PlantSimLab - a modeling and simulation web tool for plant biologists.

BACKGROUND: At the molecular level, nonlinear networks of heterogeneous molecules control many biological processes, so that systems biology provides a valuable approach in this field, building on the integration of experimental biology with mathematical modeling. One of the biggest challenges to making this integration a reality is that many life scientists do not possess the mathematical expertise needed to build and manipulate mathematical models well enough to use them as tools for hypothesis generation. Available modeling software packages often assume some modeling expertise. There is a need for software tools that are easy to use and intuitive for experimentalists. RESULTS: This paper introduces PlantSimLab, a web-based application developed to allow plant biologists to construct dynamic mathematical models of molecular networks, interrogate them in a manner similar to what is done in the laboratory, and use them as a tool for biological hypothesis generation. It is designed to be used by experimentalists, without direct assistance from mathematical modelers. CONCLUSIONS: Mathematical modeling techniques are a useful tool for analyzing complex biological systems, and there is a need for accessible, efficient analysis tools within the biological community. PlantSimLab enables users to build, validate, and use intuitive qualitative dynamic computer models, with a graphical user interface that does not require mathematical modeling expertise. It makes analysis of complex models accessible to a larger community, as it is platform-independent and does not require extensive mathematical expertise.

Genes controlling expression of defense responses in Arabidopsis--2001 status.

In the past two years, the focus of studies of the genes controlling expression of defense responses in Arabidopsis has shifted from the identification of mutants to gene isolation and the ordering of genes within branches of the signal transduction networks. It is now clear that gene-for-gene resistance can be mediated through at least three genetically distinguishable pathways. Additional genes affecting salicylic-acid-dependent signaling have been identified, and double-mutant analyses have begun to reveal the order in which they act. Genes required for jasmonic-acid-dependent signaling and for induced systemic resistance have also been identified.

Genes controlling expression of defense responses in Arabidopsis.

In the past year, two regulatory defense-related genes, EDS1l and COl1, have been cloned. Several other genes with regulatory functions have been identified by mutation, including DND1, PAD4, CPR6, and SSl1. It has become clear that jasmonate signaling plays an important role in defense response signaling, and that the jasmonate and salicylic acid signaling pathways are interconnected.

Molecular genetics of mosquito resistance to malaria parasites.

Malaria parasites are transmitted by the bite of an infected mosquito, but even efficient vector species possess multiple mechanisms that together destroy most of the parasites present in an infection. Variation between individual mosquitoes has allowed genetic analysis and mapping of loci controlling several resistance traits, and the underlying mechanisms of mosquito response to infection are being described using genomic tools such as transcriptional and proteomic analysis. Malaria infection imposes fitness costs on the vector, but various forms of resistance inflict their own costs, likely leading to an evolutionary tradeoff between infection and resistance. Plasmodium development can be successfully completed onlyin compatible mosquito-parasite species combinations, and resistance also appears to have parasite specificity. Studies of Drosophila, where genetic variation in immunocompetence is pervasive in wild populations, offer a comparative context for understanding coevolution of the mosquito-malaria relationship. More broadly, plants also possess systems of pathogen resistance with features that are structurally conserved in animal innate immunity, including insects, and genomic datasets now permit useful comparisons of resistance models even between such diverse organisms.

Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening.

To discover which components of plant defense responses make significant contributions to limiting pathogen attack, we screened a mutagenized population of Arabidopsis thaliana for individuals that exhibit increased susceptibility to the moderately virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). The 12 enhanced disease susceptibility (eds) mutants isolated included alleles of two genes involved in phytoalexin biosynthesis (pad2, which had been identified previously, and pad4, which had not been identified previously), two alleles of the previously identified npr1 gene, which affects expression of other defense genes, and alleles of seven previously unidentified genes of unknown function. The npr1 mutations caused greatly reduced expression of the PR1 gene in response to PsmES4326 infection, but had little effect on expression of two other defense genes, BGL2 and PR5, suggesting that PR1 expression may be important for limiting growth of PsmES4326. While direct screens for mutants with quantitative pathogen-susceptibility phenotypes have not been reported previously, our finding that mutants isolated in this way include those affected in known defense responses supports the notion that this type of screening strategy allows genetic dissection of the roles of various plant defense responses in disease resistance.

Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance.

We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens. Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not. Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1. Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively. The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function. PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica. The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates. Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates.

Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA.

The Arabidopsis enhanced disease susceptibility 4 (eds4) mutation causes enhanced susceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). Gene-for-gene resistance to bacteria carrying the avirulence gene avrRpt2 is not significantly affected by eds4. Plants homozygous for eds4 exhibit reduced expression of the pathogenesis-related gene PR-1 after infection by Psm ES4326, weakened responses to treatment with the signal molecule salicylic acid (SA), impairment of the systemic acquired resistance response, and reduced accumulation of SA after infection with Psm ES4326. These phenotypes indicate that EDS4 plays a role in SA-dependent signaling. SA has been shown to have a negative effect on activation of gene expression by the signal molecule jasmonic acid (JA). Two mutations that cause reduced SA levels, eds4 and pad4, cause heightened responses to inducers of JA-dependent gene expression, providing genetic evidence to support the idea that SA interferes with JA-dependent signaling. Two possible working models of the role of EDS4 in governing activation of defense responses are presented.

Genetic mapping of symbiotic loci on the Rhizobium meliloti chromosome.

