Relative Importance of Bacteriocin-Like Genes in Antagonism of Xanthomonas perforans Tomato Race 3 to Xanthomonas euvesicatoria Tomato Race 1 Strains

In a previous study, tomato race 3 (T3) strains of Xanthomonas perforans became predominant in fields containing both X. euvesicatoria and X. perforans races T1 and T3, respectively. This apparent ability to take over fields led to the discovery that there are three bacteriocin-like compounds associated with T3 strains. T3 strain 91-118 produces at least three different bacteriocin-like compounds (BCN-A, BCN-B, and BCN-C) antagonistic toward T1 strains. We determined the relative importance of the bacteriocin-like compounds by constructing the following mutant forms of a wild-type (WT) T3 strain to evaluate the antagonism to WT T1 strains: Mut-A (BCN-A⁻), Mut-B (BCN-B⁻), Mut-C (BCN-C⁻), Mut-AB, Mut-BC, and Mut-ABC. Although all mutant and WT T3 strains reduced the T1 populations in in planta growth room experiments, Mut-B and WT T3 were significantly more effective. Mutants expressing BCN-B and either BCN-A or BCN-C reduced T1 populations less than mutants expressing only BCN-A or BCN-C. The triple-knockout mutant Mut-ABC also had a significant competitive advantage over the T1 strain. In pairwise-inoculation field experiments where plants were coinoculated with an individual mutant or WT T3 strain and the T1 strain, the mutant strains and the WT T3 strain were reisolated from more than 70% of the lesions. WT T3 and Mut-B were the most frequently reisolated strains. In field experiments where plants were group inoculated with Mut-A, Mut-B, Mut-C, Mut-ABC, and WT T1 and T3 strains, Mut-B populations dominated all three seasons. In greenhouse and field experiments, the WT and mutant T3 strains had a selective advantage over T1 strains. Bacterial strains expressing both BCN-A and BCN-C appeared to have a competitive advantage over all other mutant and WT strains. Furthermore, BCN-B appeared to be a negative factor, with mutant T3 strains lacking BCN-B having a selective advantage in the field.     

Calcicludine binding to the outer pore of L-type calcium channels is allosterically coupled to dihydropyridine binding

How dihydropyridines modulate L-type voltage-gated Ca2+ channels is not known. Dihydropyridines bind cooperatively with Ca2+ binding to the selectivity filter, suggesting that they alter channel activity by promoting structural rearrangements in the pore. We used radioligand binding and patch-clamp electrophysiology to demonstrate that calcicludine, a toxin from the venom of the green mamba snake, binds in the outer vestibule of the pore and, like Ca2+, is a positive modulator of dihydropyridine binding. Data were fit using an allosteric scheme where dissociation constants for dihydropyridine and calcicludine binding, KDHP and KCaC, are linked via the coupling factor, alpha. Nine acidic amino acids located within the S5-Pore-helix segment of repeat III were sequentially changed to alanine in groups of three, resulting in the mutant channels, Mut-A, Mut-B, and Mut-C. Mut-A, whose substitutions are proximal to IIIS5, exhibits a 4.5-fold reduction in dihydropyridine binding and is insensitive to calcicludine binding. Block of Mut-A currents by calcicludine is indistinguishable from wild-type, indicating that KCaC is unchanged and that the coupling between dihydropyridine and calcicludine binding (i.e., alpha) is disrupted. Mut-B and Mut-C possess KDHP values that resemble that of the wild type. Mut-C, the most C-terminal of the mutant channels, is insensitive to calcicludine binding and block. KCaC values for the Mut-C single mutants, E1122A, D1127A, and D1129A, increase from 0.3 (wild type) to 1.14, 2.00, and 20.5 microM, respectively. Together, these findings suggest that dihydropyridine antagonist and calcicludine binding to L-type Ca2+ channels promote similar structural changes in the pore that stabilize the channel in a nonconducting, blocked state.     

