Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance.

Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes. Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes. Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.

Perception and response in plant disease resistance.

Plants express sophisticated mechanisms for recognizing pathogens. The functionally defined repertoire of non-self perception is large; the number and nature of subsequent molecular events required for resistance is unknown. Recent cloning of disease resistance genes, and genetic identification of loci required for their function, allows dissection of the structure, evolution, and deployment within populations of pathogen-perception mechanisms. Roles for reactive oxygen species and programmed cell death in resistance have also been suggested recently. New results document a role for salicylic acid as a lynchpin in the establishment and maintenance of the 'effector functions' of disease resistance, and strategies for engineered plant protection are moving closer to reality.

Biofilm adaptation to iron availability in the presence of biotite and consequences for chemical weathering.

Bacteria in nature often live within biofilms, exopolymeric matrices that provide a favorable environment that can differ markedly from their surroundings. Biofilms have been found growing on mineral surfaces and are expected to play a role in weathering those surfaces, but a clear understanding of how environmental factors, such as trace-nutrient limitation, influence this role is lacking. Here, we examine biofilm development by Pseudomonas putida in media either deficient or sufficient in Fe during growth on biotite, an Fe rich mineral, or on glass. We hypothesized that the bacteria would respond to Fe deficiency by enhancing biotite dissolution and by the formation of binding sites to inhibit Fe leaching from the system. Glass coupons acted as a no-Fe control to investigate whether biofilm response depended on the presence of Fe in the supporting solid. Biofilms grown on biotite, as compared to glass, had significantly greater biofilm biomass, specific numbers of viable cells (SNVC), and biofilm cation concentrations of K, Mg, and Fe, and these differences were greater when Fe was deficient in the medium. Scanning electron microscopy (SEM) confirmed that biofilm growth altered the biotite surface, smoothing the rough, jagged edges of channels scratched by hand on the biotite, and dissolving away small, easy-to-access particles scattered across the planar surface. High-resolution magic angle spinning proton nuclear magnetic resonance (HRMAS 1 H NMR) spectroscopy showed that, in the Fe-deficient medium, the relative amount of polysaccharide nearly doubled relative to that in biofilms grown in the medium amended with Fe. The results imply that the bacteria responded to the Fe deficiency by obtaining Fe from biotite and used the biofilm matrix to enhance weathering and as a sink for released cation nutrients. These results demonstrate one mechanism by which biofilms may help soil microbes overcome nutrient deficiencies in oligotrophic systems.

Biochemical consequences of resistance to a recently discovered IMP dehydrogenase inhibitor, benzamide riboside, in human myelogenous leukemia K562 cells.

Benzamide riboside (BR) exhibits potent antitumor activity in a variety of cultured human tumor cells. The drug is metabolized to benzamide adenine dinucleotide (BAD), which in turn functions as a selective inhibitor of IMP dehydrogenase (IMPDH) activity with a Ki of 0.118 microM. In vitro, BR is a more potent antitumor inhibitor of IMPDH than tiazofurin, another IMPDH inhibitor which has shown significant oncolytic activity in adult patients with end-stage leukemia. To elucidate the mechanism of resistance, a variant of human myelogenous leukemia K562 cells was developed by subculturing sensitive cells in sublethal concentrations of BR over 60 generations. The BR resistant line that emerged exhibited an IC50 (a concentration producing 50% reduction in cell proliferation) of 148 microM, compared to the sensitive line which had an IC50 of 1.6 microM. The activity of the target enzyme, IMPDH, was increased 3-fold in the resistant variant. Studies on BR metabolism revealed that resistant cells formed only 18% of the active metabolite, BAD, compared to sensitive cells. This finding, in turn, correlated with the specific activity of NAD pyrophosphorylase (the enzyme responsible for the synthesis of BAD) which was reduced to undetectable levels in the resistant variant. The basal levels of NAD and guanylates were also significantly decreased to 41% and 48%, respectively, in the resistant line compared to the parent line. Additionally, after treatment with BR a decrease in guanylate level was observed only in the sensitive cells. Sensitive and resistant cells exhibit comparable cytotoxicity to agents outside the tiazofurin family, suggesting that a multidrug resistance was unlikely to explain the resistance to BR. Moreover, BR resistant cells exhibit collatoral sensitivity to 6-aminopurine, cytarabine and 5-fluorouracil, which have different mechanisms of action. In conclusion, these studies establish that the primary mechanism of resistance to BR involves an increase in IMPDH (target enzyme) activity with a concurrent decrease in NAD pyrophosphorylase (BAD synthetic enzyme) activity.

Simulating in vitro epithelial morphogenesis in multiple environments.

In vitro studies of epithelial cell morphogenesis have demonstrated the influence of environment composition and orientation in the development of multicellular epithelial structures such as tubules and cysts. We have constructed a low resolution, discrete event simulation model and report on its use to explore how experimentally observed morphogenetic phenomena under four growth conditions might be generated and controlled. We identified simulation attributes that may have in vitro counterparts. We studied how changes in the logic governing simulated epithelial cell behavior might cause abnormal growth. Simulation results support the importance of a polarized response to the environment to the generation of a normal epithelial phenotype and show how disruptions of tight mechanistic control lead to aberrant growth characteristics.

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase.

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (greater than 90%) and nucleotide (greater than 80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.

The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus).

An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A)+ RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A)+ RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23% identical and 66% similar (allowing for conservative substitutions) to a human glycosylasparaginase.

Independent deletions of a pathogen-resistance gene in Brassica and Arabidopsis.

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with approximately 81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.

Molecular cloning of the gene encoding developing seed L-asparaginase from Lupinus angustifolius.

A genomic sequence encoding Lupinus angustifolius L-asparaginase has been obtained, and is the first report of this gene from a plant source. The 3.2 kb of DNA sequenced contains a 1136 bp 5' flanking sequence, four exons and three introns. Intron-exon borders were mapped by comparing the genomic sequence with that of a L. arboreus cDNA. Primer extension analysis revealed transcription start sites 16 bp and 13 bp 5' of the initiating ATG for L. angustifolius and L. arboreus, respectively. The 5' flanking region contained sequences associated with seed-specific expression.