Contributions of the effector gene hopQ1-1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

To study the role of type III-secreted effectors in the host adaptation of the tobacco ( Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci , a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean ( Phaseolus vulgaris ) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1-1 _Pph1448A were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1-1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1-1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine-tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non-host species.     

Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: modeling the impact of superoxide dismutase and nitric oxide diffusion

Tyrosine nitration is a posttranslational modification observed in many pathologic states that can be associated with peroxynitrite (ONOO(-)) formation. However, in vitro, peroxynitrite-dependent tyrosine nitration is inhibited when its precursors, superoxide (O(2)*(-)) and nitric oxide ((*)NO), are formed at ratios (O(2)*(-)/(*)NO) different from one, severely questioning the use of 3-nitrotyrosine as a biomarker of peroxynitrite-mediated oxidations. We herein hypothesize that in biological systems the presence of superoxide dismutase (SOD) and the facile transmembrane diffusion of (*)NO preclude accumulation of O(2)*(-) and (*)NO radicals under flux ratios different from one, preventing the secondary reactions that result in the inhibition of 3-nitrotyrosine formation. Using an array of reactions and kinetic constants, computer-assisted simulations were performed in order to assess the flux of 3-nitrotyrosine formation (J(NO(2(-))Y)) during exposure to simultaneous fluxes of superoxide (J(O(2)*(-))) and nitric oxide (J((*)NO)), varying the radical flux ratios (J(O(2)*(-))/ J((*)NO)), in the presence of carbon dioxide. With a basic set of reactions, J(NO(2(-))Y) as a function of radical flux ratios rendered a bell-shape profile, in complete agreement with previous reports. However, when superoxide dismutation by SOD and (*)NO decay due to diffusion out of the compartment were incorporated in the model, a quite different profile of J(NO(2(-))Y) as a function of the radical flux ratio was obtained: despite the fact that nitration yields were much lower, the bell-shape profile was lost and the extent of tyrosine nitration was responsive to increases in either O(2)*(-) or (*)NO, in agreement with in vivo observations. Thus, the model presented herein serves to reconcile the in vitro and in vivo evidence on the role of peroxynitrite in promoting tyrosine nitration.     

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.     

Stimulation of nitric oxide synthase in cerebral cortex due to elevated partial pressures of oxygen: an oxidative stress response

The purpose of this investigation was to determine the impact of elevated partial pressures of O(2) on the steady state concentration of nitric oxide ((*)NO) in the cerebral cortex. Rodents with implanted O(2)- and (*)NO-specific microelectrodes were exposed to O(2) at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA) for up to 45 min. Elevations in (*)NO concentration occurred with all partial pressures above that of ambient air. In rats exposed to 2.8 ATA O(2) the increase was 692 +/- 73 nM (S.E., n = 5) over control. Changes were not associated with alterations in concentrations of nitric oxide synthase (NOS) enzymes. Based on studies with knock-out mice lacking genes for neuronal NOS (nNOS) or endothelial NOS (eNOS), nNOS activity contributed over 90% to total (*)NO elevation due to hyperoxia. Immunoprecipitation studies indicated that hyperoxia doubles the amount of nNOS associated with the molecular chaperone, heat shock protein 90 (Hsp90). Both (*)NO elevations and the association between nNOS and Hsp90 were inhibited in rats infused with superoxide dismutase. Elevations of (*)NO were also inhibited by treatment with the relatively specific nNOS inhibitor, 7 nitroindazole, by the ansamycin antibiotics herbimycin and geldanamycin, by the antioxidant N-acetylcysteine, by the calcium channel blocker nimodipine, and by the N-methyl-D-aspartate inhibitor, MK 801. Hyperoxia did not alter eNOS association with Hsp90, nor did it modify nNOS or eNOS associations with calmodulin, the magnitude of eNOS tyrosine phosphorylation, or nNOS phosphorylation via calmodulin kinase. Cerebral cortex blood flow, measured by laser Doppler flow probe, increased during hyperoxia and may be causally related to elevations of steady state (*)NO concentration. We conclude that hyperoxia causes an increase in (*)NO synthesis as part of a response to oxidative stress. Mechanisms for nNOS activation include augmentation in the association with Hsp90 and intracellular entry of calcium.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Mechanism of superoxide dismutase/H(2)O(2)-mediated nitric oxide release from S-nitrosoglutathione--role of glutamate