Suppression of the Bacterial Spot Pathogen Xanthomonas euvesicatoria on Tomato Leaves by an Attenuated Mutant of Xanthomonas perforans

A bacteriocin-producing strain of the bacterial spot of tomato plant pathogen, Xanthomonas perforans, with attenuated pathogenicity was deployed for biocontrol of a bacteriocin-sensitive strain of the genetically closely related bacterial spot of tomato plant pathogen, X. euvesicatoria. The attenuated mutant (91-118ΔopgHΔbcnB) of X. perforans was tested in leaf tissue and shown to significantly inhibit internal populations of the wild-type X. euvesicatoria strain although significantly less than the wild-type 91-118 strain, whereas in a phyllosphere inhibition assay, the mutant strain reduced epiphytic populations comparably to 91-118. Thus, the attenuated mutant limited the sensitive bacterium more efficiently on the leaf surface than inside the leaf. In field experiments, weekly application of 91-118ΔopgHΔbcnB significantly reduced X. euvesicatoria populations compared to the growers’ standard control (copper hydroxide and mancozeb applied weekly and acibenzolar-S-methyl applied every 2 weeks). The biological control agent, 91-118ΔopgHΔbcnB, applied every 2 weeks also significantly reduced X. euvesicatoria populations in one season but was not significantly different from the growers’ standard control. Potentially, attenuated pathogenic strains could be deployed as biological control agents in order to improve disease control of foliar plant pathogens.Bacterial spot of tomato is incited by several Xanthomonas spp., including Xanthomonas euvesicatoria, X. perforans, and X. vesicatoria (13). On tomato plants, three races, designated tomato race 1 (T1), T2, and T3, were originally identified based on their reactions on three tomato genotypes (22, 23, 33, 37). These races, T1, T2, and T3, are principally members of X. euvesicatoria, X. vesicatoria, and X. perforans, respectively. Control of bacterial spot of tomato is extremely difficult when moderate-to-high temperatures and high moisture conditions exist. The disease causes significant damage to the crop, resulting in major losses (27). Management is primarily limited to bactericides, such as fixed coppers (3, 20, 32); however, copper-tolerant strains have become prevalent (31, 32) and chemical control alone is insufficient to control the disease under optimal weather conditions. Additionally, the use of copper compounds has led to soil contamination in some instances (16).Recently, there has been increased interest in integrated biological control strategies for bacterial diseases (5, 7, 11, 19, 23). However, optimization of biocontrol agents for consistent disease suppression for many bacterial diseases has been difficult. Studies are increasing our understanding of the ecology of nonpathogenic saprophytes as biocontrol agents, but their selection is limited to labor-intensive protocols. New biological control strategies are currently being sought, including the use of bacteriocins, bacteriophages, and attenuated plant pathogens (4, 5, 7, 9, 11, 23, 26, 35, 38).For many years, only X. euvesicatoria (T1) was present on tomato plants in Florida. In 1991, X. perforans (T3) appeared in Florida tomato fields (15) and eventually became the prevalent race (14). Following that observation, Jones et al. (12) demonstrated that when X. perforans and X. euvesicatoria were coinoculated onto tomato plants in the field, X. perforans displaced X. euvesicatoria and became the predominant strain. Further studies revealed that the competitive nature of X. perforans was due in large part to its antagonism toward X. euvesicatoria strains (9, 12, 36). Tudor-Nelson et al. (36) identified three different bacteriocins, designated BCN-A, BCN-B, and BCN-C, which were found to confer the ability to inhibit X. euvesicatoria strains in plate assays. Hert et al. (9) determined that these bacteriocins provided X. perforans strains with a competitive advantage in the greenhouse and field and that a mutant X. perforans strain expressing only BCN-A and BCN-C was most effective in displacing X. euvesicatoria and outcompeted wild-type (WT) X. perforans. Field experiments conducted with a nonpathogenic Hrp⁻ strain of X. perforans as a potential biocontrol agent for controlling X. euvesicatoria resulted in marginal control (18). Although the WT X. perforans strain has a competitive advantage over X. euvesicatoria populations, Hrp⁻ mutants such as that used in the study by Liu (18) do not appear to have the necessary competitiveness to suppress X. euvesicatoria populations.Previous research has focused on colonization of the plant by biocontrol agents to determine the relationship between invasion efficiency and biological control efficacy. For example, Frey et al. (8) achieved only low-to-moderate levels of biological control of a WT bacteriocin-sensitive strain of Ralstonia solanacearum when using a bacteriocin-producing nonpathogenic Hrp⁻ mutant strain of R. solanacearum. However, control using a moderately pathogenic hrp mutant (hrcV) capable of higher levels of colonization of the root and stem achieved greater disease suppression (6). Etchebar et al. (6) suggested that there was a positive correlation between colonization of the xylem by the hrp mutant and the level of control of WT R. solanacearum. As a result of previous studies showing that nonpathogenic strains of X. perforans only provide low levels of biological control (18), we hypothesized that using an attenuated pathogenic bacteriocin-producing strain of X. perforans rather than a nonpathogenic strain may increase the efficiency of control under field conditions.In this study, our strategy was to use an attenuated mutant of X. perforans that colonizes leaf tissue more effectively than nonpathogenic strains do and potentially provides more effective colonization and increases the likelihood for interaction between X. perforans and X. euvesicatoria. We selected strain 91-118ΔopgHΔbcnB as the biocontrol agent since it was previously shown that deletion of the osmoregulated periplasmic glucan gene opgH resulted in a pathogenic phenotype with a significantly reduced ability to cause disease and internal colonization in susceptible tomato tissue (22). The selected mutant also lacked BcnB activity based on a previous study in which BcnB appeared to negatively affect competitive ability in that a ΔbcnB mutant was more effective at colonizing tomato leaves in field experiments than WT X. perforans was (9).     

