Mark M. Stayton

Bacteria expressing avirulence gene D produce a specific elicitor of the soybean hypersensitive reaction

Production of the avrD elicitor by P. s. pv. glycinea cells carrying the cloned avrD gene occurred independently of the hrp genes, considered important for pathogenicity and HR induction by certain P. syringae pathovars. The results indicated that expression of avirulence gene D in P. syringae pathovars and in E. coli causes them to produce a diffusible, elicitor-active molecule which initiates cultivar-specific induction of the HR

Inhibition of gene expression in transformed plants by antisense RNA

We report the successful suppression of nopaline synthase (EC 1.5.1.19) enzymatic activity in the leaves of tobacco plants via the overproduction of RNAs complementary to the nopaline synthase (nos) mRNA. Several different regions of the nos gene were fused, in antisense orientation, to the promoter from a strongly expressed petunia chlorophyll a/b-binding protein gene. These constructions were directly introduced into a tobacco line which contained a single copy of the wild-type nos gene and transgenic plants were regenerated. The degree of nopaline synthase suppression in the leaves of the double transformants ranged up to 85% and was dependent on the particular region of the nos gene present in the antisense RNA. The most effective nos antisense sequences were derived from the 3′ half of the nos gene transcript. In addition, we report a new sensitive method for the detection and quantitation of nopaline synthase activity in crude plant extracts.

Nucleotide sequence and chromosomal location of Cab -7, the tomato gene encoding the type II chlorophyll a/b-binding polypeptide of photosystem I

Eran Pichersky, 1 Steven D. Tanksley, 2 Birgit Piechulla, 3 Mark M. Stayton 4 and Pamela Dunsmuir 4 IBiology Department, University of Michigan, Ann Arbor, MI 48109, USA; 2Department of Plant Breeding and Biometry, Cornell University, Ithaca, N Y 14853, USA; 3Institut fiir Biochemie der Pflanze der Universittit G6ttingen, Untere Karspule 2, 34 G6ttingen, Federal Republic of Germany; 4Advanced Genetic Sciences, Inc., 6701 San Pablo Avenue, Oakland, CA 94608, USA

The syringolides : bacterial C-glycosyl lipids that trigger plant disease resistance

Abstract The salient structural and bioorganic properties of this new class of signal molecules are reported.

Characterization of a full-length petunia cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I.

We have isolated and characterized a full-length cDNA clone (LHCI-15) which specifies a new chlorophyll-binding protein. This protein is associated with the light-harvesting complex of photosystem I (LHCI). The DNA sequence predicts a precursor protein of 270 amino acids, which shares significant homology with the amino acid sequence of another chlorophyll-binding protein; the chlorophyll a/b-binding (Cab) protein of the photosystem II light-harvesting complex (LHCII). There are two extensive regions of homology (at least 45 residues each) which have approximately 50% amino acid sequence identity. These regions coincide with two of the proposed membrane-spanning alpha helices in the Cab proteins of the LHCII and probably include conserved chlorophyll-binding sites. The LHCI-15 cDNA hybridizes to at least 7 genomic EcoRI DNA fragments, which are very closely related at the nucleotide sequence level.

Earliest changes in the left ventricular transcriptome post-myocardial infarction

We report a genome-wide survey of early responses of the mouse heart transcriptome to acute myocardial infarction (AMI). For three regions of the left ventricle (LV), namely, ischemic/infarcted tissue (IF), the surviving LV free wall (FW), and the interventricular septum (IVS), 36,899 transcripts were assayed at six time points from 15 min to 48 h post-AMI in both AMI and sham surgery mice. For each transcript, temporal expression patterns were systematically compared between AMI and sham groups, which identified 515 AMI-responsive genes in IF tissue, 35 in the FW, 7 in the IVS, with three genes induced in all three regions. Using the literature, we assigned functional annotations to all 519 nonredundant AMI-induced genes and present two testable models for central signaling pathways induced early post-AMI. First, the early induction of 15 genes involved in assembly and activation of the activator protein-1 (AP-1) family of transcription factors implicates AP-1 as a dominant regulator of earliest post-ischemic molecular events. Second, dramatic increases in transcripts for arginase 1 (ARG1), the enzymes of polyamine biosynthesis, and protein inhibitor of nitric oxide synthase (NOS) activity indicate that NO production may be regulated, in part, by inhibition of NOS and coordinate depletion of the NOS substrate, L-arginine. ARG1 was the single-most highly induced transcript in the database (121-fold in IF region) and its induction in heart has not been previously reported.

Avirulence gene D of Pseudomonas syringae pv. tomato may have undergone horizontal gene transfer

Avirulence gene D (avrD) is carried on the B‐plasmid of the plant pathogen Pseudomonas syringae pv. tomato with plasmid‐borne avrD homologs widely distributed among the Pseudomonads. We now report sequences in the soft rot pathogen Erwinia carotovora that cross‐hybridize to avrD suggesting a conserved function beyond avirulence. Alternatively, avrD may have been transferred horizontally among species: (i) DNA linked to avrD shows evidence of class II transpositions and contains a novel IS3‐related insertion sequence, and (ii) short sequences linked to avrD are similar to pathogenicity genes from a variety of unrelated pathogens. We have also identified the gene cluster that controls B‐plasmid stability.

Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines. A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45. Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level. A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism. The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria. The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme. Michaelis constants for both the wild-type and overproduced enzyme preparations were the same. And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.

A computer analysis of the validity of the integrated Michaelis-Menten equation.

Abstract The validity of assumptions made in integrating the Michaelis-Menten equation (i.e. the steady-state assumption) was examined by simulating the model on the digital computer. Time courses thus obtained by numerical integration were compared with data generated by the three most common forms of the integrated equation. Agreement was good within specified limits, the chief exception being the early transient phase of the reaction. The observed differences were very much less than theoretical estimates of the maximum error, even when rate constants were chosen that should exaggerate that error.

Avirulence Gene D from Pseudomonas Syringae pv. Tomato and Its Interaction with Resistance Gene Rpg4 in Soybean

Certain pathogen avirulence genes have been associated with production by the pathogen of specific elicitors, chemicals that initiate hypersensitive defense responses (HR) only in those plant cultivars which carry complementary disease resistance genes. We have cloned and characterized one of these avirulence genes, avrD, from Pseudomonas syringae pv. tomato. Various Gram-negative bacteria containing this cloned avirulence gene produce a low molecular weight compound(s) that causes the HR exclusively in soybean cultivars carrying the disease resistance gene, Rpg4. We isolated two major avrD elicitor-active molecules and have partially characterized them as homologous C13 and C15 hydrocarbons that are heavily substituted with oxygen atoms and cyclized at one end. The physiologic role of the avrD elicitor molecules in P.s. pv. tomato remains unclear since mutants deficient in avrD retained virulence in tomato plants. However, the fact that expression of avrD was greatly stimulated when P.s. pv. tomato was grown on plant leaves or in cell suspension cultures suggests that the gene may be important for the survival of the bacteria on or in plant leaves. The purified elicitor-active molecules specifically induced PAL and CHS mRNAs in the soybean cultivar Norchief, which carries the Rpg4 resistance gene, but not in Acme which lacks this gene. Thus, gene-for-gene specificity mediated by the elicitor was reflected at the level of defense gene expression. The avrD specific elicitor preparations also elicited necrosis and phytoalexin production in soybean callus and cotyledons. The evidence therefore indicates that the avrD elicitor molecules are the signal which elicits the HR in the avrD-Rpg4 gene-for-gene interaction.