S-Nitrosoglutathione (GSNO), a physiologically relevant nitric oxide ((*)NO) donor, exhibits antioxidant, anti-ischemic, and antiplatelet properties. The exact mechanism of (*)NO release from GSNO in biological systems has not been determined. Both copper ions and copper-containing enzymes have been shown to catalyze (*)NO release from GSNO. In this study we observed that copper-zinc superoxide dismutase (Cu,ZnSOD) in the presence of H(2)O(2) caused a rapid decomposition of GSNO, forming oxidized glutathione (GSSG) and (*)NO. The cupric ions (Cu(2+)) released from Cu,ZnSOD were bound to the glutamate moiety of GSNO, yielding a 2:1 (GSNO)(2)Cu(2+) complex. Strong chelators of cupric ions, such as histidine and diethylenetriaminepentaacetic acid, inhibited the formation of (GSNO)(2)Cu(2+) complex, GSSG, and (*)NO. GSSG alone inhibited Cu(2+)-induced decomposition of GSNO. This effect is attributed to complexation of copper by GSSG. We conclude that binding of copper to GSNO is obligatory for (*)NO release from GSNO; however, the rate of this reaction was considerably slowed due to binding of Cu(2+) by GSSG. The glutamate moiety in GSNO and GSSG controls copper-catalyzed (*)NO release from GSNO. Cu,ZnSOD and H(2)O(2) enhanced peroxidation of unsaturated lipid that was inhibited by GSNO. The antioxidant function of GSNO is related to the sequestering of copper by GSNO and its ability to slowly release (*)NO. Implications of these findings are discussed in relation to GSNO-induced cardioprotection and to neuropathological processes.     

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.     

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.     

Post-translational modification of flagellin determines the specificity of HR induction

Flagellin, a constituent of the flagellar filament, is a potent elicitor of hypersensitive cell death in plant cells. Flagellins of Pseudomonas syringae pvs. glycinea and tomato induce hypersensitive cell death in their non-host tobacco plants, whereas those of P. syringae pv. tabaci do not remarkably induce it in its host tobacco plants. However, the deduced amino acid sequences of flagellins from pvs. tabaci and glycinea are identical, indicating that post-translational modification of flagellins plays an important role in determining hypersensitive reaction (HR)-inducibility. To investigate genetically the role of modification of flagellin in HR-induction, biological and phytopathological phenotypes of a flagella-defective Delta fliC mutant and Delta fliC mutants complemented by the introduction of the flagellin gene (fliC) from different pathovars of P. syringae were investigated. The Delta fliC mutant of pv. tabaci lost flagella, motility, the ability to induce HR cell death in non-host tomato cells and virulence toward host tobacco plants, whereas all pv. tabaci complemented by the introduction of the fliC gene of pvs. tabaci, glycinea or tomato recovered all the abilities that the Delta fliC mutant had lost. These results indicate that post-translational modification of flagellins is strongly correlated with the ability to cause HR cell death.     

Early determinants of H2O2-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.     

Formation of peroxynitrite from reaction of nitroxyl anion with molecular oxygen.

Peroxynitrite (ONOO(-)/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O(2)) and nitric oxide ((*)NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO(-)), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate, (-)ON=NO(2)(-)), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO(-) in the presence of oxygen is mainly characterized by peroxynitrite and not by HO( small middle dot) radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of (*)NO and O(2), probably by an electron-transfer mechanism. This prediction is supported by the fact that H(2)O(2) is formed during the decay of NO(-) in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO(-) in addition to the "classical" ( small middle dot)NO + O(2)() pathway.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

Expression of the Tomato Pto Gene in Tobacco Enhances Resistance to Pseudomonas syringae pv tabaci Expressing avrPto