To facilitate genetic analyses of Rhizobium meliloti genes that are involved in symbiosis, we determined the map positions of 11 symbiotic loci on the R. meliloti chromosome by using a combination of the Tn5-Mob conjugational transfer method described by Klein et al. (S. Klein, K. Lohmann, G. C. Walker, and E. R. Signer. J. Bacteriol. 174:324-326, 1992) and co-transduction of genetic markers by bacteriophage phi M12. Loci involved in effective nodule formation (fix-379, fix-382, fix-383, fix-385, and fix-388), polysaccharide synthesis (exoR, exoS, exoC, and ndvB), nodule invasion (exoD), and nitrogen regulation (ntrA) were ordered with respect to previously mapped markers and each other. The positions of two other loci, degP and pho-1, were also determined.

Mode of action of the Arabidopsis thaliana phytoalexin camalexin and its role in Arabidopsis-pathogen interactions.

The virulent Arabidopsis thaliana pathogen Pseudomonas syringae pv. maculicola strain ES4326 (Psm ES4326) and other gram-negative bacteria are sensitive to camalexin (3-thiazol-2'-yl-indole), the Arabidopsis phytoalexin. Furthermore, Psm ES4326 is unable to degrade camalexin or to become tolerant to it. Apparently, Psm ES4326 is a successful pathogen even though it elicits synthesis of a host phytoalexin to which it is sensitive. Assays of membrane integrity revealed that, like other phytoalexins, camalexin disrupts bacterial membranes, suggesting that camalexin toxicity is a consequence of membrane disruption. A screen for camalexin-resistant mutants of Psm ES4326 yielded only partially resistant mutants, which displayed partial resistance in both killing and membrane integrity assays. These mutants were also resistant to low concentrations of tetracycline and nalidixic acid, suggesting that they were affected in components of the outer membrane. The mutants were not distinguishable from Psm ES4326 in virulence assays. Camalexin was toxic to Arabidopsis cells growing in tissue culture. However, comparison of the extent of cell death associated with disease symptoms in infected leaves of wild-type Arabidopsis and a camalexin-deficient mutant suggested that camalexin does not contribute significantly to cell death in infected tissue.

Sequence analysis of the pyr-4 (orotidine 5'-P decarboxylase) gene of Neurospora crassa.

The pyr-4 gene of Neurospora crassa encodes orotidine-5' -phosphate decarboxylase, which catalyses the sixth step in the pyrimidine biosynthetic pathway. The complete nucleotide sequence of a 1.8-kb genomic fragment containing the pyr-4 gene has been determined. Using transposon mutagenesis, the coding region has been identified, and the amino acid (aa) sequence deduced. Comparison of the pyr-4 aa sequence with URA3, the equivalent gene of Saccharomyces cerevisiae, showed extensive blocks of homology, with non-homologous sequences between these blocks being generally much longer in Neurospora than in yeast. Computer-predicted protein secondary structure of pyr-4 and URA3 was conserved within equivalent blocks. Upstream sequences of pyr-4 were compared with other sequenced Neurospora genes and possible promoter sequences identified.

Diagnostic reagents for hepatitis C virus.

The development of diagnostic methods for hepatitis C virus is presented. Special attention is paid to the selection of antigenic markers, the type of assay selected and the interpretation of results. A few of the pitfalls and ambiguities of various assays are discussed and possible future methods are described.

Structural studies of a novel exopolysaccharide produced by a mutant of Rhizobium meliloti strain Rm1021.

The structure of a novel expolysaccharide obtained from a mutant of Rhizobium meliloti strain Rm1021 was elucidated by a combination of enzymic, chemical, and spectroscopic methods. The polysaccharide is composed of a disaccharide repeating-unit, beta-D-Glcp-(1----3)-alpha-D-Galp-(1----3), having a 6-O-acetyl group attached to most D-glucose residues and a 4,6-O-(1-carboxyethylidene) group attached to every D-galactose residue.

Genetic analyses of Rhizobium meliloti exopolysaccharides.

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.

Studies on cardiovascular fluke (Digenea: Spirorchiidae) infections in sea turtles from the Great Barrier Reef, Queensland, Australia.

Twenty-seven sea turtles (23 Chelonia mydas and four Eretmochelys imbricata) from northeast Queensland were found to be infected with cardiovascular flukes and/or their eggs. Five had originated from turtle farms in the Torres Strait, five from an oceanarium on Magnetic Island (146 degrees 56'E, 19 degrees S) and the remainder from coral reefs in the Torres Strait or near Townsville. The incidence of flukes and/or eggs in the groups was 4.8 per cent (5 of 104), 33.3 per cent (5 of 15) and 72.2 per cent (17 of 22), respectively. Affected animals ranged in size from 18 to 108 cm (curved carapace length) and weighed between 0.5 and 77 kg. The average number of flukes per host was 47. Flukes were recovered from the three chambers of the heart and major vessels (right aortic arch and brachiocephalic artery), where they were attached to the walls or free in the lumen. They were subsequently identified as Haplotrema spp. and/or Learedius spp. In 59.2 per cent (16 of 27) of turtles, flukes were not found, although their eggs were detected microscopically. Gross pathological changes associated with the presence of flukes included thickening and hardening of arterial walls (four turtles), thrombus formation (three), chronic pneumonia (two) and an excess of pericardial or peritoneal fluid (four). Microscopically, the essential changes was that of chronic inflammation, as evidenced by the proliferation of epithelial cells, reticulo-endothelial cells and fibroblasts in areas accessible to flukes and/or eggs. Multiple diffuse egg granulomas were a prominent feature of most organs, the spleen and lungs being predilection sites. Proliferative changes had occurred in the endocardium and in the endothelium of vessels supplying the spleen, stomach, intestine and pancreas (18 turtles). The walls of major arteries, lungs, liver, brain, crop and stomach were also acutely inflamed (eight turtles). Haemorrhage was recorded in the lungs and/or brain of eight turtles with heavy fluke infestations. Other vascular changes, viz. congestion, oedema and hypertrophy of arterial/arteriolar walls, resulted from the inhibition of blood flow by parasitic emboli.