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Genetic mapping of resistance genes for sorghum downy mildew (SDM) in maize revealed multiple-locus inheritance. A combination of AFLP (amplified fragment length polymorphism) technique with bulked segregant analysis (BSA) was applied to map the genes involved in the resistance to SDM (Peronosclerospora sorghi) in a recombinant inbred population. Three AFLP markers were identified and mapped to chromosomes 1 and 9, in regions previously associated with SDM resistance. One other AFLP marker was found to be associated with disease susceptibility but could not be linked to any chromosome. These four AFLP fragments were isolated, cloned and sequenced. A BLAST search of the GenBank database showed that none of these four sequences was closely related to resistance genes that have been reported previously. Sequence-characterized amplified regions (SCARs) were produced and used to assess the presence of SDM resistance genes and characterize specific genotypes. These markers may be useful in marker-assisted breeding programs.

The oomycete grape downy mildew (Plasmopara viticola Berk. & Curt. Ex de Bary) is a serious pathogen of grapevine and spreads by extremely efficient cycles of asexual propagation. The high efficiency must involve efficient sensing of the host. We therefore analyzed the time course and morphology of the early development of this pathogen in a host system, by infection of leaf discs of grapevine (Vitis vinifera L. cv. Müller-Thurgau), and in a host-free system. Host factors were demonstrated to influence pathogen development in the following ways: (i) the release of zoospores from mature sporangia was accelerated, (ii) the morphogenesis of the germ tube was coordinated, and (iii) the zoospores were targeted to the stomata by factors that depended on stomata closure. The findings show that the early development of P. viticola is regulated, specifically and coordinately, by factors originating from the host plant.

Objective: To screen effectual fungicides for field control because of the seriousness of opium poppy mildew and importance of chemical control on plant diseases., Method: Seven fungicides were screened in Lab experiment and field test during 1996-1997., Result and Conclusion: All of them and their different dosages were effective to control conidia of Peronospora arborescens. Among them, 72.2% propamocarb of 1203 and 902.5 ppm were the most effective both in Lab experiment and field test with efficacy 79.91% and 79.33% respectively in field test, and the efficacy of other fungicides was over 50%. Seven fungicides tested can be used to control nonsystematic symptom of opium poppy mildew.

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.

The Arabidopsis genome contains 28 genes with sequence homology to the Arabidopsis NDR1 gene and the tobacco HIN1 gene. Expression analysis of eight of these genes identified two (NHL25 and NHL3 for NDR1/HIN1-like) that show pathogen-dependent mRNA accumulation. Transcripts did not accumulate during infection with virulent Pseudomonas syringae pv. tomato DC3000 but did accumulate specifically when the bacteria carried any of the four avirulence genes avrRpm1, avrRpt2, avrB, or avrRps4. Furthermore, expression of avrRpt2 in plants containing the corresponding resistance gene, RPS2, was sufficient to induce transcript accumulation. However, during infection with an avirulent oomycete, Peronospora parasitica isolate Cala-2, only NHL25 expression was reproducibly induced. Salicylic acid (SA) treatment can induce expression of NHL25 and NHL3. Studies performed on nahG plants showed that, during interaction with avirulent bacteria, only the expression of NHL25 but not that of NHL3 was affected. This suggests involvement of separate SA-dependent and SA-independent pathways, respectively, in the transcriptional activation of these genes. Bacteria-induced gene expression was not abolished in ethylene- (etrl-3 and ein2-1) and jasmonate- (coil-1) insensitive mutants or in mutants impaired in disease resistance (ndr1-1 and pad4-1). Interestingly, NHL3 transcripts accumulated after infiltration with the avirulent hrcC mutant of Pseudomonas syringae pv. tomato DC3000 and nonhost bacteria but not with the virulent Pseudomonas syringae pv. tomato DC3000, suggesting that virulent bacteria may suppress NHL3 expression during pathogenesis. Hence, the expression patterns and sequence homology to NDR1 and HIN1 suggest one or more potential roles for these genes in plant resistance.