The Pto gene encodes a serine-threonine kinase that confers resistance in tomato to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. We examined the ability of Pto to function in tobacco, a species that is sexually incompatible with tomato. Evidence that a heterologous Pto-like signal transduction pathway is present in tobacco was suggested by the fact that tobacco line Wisconsin-38 exhibits a hypersensitive response after infection with P. syringae pv tabaci expressing avrPto. We introduced a Pto transgene into cultivar Wisconsin-38 and assessed the ability of transformed plants to further inhibit growth of the P. s. tabaci strain expressing avrPto. The Pto-transformed tobacco plants exhibited a significant increase in resistance to the avirulent P. s. tabaci strain compared with wild-type tobacco as indicated by (1) more rapid development of a hypersensitive resistance response at high inoculum concentrations (108 colony-forming units per mL); (2) lessened severity of disease symptoms at moderate inoculum concentrations (106 and 107 colony-forming units per mL); and (3) reduced growth of avirulent P. s. tabaci in inoculated leaves. The results indicate that essential components of a Pto-mediated signal transduction pathway are conserved in tobacco and should prompt examination of resistance gene function across even broader taxonomic distances.     

Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes

The ability of nitric oxide ((*)NO) to inhibit propagative lipid peroxidation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [(14)C]cholesterol (Ch), and a nonregenerable singlet oxygen-derived primer, 5alpha-hydroperoxycholesterol (5alpha-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelate at 37 degrees C resulted in an exponential decay of 5alpha-OOH and accumulation of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH), as detected by high-performance liquid chromatography with electrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently in egg PC-containing LUVs. Including the (*)NO donor spermine NONOate (SPNO, 5-50 microM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 microM) in the reaction mixture had no effect on 5alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TBARS and 7alphabeta-OOH accumulation in a strongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that (*)NO was the responsible agent. Accumulation of several [(14)C]Ch oxidation products, detected by high-performance thin-layer chromatography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioactivity of which increased as a function of incubation time and (*)NO donor concentration. RCh.4 material was also generated via direct iron/ascorbate reduction of 7alpha-OOH in the presence of (*)NO, consistent with 7alpha-nitrite (7alpha-ONO) identity. However, various other lines of evidence suggest that RCh.4 is not 7alpha-ONO, but rather 5alpha-hydroxycholesterol (5alpha-OH) generated by reduction of 5alpha-ONO arising from 7alpha-ONO rearrangement. 5alpha-OH was only detected when (*)NO was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (*)NO chain-breaking activity.     

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.     

Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide

4,5 diaminofluorescein (DAF-2) is increasingly utilized as a fluorescent detector for nitric oxide (*NO) in cells and tissues. In oxygenated solutions, reactive nitrogen species derived from (*) NO autoxidation nitrosate DAF-2 to yield the highly fluorescent DAF-2 triazole. In the present study, we investigated the nitrosation of DAF-2 at a neutral pH by absorption and fluorescence spectroscopy using NONOates as chemical sources of (*) NO. We found that both chemically synthesized peroxynitrite and horseradish peroxidase in the presence of hydrogen peroxide (H(2)O(2)) oxidized DAF-2 to a relatively stable nonfluorescent intermediate (t(1/2) approximately 90 s). Oxidation of DAF-2 prior to the addition of the z.rad;NO donor DEA/NO resulted in an increase in fluorescence that was approximately 7-fold higher than treatment with DEA/NO alone. The increase in DAF-2 triazole formation upon oxidation of DAF-2 was confirmed by high performance liquid chromatography. Peroxynitrite generated in situ from the equimolar production of (*) NO and superoxide (O(2)(*-)) also increased the yields of DAF-2 triazole formation, which was completely inhibited when O(2)(*-) was in excess of (*) NO. We propose that DAF-2 is oxidized to a free radical intermediate that directly reacts with (*) NO, thereby bypassing the requirement for (*)NO autoxidation for the formation of DAF-2 triazole. Our findings indicate that DAF-2 fluorometric assays are quantitatively difficult to interpret in cells and in solution when oxidants and (*) NO are co-generated.