Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides.

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides, converting four herbicides of this class (fluometuron, linuron, chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to fluometuron, linuron, and diuron, and as a ring-methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10-mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants.

Biotin synthase from Arabidopsis thaliana. cDNA isolation and characterization of gene expression.

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli.

Complementation of an Arabidopsis thaliana biotin auxotroph with an Escherichia coli biotin biosynthetic gene.

The Arabidopsis thaliana biotin auxotroph biol was rendered prototrophic by transformation with a chimeric transgene containing the Escherichia coli bio A gene driven by a constitutive promoter. The bio A gene encodes the biotin biosynthetic enzyme 7, 8-diaminopelargonic acid aminotransferase. Unlike the untransformed control plants, transgenic plants expressing the bacterial transgene synthesized biotin and grew to maturity without biotin-deficiency symptoms. These findings demonstrate that bio1/bio1 mutant plants are defective in the gene encoding 7,8-diaminopelargonic acid aminotransferase.

Arabidopsis mutants simulating disease resistance response.

We describe six Arabidopsis mutants, defining at least four loci, that spontaneously form necrotic lesions on leaves. Lesions resemble those resulting from disease, but occur in the absence of pathogen. In five mutants, lesion formation correlates with expression of histochemical and molecular markers of plant disease resistance responses and with expression of genes activated during development of broad disease resistance in plants (systemic acquired resistance [SAR]). We designate this novel mutant class Isd (for lesions simulating disease resistance response). Strikingly, four Isd mutants express substantial resistance to virulent fungal pathogen isolates. Isd mutants vary in cell type preferences for lesion onset and spread. Lesion formation can be conditional and can be induced specifically by biotic and chemical activators of SAR in Isd1 mutants.

Emerging strategies for enhancing crop resistance to microbial pathogens.

There are marked differences in the pattern of host gene expression in incompatible plant:microbial pathogen interactions compared with compatible interactions, associated with the elaboration of inducible defenses. Constitutive expression of genes encoding a chitinase or a ribosome-inactivating protein in transgenic plants confers partial protection against fungal attack, and a large repertoire of such antimicrobial genes has been identified for further manipulation. In addition, strategies are emerging for the manipulation of multigenic defenses such as lignin deposition and synthesis of phytoalexin antibiotics by overexpression of genes encoding rate determining steps, modification of transcription factors or other regulatory genes, and engineering production of novel phytoalexins by interspecies transfer of biosynthetic genes. The imminent cloning of disease resistance genes, further molecular dissection of stress signal perception and transduction mechanisms, and identification of genes that affect symptom development will provide attractive new opportunities for enhancing crop protection. Combinatorial integration of these novel strategies into ongoing breeding programs should make an important contribution to effective, durable field resistance.

Coordinate Gene Activity in Response to Agents That Induce Systemic Acquired Resistance.

In a variety of plant species, the development of necrotic lesions in response to pathogen infection leads to induction of generalized disease resistance in uninfected tissues. A well-studied example of this "immunity" reaction is systemic acquired resistance (SAR) in tobacco. SAR is characterized by the development of a disease-resistant state in plants that have reacted hypersensitively to previous infection by tobacco mosaic virus. Here, we show that the onset of SAR correlates with the coordinate induction of nine classes of mRNAs. Salicylic acid, a candidate for the endogenous signal that activates the resistant state, induces expression of the same "SAR genes." A novel synthetic immunization compound, methyl-2,6-dichloroisonicotinic acid, also induces both resistance and SAR gene expression. These observations are consistent with the hypothesis that induced resistance results at least partially from coordinate expression of these SAR genes. A model is presented that ties pathogen-induced necrosis to the biosynthesis of salicylic acid and the induction of SAR.

Differential Regulation of beta-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection.

The acidic, extracellular, glucan endo-1,3-beta-glucosidases (EC 3.2.1.39; beta-1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-O) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-O. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of beta-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the beta-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of beta-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.

Isolation of single-copy-sequence clones from a yeast artificial chromosome library of randomly-sheared Arabidopsis thaliana DNA.

We describe the construction of a yeast artificial chromosome (YAC) library from the Arabidopsis thaliana genome. Randomly sheared high molecular weight source DNA was extracted from frozen, ground leaf tissue and blunt-end-ligated to the vector pYAC3. By size-fractionating the ligation products, we achieved an average clone size of 150 kb. Approximately 6% of the YACs contained inserts from the chloroplast genome. We screened clones equivalent to greater than four A. thaliana haploid nuclear genomes and isolated YACs homologous to five single-copy-sequence probes. The library should be useful for chromosome walking and genome mapping experiments. In addition, the approach used for its construction should be applicable to other higher plant species.

Integration of multiple copies of a foreign sequence into the Ti plasmid of Agrobacterium tumefaciens.

A method for constructing Ti plasmids bearing multiple copies of a sequence integrated in tandem is described. A small plasmid that confers tetracycline resistance (TcR), contains homology to a Ti plasmid, and is unable to replicate in Agrobacterium tumefaciens, was mobilized from Escherichia coli to A. tumefaciens. Ti plasmids of exconjugants selected for resistance to 12-14 micrograms Tc/ml all contained multiple tandem repeats of the integrative plasmid. Tc-sensitive variants with fewer integrated copies arose spontaneously at low frequency in the absence of Tc selection, or could be enriched for by selection on Tc in combination with the bactericidal antibiotic augmentin. Variants having an increased number of integrated copies were obtained by growth on high Tc concentrations. Tandem repeats integrated between border sequences provide, in principle, a way to reproducibly introduce many linked copies of any foreign gene into plants.

Colonization and infection of newborn infants caused by bacteriophage-group II Staphylococcus aureus strains.

A staphylococcal strain which exhibited weak lytic reaction with group II phages was isolated from a newborn infant with a skin infection. Subsequent investigations established that this weakly reacting strain was responsible for an endemic level of infection and colonization within the hospital nursery. The use of consistently appearing weak lytic reactions in the evaluation of this episode is described.

New Staphylococcus aureus phage type 94/96(292) associated with a fatal septicemia.

A fulminating septicemia due to Staphylococcus aureus phage type 94/96(292) resulting in the death of a patient with no previous history of illness. This newly characterized strain is identified by an additional typing reaction with experimental phage 292. The prevalence of this strain is discussed.

Delayed-type hypersensitivity responses to a cell wall fraction of the mycelial phase of Coccidioides immitis.

A skin test-active fraction was isolated from the mycelial-phase cell walls of Coccidioides immitis. This alkali-soluble, water-soluble antigen (C-ASWS) elicited positive reactions in 22 of 24 (92%) of the Coccidioides-sensitized guinea pigs whereas only 14 (54%) of the same guinea pigs reacted to commercial coccidioidin (BioCox). None of the 21 Histoplasma-sensitized guinea pigs cross-reacted with the C-ASWS antigen. Footpad tests in mice actively infected with Coccidioides further established the efficacy of the C-ASWS antigen in eliciting a delayed-type hypersensitivity response. One-microgram doses of C-ASWS produced reactions comparable to 100-mug doses of nondialyzable coccidioidin (Smith's lot 64 D4). The C-ASWS fractions isolated from three different C. immitis strains showed similar reactivity in terms of the number of positive reactions produced in Coccidioides-sensitized guinea pigs. However, the induration responses (diameter in millimeters) elicited by the C-ASWS fraction of one strain were significantly less than those elicited by the C-ASWS fractions of the other two C. immitis strains.