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 avrGf 1. Xac-A^wΔ avrGf 1 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.     

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.     

Assessment of mutant tomato lines as a starting material for breeding varieties resistant to Alternaria alternata

An assessment of 27 mutant tomato lines from four countries (Germany, USA, Russia, Bulgaria) was carried out for resistance to five Alternaria alternata strains under conditions of the South of Russia. Five strains of the A. alternata fungus were isolated from naturally infected plants selected in five agroclimatic zones of Krasnodar Krai: Central - strain 1, Western - strain 6, North - strain 11, South Foothill - strain 12, Chernomorskaya - strain 13. The assessment was carried out in the field during 2018–2020, in a greenhouse and under the laboratory conditions three times for each studied strain. In the field, the plants were treated every year with a spore suspension of A. alternata strain 1. Mutant lines obtained from the United States: 868, 663, 533, 544 and 898 showed the greatest resistance to Alternaria in 2018–2020, the lesion of which averaged 4.5–8.0% over three years. 13 mutant lines: 17, 40, 688, 722 (Germany), 311, 394, 418, 542, 728, 743, 917 (USA), 322 (Russia), 159 (Bulgaria) showed average resistance with the development of the disease 10.2–24.9% over three years of the research. Mutant lines 743, 663, 868, 544 obtained from the USA possessed relatively high resistance to all the studied strains under greenhouse conditions; moreover, no signs of damage with strains 1 and 11 were observed on Mo 868, signs of damage by strain 11 of A. alternata were not observed on Mo 743. Under laboratory conditions, mutant lines 663, 743, 868, obtained from the United States, were most resistant. Mo 663 showed resistance to strains 1, 13; line 743 - to strains 11, 12; line 868 - to strains 1, 11. There was a predominantly positive correlation between the results of field, greenhouse and laboratory assessments, which indicates a strong connection between them and the possibility of using these methods to assess the resistance of tomato samples to Alternaria independently of each other.     

Proximity-dependent inhibition in Escherichia coli isolates from cattle

We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-μm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.     

Bacteria competing with the adhesion and biofilm formation by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The activity of antagonistic substances produced by Pseudomonas aeruginosa and Lactobacillus acidophilus against the planktonic and sessile populations of Staphylococcus aureus strains was demonstrated. The strongest effects were caused by probiotic L. acidophilus strain — bacteriocin-like inhibitory substances (BLIS) positive. However, the S. aureus A3 growth, adhesion and biofilm formation was also limited by cell-free supernatant of L. acidophilus H-1 (BLIS negative). Moreover, competitive direct interactions were observed between staphylococci and the above bacteria, which influenced the formation of dualspecies aggregates on the surface.     