Animal SGT1 is a component of Skp1-Cullin-F-box protein (SCF) ubiquitin ligases that target regulatory proteins for degradation. Mutations in one (SGT1b) of two highly homologous Arabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss of SGT1b function in resistance is not compensated for by SGT1a. R genes differ in their requirements for SGT1b and a second resistance signaling gene, RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively to RPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.

PCR amplification of two CHS gene fragments of the obligate biotroph Plasmopara viticola, the causal agent of downy mildew of grapevine, is described. While one fragment shows homology to fungal class IV chitin synthases, the other fragment groups with other oomycete chitin synthases to form a novel class of chitin synthases most closely related to class I-III. RT-PCR experiments indicate that PvCHS1 is constitutively expressed, whereas PvCHS2 is specifically transcribed in sporangiophores and sporangia. Analyses of wheat germ agglutinin labeling patterns by confocal laser scanning microscopy show that chitin is present on the surface of hyphal cell walls during in planta growth, and of sporangiophores and sporangia.

Evolution is based on genetic variability and subsequent phenotypic selection. Mechanisms that modulate the rate of mutation according to environmental cues, and thus control the balance between genetic stability and flexibility, might provide a distinct evolutionary advantage. Stress-induced mutations stimulated by unfavorable environments, and possible mechanisms for their induction, have been described for several organisms, but research in this area has mainly focused on microorganisms. We have analyzed the influence of adverse environmental conditions on the genetic stability of the higher plant Arabidopsis thaliana. Here we show that a biotic stress factor-attack by the oomycete pathogen Peronospora parasitica-can stimulate somatic recombination in Arabidopsis. The same effect was observed when plant pathogen-defense mechanisms were activated by the chemicals 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), or by a mutation (cim3). Together with previous studies of recombination induced by abiotic factors, these findings suggest that increased somatic recombination is a general stress response in plants. The increased genetic flexibility might facilitate evolutionary adaptation of plant populations to stressful environments.

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are each involved in the regulation of basal resistance against different pathogens. These three signals play important roles in induced resistance as well. SA is a key regulator of pathogen-induced systemic acquired resistance (SAR), whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance (ISR). Both types of induced resistance are effective against a broad spectrum of pathogens. In this study, we compared the spectrum of effectiveness of SAR and ISR using an oomycete, a fungal, a bacterial, and a viral pathogen. In noninduced Arabidopsis plants, these pathogens are primarily resisted through either SA-dependent basal resistance (Peronospora parasitica and Turnip crinkle virus [TCV]), JA/ET-dependent basal resistance responses (Alternaria brassicicola), or a combination of SA-, JA-, and ET-dependent defenses (Xanthomonas campestris pv. armoraciae). Activation of ISR resulted in a significant level of protection against A. brassicicola, whereas SAR was ineffective against this pathogen. Conversely, activation of SAR resulted in a high level of protection against P. parasitica and TCV, whereas ISR conferred only weak and no protection against P. parasitica and TCV, respectively. Induction of SAR and ISR was equally effective against X. campestris pv. armoraciae. These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent defenses, whereas ISR is effective against pathogens that in noninduced plants are resisted through JA/ET-dependent defenses. This suggests that SAR and ISR constitute a reinforcement of extant SA- or JA/ET-dependent basal defense responses, respectively.

The fish, Aplocheilus panchax (Hamilton-Buchanan, 1839), is recorded as a new host of Aphanomyces laevis de Bary, 1860. It is the first and only report of any oomycete causing disease on A. panchax. Aphanomyces laevis was found to be a virulent parasite of A. panchax that caused cotton-wool disease involving the skin and fins. The hyphae also penetrated deep into the tissues of the infected fish and all of them ultimately died.

