Mitochondria are both a source of ATP and a site of reactive oxygen species (ROS) production. However, there is little information on the sites of mitochondrial ROS (mROS) production or the biological role of such mROS in plants. We provide genetic proof that mitochondrial complex II (Complex II) of the electron transport chain contributes to localized mROS that regulates plant stress and defense responses. We identify an Arabidopsis mutant in the Complex II subunit, SDH1-1, through a screen for mutants lacking GSTF8 gene expression in response to salicylic acid (SA). GSTF8 is an early stressresponsive gene whose transcription is induced by biotic and abiotic stresses, and its expression is commonly used as a marker of early stress and defense responses. Transcriptional analysis of this mutant, disrupted in stress responses 1 (dsr1), showed that it had altered SA-mediated gene expression for specific downstream stress and defense genes, and it exhibited increased susceptibility to specific fungal and bacterial pathogens. The dsr1 mutant also showed significantly reduced succinate dehydrogenase activity. Using in vivo fluorescence assays, we demonstrated that root cell ROS production occurred primarily from mitochondria and was lower in the mutant in response to SA. In addition, leaf ROS production was lower in the mutant after avirulent bacterial infection. This mutation, in a conserved region of SDH1-1, is a unique plant mitochondrial mutant that exhibits phenotypes associated with lowered mROS production. It provides critical insights into Complex II function with implications for understanding Complex II's role in mitochondrial diseases across eukaryotes.plant defense | respiration | Pseudomonas syringae | Rhizoctonia solani

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An expression library was constructed from an Actinobacfllus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-* Corresponding author.

An expression library was constructed from ActinobaciUlus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in theA. pleuopneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pkuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.

The gene encoding the ActinobaciUus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70%o of the protein was expressed as an aggregate protein in Escherichia coli.The nucleotide sequences of the aJbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that ofA. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The aJbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tJbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity, with a frequency of approximately 10-4. The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.Rearrangement of bacterial DNA as a means to increase the fitness of survival of the respective bacterial population is a relatively common phenomenon. Examples are the genetic switch resulting in the expression of an antigenically distinct flagella protein (H-antigen) in Salmonella species (25), the on/off switch of type 1 fimbriae in Escherichia coli (1), and the chromosomal rearrangements leading to the expression of antigenically distinct fimbriae in Moraxella bovis (18) and Neisseria gonorrhoeae (23). More recently, the site-specific integration of the virulence plasmid of enteroinvasive E. coli and Shigellaflexneri into the chromosome has been described; this results in a noninvasive and thus avirulent phenotype (36). Also, high-frequency excision of mobile genetic elements under conditions of nutritional stress has been described; this restored the relevant catabolic genes to normal function (8,24). Insertion and excision events are also encountered in combination with horizontal transfer of virulence factors. Thus, the heat-stable toxins of enterotoxigenic E. coli (STI and STII) can be carried by transposable elements, and the inverted repeats in these elements are composed of insertion sequences IS] and IS2, respectively (11,15,27,28).The cytolysins or RTX toxins form a large family of calcium-dependent, pore-forming toxins which can be found in different genera of the families Enterobacteriaceae and Pasteurellaceae (34). Genetic investigation of these toxins has shown that the toxin-encoding gene (hlyA in E. coli) is commonly clustered with at least three other genes encoding an activator (HlyC) and proteins involved in membrane transport (HlyB and HlyD [5,35]). A comparison of the nucleotide and amino acid sequences of RTX toxins from different species shows a high degree of similarity, thus suggesting the occurrence of horizontal transfer rather than parallel evolutionary evolvement (2,5,6,16,34).In Actinobacillus pleuropneumoniae at least three immunologically and functionally distinct cytolysins of + 110 x 103, + 105 x 103, and + 103 x 103 Da (+110K, 105K, and * Corresponding author. t VIDO Journa...

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