Identification of CiaR Regulated Genes That Promote Group B Streptococcal Virulence and Interaction with Brain Endothelial Cells

Group B Streptococcus (GBS) is a major causative agent of neonatal meningitis due to its ability to efficiently cross the blood-brain barrier (BBB) and enter the central nervous system (CNS). It has been demonstrated that GBS can invade human brain microvascular endothelial cells (hBMEC), a primary component of the BBB; however, the mechanism of intracellular survival and trafficking is unclear. We previously identified a two component regulatory system, CiaR/H, which promotes GBS intracellular survival in hBMEC. Here we show that a GBS strain deficient in the response regulator, CiaR, localized more frequently with Rab5, Rab7 and LAMP1 positive vesicles. Further, lysosomes isolated from hBMEC contained fewer viable bacteria following initial infection with the ΔciaR mutant compared to the WT strain. To characterize the contribution of CiaR-regulated genes, we constructed isogenic mutant strains lacking the two most down-regulated genes in the CiaR-deficient mutant, SAN_2180 and SAN_0039. These genes contributed to bacterial uptake and intracellular survival. Furthermore, competition experiments in mice showed that WT GBS had a significant survival advantage over the Δ2180 and Δ0039 mutants in the bloodstream and brain.     

Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.     

Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans

The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.     

Role of the reversible xanthophyll cycle in the photosystem II damage and repair cycle in Dunaliella salina

The Dunaliella salina photosynthetic apparatus organization and function was investigated in wild type (WT) and a mutant (zea1) lacking all beta,beta-epoxycarotenoids derived from zeaxanthin (Z). The zea1 mutant lacked antheraxanthin, violaxanthin, and neoxanthin from its thylakoid membranes but constitutively accumulated Z instead. It also lacked the so-called xanthophyll cycle, which, upon irradiance stress, reversibly converts violaxanthin to Z via a de-epoxidation reaction. Despite the pronounced difference observed in the composition of beta,beta-epoxycarotenoids between WT and zea1, no discernible difference could be observed between the two strains in terms of growth, photosynthesis, organization of the photosynthetic apparatus, photo-acclimation, sensitivity to photodamage, or recovery from photo-inhibition. WT and zea1 were probed for the above parameters over a broad range of growth irradiance and upon light shift experiments (low light to high light shift and vice versa). A constitutive accumulation of Z in the zea1 strain did not affect the acclimation of the photosynthetic apparatus to irradiance, as evidenced by indistinguishable irradiance-dependent adjustments in the chlorophyll antenna size and photosystem content of WT and zea1 strain. In addition, a constitutive accumulation of Z in the zea1 strain did not affect rates of photodamage or the recovery of the photosynthetic apparatus from photo-inhibition. However, Z in the WT accumulated in parallel with the accumulation of photodamaged PSII centers in the chloroplast thylakoids and decayed in tandem with a chloroplast recovery from photo-inhibition. These results suggest a role for Z in the protection of photodamaged and disassembled PSII reaction centers, apparently needed while PSII is in the process of degradation and replacement of the D1/32-kD reaction center protein.     

Investigation of the basis for Ni tolerance conferred by the expression of TjZnt1 and TjZnt2 in yeast strains.

Introduction of ZIP family transporter gene homologues TjZnt1 and TjZnt2 (metal ion transporters) into yeast strains conferred increased Ni(II) tolerance in that species. The action of ZIP family transporter homologues, however, could not explain the Ni resistance of yeast strains transformed with TjZnt1 and TjZnt2. To elucidate the mechanism of Ni tolerance conferred by TjZnt1 and TjZnt2 in yeast strains, we made a series of investigations based upon three hypotheses, including (1) cellular Ni efflux, (2) exclusion of Ni due to competitive uptake of other metals, and (3) Ni binding to histidine-rich domains (chelation). The critical Ni tolerance level of TjZnt2 expressing yeast strains was 1.4mM, whereas, the TjZnt1 expressing yeast strains were tolerant of Ni concentrations as high as 2.0mM. The TjZnt1 expressing yeast strain had significantly lower Ni content and significantly higher Zn content than the control and TjZnt2 expressing yeast strain. Effects of the deletion of histidine-rich domain HRD1 or HRD2, or deletion of the region from HRD1 to HRD2, resulted in the same or slightly less Ni(II) tolerance in the TjZnt1 expressing yeast strain. These data indicate that Ni tolerance of the TjZnt2 expressing yeast strain is not correlated with binding to HRDs (Hypothesis 3). Ni tolerance of TjZnt1 expressing yeast strain was, however, partially correlated with Zn influx, which suppressed Ni influx, therefore Ni influx (Hypothesis 1) and competitive inhibition of Ni influx by other metals (Hypothesis 2), remain viable hypotheses which will be subject to further testing.     