Epizootic ulcerative syndrome (EUS) is one of the most destructive diseases of fresh and brackish water farmed and wild fish in the Asia-Pacific region. The in vitro germination and growth of the propagules of the EUS pathogen, Aphanomyces invadans (= A. piscicida), were assessed in the presence of the head-kidney macrophages, serum, and serum heated to inactivate complement proteins, of three EUS-susceptible and one resistant fish species. The susceptible species were: striped snakehead (Channa striata), giant gourami (Osphronemus gouramy) and silver barb [Barbodes (= Puntius) gonionotus], and the resistant species was Nile tilapia (Oreochromis niloticus). Fish of all species were acclimatised to either low temperature (20 degrees C +/- 1.6) at which EUS is known to occur, or to high temperature (32 degrees C +/- 5.0) at which EUS does not occur, except for giant gouramis which were only studied at low temperature. The respiratory burst of the macrophages was assessed in the presence of A. invadans or the stimulant phorbol myristate acetate (PMA), and compared to that of controls. Anti-A. invadans antibody concentrations were assessed in all species except silver barbs. All assays were carried out at the same temperature, regardless of the temperature that the fish were kept at. Macrophages of all species other than snakeheads inhibited fungal germination at both temperatures, though only silver barb and gourami macrophages could inhibit germling growth. PMA increased the respiratory burst in nearly all cases. The respiratory burst in the presence of A. invadans was consistently lower than that of controls, though the difference was only significant in the case of snakeheads. The respiratory burst of all macrophage treatments was higher at a low temperature. Except in the case of PMA-stimulated macrophages, regressions between respiratory burst and inhibitory action were only found in susceptible species, suggesting that the respiratory burst is important in those species, but is unable to prevent the proliferation of A. invadans. Serum inhibited fungal germination in all cases other than low temperature tilapia, indicating that the EUS resistance of tilapia is not due to the serum. Inhibition of germling growth by serum only occurred in silver barbs and gourami. Heated serum did not inhibit germination in any case except that of high temperature snakehead, and in fact stimulated germination in the case of tilapia. Heating serum did not affect the growth inhibiting activity of silver barbs and gouramis, but it stimulated growth in some groups. At high temperatures snakeheads had high anti-A. invadans antibody concentrations, which may explain the inhibitory activity of their heated serum. A role for complement and antibodies in defence against A. invadans in susceptible species is suggested.

The copines are a newly identified class of calcium-dependent, phospholipid binding proteins that are present in a wide range of organisms, including Paramecium, plants, Caenorhabditis elegans, mouse, and human. However, the biological functions of the copines are unknown. Here, we describe a humidity-sensitive copine mutant in Arabidopsis. Under nonpermissive, low-humidity conditions, the cpn1-1 mutant displayed aberrant regulation of cell death that included a lesion mimic phenotype and an accelerated hypersensitive response (HR). However, the HR in cpn1-1 showed no increase in sensitivity to low pathogen titers. Low-humidity-grown cpn1-1 mutants also exhibited morphological abnormalities, increased resistance to virulent strains of Pseudomonas syringae and Peronospora parasitica, and constitutive expression of pathogenesis-related (PR) genes. Growth of cpn1-1 under permissive, high-humidity conditions abolished the increased disease resistance, lesion mimic, and morphological mutant phenotypes but only partially alleviated the accelerated HR and constitutive PR gene expression phenotypes. The disease resistance phenotype of cpn1-1 suggests that the CPN1 gene regulates defense responses. Alternatively, the primary function of CPN1 may be the regulation of plant responses to low humidity, and the effect of the cpn1-1 mutation on disease resistance may be indirect.

To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.

The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA). An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1. In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2. High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype. The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1. These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1. This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1. Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.

