears to be governed by more than one locus.

Resistance of Tomato and Pepper to T3 Strains of Xanthomonas campestris pv. vesicatoria Is Specified by a Plant-Inducible Avirulence Gene

Tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes. One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants. A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced. The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene. Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system. Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein. Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.

Two genetically diverse groups of strains are included in Xanthomonas campestris pv. vesicatoria.

Two genetically diverse groups of strains were identified among cultures of Xanthomonas campestris pv. vesicatoria isolated from plants with bacterial spot of pepper and tomato. Group A strains do not pit pectate gels or hydrolyze starch, whereas group B strains are strongly positive for these reactions. Group A strains cause a hypersensitive reaction in plants of tomato breeding line Hawaii 7998, but group B strains do not. Other differences between the two groups of strains were discovered in tests for utilization of carbon compounds, serology, fatty acid profiles, silver-stained protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and DNA restriction enzyme digestion profiles. The levels of DNA homology between strains belonging to the same group were more than 74%, but the levels of DNA homology between strains belonging to different groups were less than 46%. The two groups of strains have different genetic backgrounds, but cause essentially the same disease of tomato and pepper.

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.

Durability of Resistance in Tomato and Pepper to Xanthomonads Causing Bacterial Spot

Both hypersensitive and quantitative forms of resistance to the bacterial spot pathogens (Xanthomonas spp.) occur in pepper and tomato. Five resistance genes involved in hypersensitivity in pepper and four in tomato have been identified so far. The corresponding pathogen avirulence genes have been cloned and characterized, and features, including a propensity for accumulating mutations and at times, loss of plasmid-borne avirulence genes, are known to occur. The frequency of these changes affects race composition among pathogen populations and determines the durability of the corresponding plant resistance. At least four different species of Xanthomonas are known to cause bacterial spot, and these can differ in specific avirulence gene content. Quantitative or multigenic resistance has also more recently been researched and appears to be more durable than the hypersensitive resistance. Two recessive genes have been identified that yield a nonhypersensitive form of resistance in pepper and together can provide strong resistance. More emphasis is being given to transfer of quantitative trait resistance to commercial cultivars of both tomato and pepper.

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and the 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.

RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

Identification of Xanthomonas citri ssp. citri host specificity genes in a heterologous expression host.

We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-A(w), a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans, as a recipient of the Xac-A(w) genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf1. Xac-A(w)DeltaavrGf1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-A(w) hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw. This gene, when expressed in a pustule-minus Xac-A(w), did not complement pustule formation; however, pthA(w), a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-A(w) contains other unidentified factors that suppress development in grapefruit.

Detection of Xanthomonas campestris pv. vesicatoria associated with pepper and tomato seed by DNA amplification.

Detection of Xanthomonas campestris pv. vesicatoria associated with pepper and tomato seed was achieved by amplification of a DNA fragment of the bacterium by the polymerase chain reaction. Oligonucleotide primers specific for the hypersensitive reaction and pathogenicity (hrp) gene cluster were used in the reaction. The method includes extraction of total DNA from buffered seed washings to which sodium ascorbate and insoluble polyvinylpolypyrrolidone were added. The hrp fragments were amplified from the DNA preparations if cells of X. c. pv. vesicatoria were added to seed washes. Identification of an hrp fragment as that from X. c. pv. vesicatoria was attained by restriction enzyme analyses of the amplified fragment. The minimum number of cells that could be detected in washes from pepper or tomato seed was from 10 2 to 10 3 cfu per ml. This was about 1,000 times fewer than the minimum number of cells detected with an enzyme-linked immunosorbent assay. The pathogen was also detected in washes obtained from several lots of naturally contaminated pepper and tomato seed, one of which contained background bacterial microflora greater than 10 7 cfu per g of seed.