Polyomavirus mutation that confers a cell-specific cis advantage for viral DNA replication.

The structural and biological properties of a polyomavirus mutant selected in Friend erythroleukemic cells were investigated. The growth efficiency of this mutant (PyFL78) was compared with that of the parental PyA2 strain by a growth competition assay in Friend erythroleukemic and 3T3 (or 3T6) cell lines. The results reveal that PyFL78 displays a cis-acting growth advantage over the PyA2 parental strain in Friend erythroleukemic cells but not in 3T3 or 3T6 cells. This cell-specific cis advantage is shown to be due to modifications within the polyomavirus noncoding regulatory region.     

Population Dynamics of Pseudomonas syringae pv. tomato Strains on Tomato Cultivars Rio Grande and Rio Grande-Pto under Field Conditions

We examined the effects of the Pto resistance locus on the population dynamics of Pseudomonas syringae pv. tomato (Pst) strains in field experiments with the nearly isogenic tomato lines Rio Grande (RG, susceptible to Pst races 0 and 1) and Rio Grande-Pto (RG-Pto, resistant to Pst race 0, susceptible to Pst race 1). Pst strain SM78-1Sm^r (race 0) grew well under field conditions and caused ample bacterial speck disease on susceptible RG plants. In contrast, strain DC3000 failed to establish large populations when inoculated onto field grown RG plants. Mean population sizes of SM78-1Sm^r were 4–5 orders of magnitude larger on RG than RG-Pto plants indicating that RG-Pto plants were highly effective in attenuating pathogen population development. Most of the sampled leaflets from RG-Pto field plots harboured small numbers of SM78-1Sm^r. However, population sizes SM78-1Sm^r as large as 10⁵–10⁶ CFU were found on a few leaflets. Bacteria isolated from these leaflets had phenotypes characteristic of Pst race 1 strains. In growth chamber plant assays, the bacterial strains grew well and caused typical speck lesions on RG-Pto plants. The strains appeared to be race-shift mutants of SM SM78-1Sm^r. Interestingly, results from DNA hybridization experiments demonstrated that the race-shift mutants were deleted for the avirulence gene, avrPto but not for avrPtoB.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Evaluation of tomato wild species for resistance to race T1 nad T3 of Xanthomonas vesicatoria

Thirty six tomato wild species accessions of the subgenera Eulycopersicon and Eriopersicon of the genus Lycopersicon were inoculated with race T1 and T3 of Xanthomonas vesicatoria by vaccum infiltration method. Degree of diseases was evaluated by scale of 0 to 4. It was established that some accessions showed low degree of disease to race T1 and others to race T3. LA 2623 indicated very low degree of disease to race T1 and was immune in inoculation with T3. LA 386 and LA 1297 manifested hypersensitive reaction to both races and PI 127826 to race T3 only. The accessions possessing low degree of disease or hypersensitive reaction to race T1 and race T3 are new promising sources of resistance to Xanthomonas vesicatoria.     

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.     

Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy

During growth of Pseudomonas aeruginosa strain PAO1 with the toxic detergent SDS, a part of the population actively formed macroscopic cell aggregates while the other part grew as freely suspended cells. The physiological function of aggregation for growth with SDS was investigated. Three mutants growing with SDS without aggregation were isolated: the spontaneous mutant strain N and two mutants with transposon insertions in the psl operon for exopolysaccharide synthesis. SDS-induced aggregation in strain N but not in a pslJ mutant was restored by complementation with two genes encoding diguanylate cyclases responsible for synthesis of cyclic-di-guanosine monophosphate (c-di-GMP). By expressing a c-di-GMP-specific phosphodiesterase SDS-induced aggregation of strain PAO1 was reduced. Upon exposure to SDS in the presence of the uncoupler carbonyl cyanide chlorophenylhydrazone, the aggregating strains had ca. 500-fold higher survival rates than the non-aggregating strains. Co-incubation experiments revealed that strain N could integrate into aggregates of strain PAO1 and thereby increase its survival rate more than 1000-fold. These results showed that SDS-induced aggregation involved c-di-GMP signalling with the psl operon as a possible target. Cell aggregation could serve as a pre-adaptive strategy ensuring survival and growth of P. aeruginosa populations in environments with multiple toxic chemicals.