The Arabidopsis EDS1 and PAD4 genes encode lipase-like proteins that function in resistance (R) gene-mediated and basal plant disease resistance. Phenotypic analysis of eds1 and pad4 null mutants shows that EDS1 and PAD4 are required for resistance conditioned by the same spectrum of R genes but fulfil distinct roles within the defence pathway. EDS1 is essential for elaboration of the plant hypersensitive response, whereas EDS1 and PAD4 are both required for accumulation of the plant defence-potentiating molecule, salicylic acid. EDS1 is necessary for pathogen-induced PAD4 mRNA accumulation, whereas mutations in PAD4 or depletion of salicylic acid only partially compromise EDS1 expression. Yeast two-hybrid analysis reveals that EDS1 can dimerize and interact with PAD4. However, EDS1 dimerization is mediated by different domains to those involved in EDS1-PAD4 association. Co-immunoprecipitation experiments show that EDS1 and PAD4 proteins interact in healthy and pathogen-challenged plant cells. We propose two functions for EDS1. The first is required early in plant defence, independently of PAD4. The second recruits PAD4 in the amplification of defences, possibly by direct EDS1-PAD4 association.

Lagenidium giganteum was cultivated on solid media in the absence of free water and evaluated for efficacy against second-instar Aedes aegypti mosquito larvae in 100-ml bioassays. Bioassay variables included level of media addition, CO(2) evolution rate (CER) and cell density. Logistic regression was performed on bioassay infection observations to determine if the tested variables were correlated to the probability of attaining at least 80% infection. Both CER (p=0.003) and number of cells (p=0.017) were significantly correlated, while level of media addition was not (p=0.42). Although media addition did not correlate with efficacy, media levels greater than 2 g/l reduced water clarity and infection. Media from cultures younger than 3 days performed poorly under all conditions tested.

The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.

Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.

RPP13-Nd-mediated resistance prevents parasitism by five isolates of Peronospora parasitica (At) in a transgenic Arabidopsis. Columbia background. We tested the effect of a number of known disease resistance mutations on the RPP13-Nd function and found that resistance remained unaltered in plants carrying mutations in either EDS1 or NDR1 and in double ndr1-1/eds1-2 mutant lines. Furthermore, we found that pbs2, pad4-1, npr1-1, and rps5-1, which compromise resistance to a number of P. parasitica (At) isolates, had no affect on RPP13-Nd function. In addition, RPP13-Nd-mediated resistance remained unchanged in a background of salicylic acid depletion (nahG). We conclude that RPP13-Nd is the first Arabidopsis R gene product reported to act via a novel signaling pathway that is independent of salicylic acid-mediated responses and is completely independent of NDR1 and EDS1.

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.

Screening chemical compounds, we found that a xenoestrogen, bisphenol A, showed potent repellent activity against the zoospores of Aphanomyces cochlioides. Based on this finding, we tested a number of androgenic and estrogenic compounds (e.g. testosterone, progesterone, estradiols, diethylstilbestrol, estrone, estriol, pregnenolone, dienestrol etc.) on the motility behavior of A. cochlioides zoospores. Interestingly, most of the estrogenic compounds exhibited potent repellent activity (1 microg/ml or less by the "particle method") toward the motile zoospores of A. cochlioides. We derivatized some of the estrogens and discussed the relationship between the structure of active molecules and their repellent activity. Apparently, aromatization of the A ring with a free hydroxyl group at C-3 position of a steroidal structure is necessary for higher repellent activity. Interestingly, methylation of diethylstilbestrol (DES) yielded completely different activity i.e. both mono- and di-methyl ethers of DES showed attractant activity. Moreover, the attracted zoospores were encysted and then germinated in the presence of di-methyl ether of DES. The potential usefulness of this repellent test is discussed for the detection of estrogenic activity of naturally occurring compounds, and the possible role of phytoestrogens in host/parasite interactions. So far, this will be the first report of repellent activity of estrogenic compounds toward trivial fungal zoospores.

