Robert E. Stall

Ethylene regulates the susceptible response to pathogen infection in tomato

Ethylene evolution occurs concomitantly with the progression of disease symptoms in response to many virulent pathogen infections in plants. A tomato mutant impaired in ethylene perception—Never ripe—exhibited a significant reduction in disease symptoms in comparison to the wild type after inoculations of both genotypes with virulent bacterial (Xanthomonas campestris pv vesicatoria and Pseudomonas syringae pv tomato) and fungal (Fusarium oxysporum f sp lycopersici) pathogens. Bacterial spot disease symptoms were also reduced in tomato genotypes impaired in ethylene synthesis (1-aminocyclopropane-1-carboxylic acid deaminase) and perception (14893), thereby corroborating a reducing effect for ethylene insensitivity on foliar disease development. The reduction in foliar disease symptoms in Never ripe plants was a specific effect of ethylene insensitivity and was not due to reductions in bacterial populations or decreased ethylene synthesis. PR-1B1 mRNA accumulation in response to X. c. vesicatoria infection was not affected by ethylene insensitivity, indicating that ethylene is not required for defense gene induction. Our findings suggest that broad tolerance of diverse vegetative diseases may be achieved via engineering of ethylene insensitivity in tomato.

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

SummaryThe avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.

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.

Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions.

The taxonomy and evolutionary relationships among members of the genus Xanthomonas associated with tomato and pepper have been a matter of considerable controversy since their original description in 1921. These bacteria, which are a major affliction of tomato and pepper crops in warm and humid regions, were originally described as a single species, but subsequent research has shown the existence of at least two genetic groups differentiated by physiological, biochemical and pathological characteristics. This work synthesizes the findings from several approaches, including pathogenicity tests, enzymic activity, restriction fragment analysis of the entire genome, DNA-DNA hybridization and RNA sequence comparisons based on a 2097 base sequence comprising the 16S rRNA gene, the intergenic spacer located between the 16S and 23S rRNA genes and a small region of the 23S rRNA gene. Within the group of xanthomonads pathogenic on pepper and tomato four distinct phenotypic groups exist, of which three form distinct genomic species. These include Xanthomonas axonopodis pv. vesicatoria (A and C group), Xanthomonas vesicatoria (B group) and Xanthomonas gardneri (D group). On the basis of phenotypic and genotypic differences between A- and C-group strains, the C strains should be considered as a subspecies within Xanthomonas axonopodis pv. vesicatoria.

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.

Phenotypic and genotypic characterization of human and nonhuman Escherichia coli

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.

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.