No abstract

The host range of Pseudomonas avenae is wide among monocotyledonous plants, but individual strains can infect only one or a few host species. The resistance response of rice cells to pathogens has been previously shown to be induced by a rice-incompatible strain, N1141, but not by a rice-compatible strain, H8301. To clarify the molecular mechanism of the host specificity in P. avenae, a strain-specific antibody that was raised against N1141 cells and then absorbed with H8301 cells was prepared. When a cell extract of strain N1141 was separated by SDS-polyacrylamide gel electrophoresis and immunostained with the N1141 strain-specific antibody, only a flagellin protein was detected. Purified N1141 flagellin induced the hypersensitive cell death in cultured rice cells within 6 h of treatment, whereas the H8301 flagellin did not. The hypersensitive cell death could be blocked by pretreatment with anti-N1141 flagellin antibody. Furthermore, a flagellin-deficient N1141 strain lost not only the induction ability of hypersensitive cell death but also the expression ability of the EL2 gene, which is thought to be one of the defenserelated genes. These results demonstrated that the resistance response in cultured rice cells is induced by the flagellin existing in the incompatible strain of P. avenae but not in the flagellin of the compatible strain.

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Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.

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Organic radical chemistry has blossomed in recent years in part due to a greatly increased knowledge of radical reaction rate constants. Knowledge of absolute or relative rate constants is critically important for radical-based synthetic methods because most useful reactions are chain processes in which undesired pathways compete with the desired ones. In mechanistic studies, the availability of rate constants changes a qualitative mechanistic probe study into a more quantitative radical clock 1 study. The cyclization of the 5-hexenyl radical (1) and the ring opening of the cyclopropylcarbinyl radical (2) are archetypal radical rearrangements whose rate constants serve as the linchpins for an alkyl radical kinetic scale that spans 8 orders of magnitude, 2 but, ironically, the "best" kinetic values of these reactions at room temperature (and of many other radical processes employed as radical clocks) were determined from indirect competition kinetic studies in part because the educt and product radicals lack useful UV chromophores. Such rate constants contain (1) experimental uncertainties of the initially measured second-order trapping reactions employed as basis reactions, (2) uncertainties from the actual competition kinetic studies used to calibrate the clock, and (3) an unknown degree of uncertainty due to the necessary assumption that the rate constants for reactions of model radicals employed in the calibrations of the second-order basis reactions are equal to the rate constants for reactions of radicals of interest. For cases where the functionality is removed from the radical center by several intervening methylene groups, such as in 1, there is little doubt that the latter assumption is reasonable, but this assumption is substantially less secure for radical 2 and its analogs.We discuss here a general method for direct laser flash photolysis (LFP) measurements of unimolecular radical kinetics employing ultrafast radical rearrangement reactions as UV detectable reporters. The reporter groups are installed synthetically via Wittig reactions, and the method can be applied to a wide range of radicals including those for which absolute basis kinetic values are not available.The general concept is illustrated with radicals 3 which produce radicals 4 by 5-exo cyclizations. Radicals 4 are phenylsubstituted cyclopropylcarbinyl radicals that will ring open to radicals 5, which are UV-detectable benzylic and diphenylalkyl radicals, with rate constants exceeding 1 × 10 11 s -1 at room temperature. 3 Thus, in LFP experiments, the slow rate constants for cyclizations of radicals 3 are obtained by following the formation of radicals 5.In practice, the observed rate constants for cyclizations of (E)-3a and 3b (5:1, (Z):(E)), produced by LFP of the corresponding PTOC ester 4,5 precursors, at 25°C were 8.3 and 5.0 × 10 5 s -1 , respectively, and radicals 3 can now be used as radical clocks. 6 The observed rate constants for 3 also permit an evaluation of the kinetic effects of the arylcyclopropyl reporter groups which ar...

The kinetics of ring openings of cyclopropylcarbinyl radicals containing reporter groups were measured directly by laser flash photolysis (LFP) methods. The reporter groups are aryl-substituted cyclopropanes at the position of the radical center in the incipient product, and the initial ring-opening reaction gives an aryl-substituted cyclopropylcarbinyl radical that opens “instantly” on the nanosecond time scale to give benzylic and diphenylalkyl radical products that are detected by UV spectroscopy. Three reporter group designs have been studied. The most useful design is that in substituted (trans-2-(2,2-diphenylcyclopropyl)cyclopropyl)methyl radicals (7). The important synthetic intermediate for production of this series is N-methoxy-N-methyl-trans-2-(2,2-diphenyl-1R*-cyclopropyl)-1S*,2R*-cyclopropanecarboxamide (15a) which has been prepared diastereomerically pure. The syntheses of 15a and a series of PTOC ester radical precursors derived from 15a are described. Laser photolysis (355 nm) of PTOC esters gave acyloxyl radicals that decarboxylated to give cyclopropylcarbinyl radicals that were studied. The substitutions at the cyclopropylcarbinyl position of radicals 7 were H, H (7a); H, Me (7b); Me, Me (7c); H, CO2Et (7d); Me, CO2Et (7e); and H, Ph (7f). The kinetics of ring openings of radicals 7 were measured in THF and in four cases in CH3CN in the temperature range −41 to +50 °C. Alkyl radicals 7a and 7b displayed no kinetic solvent effect, whereas ester-substituted radicals 7d and 7e did. The ester-substituted radicals react about as fast as the alkyl-substituted radicals due to favorable polarization in the transition states for ring opening. The phenyl-substituted radical 7e ring-opens about 3 orders of magnitude less rapidly than the parent radical 7a. The structures and energies of the ground states, rotational transition states, and ring-opening transition states for a series of substituted cyclopropylcarbinyl radicals corresponding to 7a−e were computed by the hybrid density functional theory B3LYP/6-31G*, and the ring-opening kinetics were compared to those predicted from the computational barriers. The precision in the Arrhenius functions for ring openings of radicals 7 permits meaningful comparisons of the log A terms to those predicted from thermochemical considerations.

Incompatible strains of Acidovorax avenae elicit an immune response in cultured rice cells, with immunity specifically induced by the flagellin of the incompatible strain. To identify genes regulated by flagellin perception signaling in cultured rice cells, gene expression patterns were analyzed with rice cDNA microarrays, including 3,353 independent rice cDNA clones. In all, 131 genes were differentially expressed between incompatible and compatible interactions. K-means clustering showed that 94 genes were upregulated and 32 genes were downregulated during incompatible interactions, whereas only 5 genes were upregulated during compatible interactions. Among the 126 genes that were up- or downregulated during incompatible interactions, expression of 46 genes was decreased when cultured rice cells were inoculated with a flagellin-deficient incompatible strain (delta fla1141-2), indicating that approximately 37% of the 126 genes were directly controlled by flagellin perception. Real-time reverse-transcription polymerase chain reaction analysis using flagellins purified from incompatible or compatible strains was performed to confirm flagellin-regulated expression of candidate genes selected by microarray analysis. Results showed that induction of some genes involved in the immune response is regulated not only by the flagellin perception pathway, but also by another recognition molecule-perception pathway.