Objective: To explore the possible defensive immunity of Culex pipiens quinquefasciatus larvae after being infected by Lagenidium giganteum., Methods: To count and analyze morphologically hemocytes of the mosquito larvae with Giemsa's stain and by both phase contrast microscope and ordinary microscope., Results: 1. Following the prolonged duration of infection, the number of hemocytes featured as increase, intense increase and gradual decrease. 2. 48 and 72 h after infection the proportion of plasmatocytes and granulocytes increased significantly, but that of prohemocytes showed a relatively slow increase, while 96 and 120 h after infection the proportion of spherulocytes and oenocytoids increased gradually and that of plasmatocyte and granulocytes showed a slow decrease. 3. After L. giganteum infection, vacuolation and morpholysis were found in plasmatocytes., Conclusion: The changes of hemocytes in amount, classification and morphology after infection revealed that Lagenidium giganteum can induce some defensive immune reactions in the larvae of Culex pipiens quinquefasciatus possibly due to a release of antigens or toxic substances by the fungus.

A white rust infected leaf of Brassica juncea var. Varuna bearing non-erumpent zoosporangial blisters was used as explant to grow a dual culture of Albugo candida and Brassica juncea on MS medium supplemented with NAA (1.0 mg/L), BAP (1.0 mg/L), biotin (1.0 mg/L), ascorbic acid (25.0 mg/L), thiamin hydrochloride (1.0 mg/L), glycine (0.5 mg/L) and casein hydrolysate (1.0 mg/L). The host callus and the pathogen established a complete balance in culture. The morphology of the mycelium, haustoria, zoosporangia, antheridia, oogonia and oospores in dual culture was identical to that of infected intact plant. Oospore formation was favoured over that of sporangia. Oospore germination by germ-tube was evident. Pathogenicity test of the fungus in dual culture further confirmed the viability of the fungus. Rate of growth of dual culture was faster than normal callus. Although the fungus grew on the substratum for a short distance away from infected callus on the surface of the medium; it did not grow independently when connections with host callus was severed. Growth of dual culture was influenced by light quality, temperature, vitamins, carbohydrates and amino acids in the medium. These differential responses can be used for future studies on host pathogen interactions and for breeding of disease resistant plants.

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.

Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of B. cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots. In vitro, B. cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes. B. cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels. However, when present at high levels on seeds, B. cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied B. cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain. This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low. B. cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence. These results suggest that B. cepacia AMMDR1 controls P. aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens.

The nonprotein amino acids gamma-aminobutyric acid (GABA) and beta-aminobutyric acid (BABA) have known biological effects in animals and plants. Their mode of action has been the object of thorough research in animals but remains unclear in plants. Our objective was to study the mode of action of BABA in the protection of Arabidopis plants against virulent pathogens. BABA protected Arabidopsis against the oomycete pathogen Peronospora parasitica through activation of natural defense mechanisms of the plant such as callose deposition, the hypersensitive response, and the formation of trailing necroses. BABA was still fully protective against P. parasitica in transgenic plants or mutants impaired in the salicylic acid, jasmonic acid, and ethylene signaling pathways. Treatment with BABA did not induce the accumulation of mRNA of the systemic acquired resistance (SAR)-associated PR-1 and the ethylene- and jasmonic acid-dependent PDF1.2 genes. However, BABA potentiated the accumulation of PR-1 mRNA after attack by virulent pathogenic bacteria. As a result, BABA-treated Arabidopsis plants were less diseased compared with the untreated control. In the case of bacteria, BABA protected mutants insensitive to jasmonic acid and ethylene but was not active in plants impaired in the SAR transduction pathway. Thus, BABA protects Arabidopsis against different virulent pathogens by potentiating pathogen-specific plant resistance mechanisms. In addition, we provide evidence that BABA-mediated papilla formation after P. parasitica infection is independent of the SAR signaling pathway.

To better understand the genetic requirements for R gene-dependent defense activation in Arabidopsis, we tested the effect of several defense response mutants on resistance specified by eight RPP genes (for resistance to Peronospora parasitica) expressed in the Col-0 background. In most cases, resistance was not suppressed by a mutation in the SAR regulatory gene NPR1 or by expression of the NahG transgene. Thus, salicylic acid accumulation and NPR1 function are not necessary for resistance mediated by these RPP genes. In addition, resistance conferred by two of these genes, RPP7 and RPP8, was not significantly suppressed by mutations in either EDS1 or NDR1. RPP7 resistance was also not compromised by mutations in EIN2, JAR1 or COI1 which affect ethylene or jasmonic acid signaling. Double mutants were therefore tested. RPP7 and RPP8 were weakly suppressed in an eds1-2/ndr1-1 background, suggesting that these RPP genes operate additively through EDS1, NDR1 and as-yet-undefined signaling components. RPP7 was not compromised in coi1/npr1 or coi1/NahG backgrounds. These observations suggest that RPP7 initiates resistance through a novel signaling pathway that functions independently of salicylic acid accumulation or jasmonic acid response components.

Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.

Bioprotection of pea roots against Aphanomyces euteiches by the arbuscular mycorrhizal fungus G. mosseae was demonstrated to depend on a fully established symbiosis. This was related with induction of mycorrrhiza-related chitinolytic enzymes. Possible mechanisms implicated in bioprotection are discussed.

Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd-1, designated RPP13-Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS-LRR type R protein with a putative amino-terminal leucine zipper. The RPP13-Rld allele, cloned from the accession Rld-2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (> 95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved beta-strand/beta-turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS-LRR type R proteins, suggesting functional significance.

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties--impairment of growth and induction of defense responses--indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.

Fifteen isolates of the biotrophic oomycete Peronospora parasitica (downy mildew) were obtained from a population of Arabidopsis thaliana plants that established naturally in a garden the previous year. They exhibited phenotypic variation in a set of 12 Arabidopsis accessions that suggested that the parasite population consisted of at least six pathotypes. One isolate, Maks9, elicited an interaction phenotype of flecking necrosis and no sporulation (FN) in the Arabidopsis accession Nd-1, and more extensive pitting necrosis with no sporulation (PN) in the accession Ws-2. RPP13 was designated as the locus for a single dominant resistance gene associated with the resistance in Nd-1 and mapped to an interval of approximately 60 kb on a bacterial artificial chromosome (BAC) contig on the lower arm of chromosome 3. This locus is approximately 6 cM telomeric to RPP1, which was previously described as the locus for the PN interaction with five Peronospora isolates, including resistance to Maks9 in Ws-2. New Peronospora isolates were obtained from four other geographically distinct populations of P. parasitica. Four isolates were characterized that elicited an FN phenotype in Nd-1 and mapped resistance to the RPP13 locus. This suggests that the RPP13 locus contains either a single gene capable of multiple isolate recognition or a group of tightly linked genes. Further analysis suggests that the RPP11 gene in the accession Rld-0 may be allelic to RPP13 but results in a different recognition capability.

In Arabidopsis, systemic acquired resistance against pathogens has been associated with the accumulation of salicylic acid (SA) and the expression of the pathogenesis-related proteins PR-1, PR-2, and PR-5. We report here the isolation of two nonallelic mutants impaired in the pathway leading to SA biosynthesis. These SA induction-deficient (sid) mutants do not accumulate SA after pathogen inoculation and are more susceptible to both virulent and avirulent forms of Pseudomonas syringae and Peronospora parasitica. However, sid mutants are not as susceptible to these pathogens as are transgenic plants expressing the nahG gene encoding an SA hydroxylase that degrades SA to catechol. In contrast to NahG plants, only the expression of PR-1 is strongly reduced in sid mutants, whereas PR-2 and PR-5 are still expressed after pathogen attack. Furthermore, the accumulation of the phytoalexin camalexin is normal. These results indicate that SA-independent compensation pathways that do not operate in NahG plants are active in sid mutants. One of the mutants is allelic to eds5 (for enhanced disease susceptibility), whereas the other mutant has not been described previously.

We have used an antisense expression technology in Arabidopsis based on the yeast GAL4/UAS transactivation system (Guyer et al., Genetics, 1998; 149:633-639) to reduce levels of protoporphyrinogen IX oxidase (PPO), the last common enzyme of the biosynthesis of the haem group and chlorophyll. Plants expressing the antisense PPO gene presented growth alterations and their leaves showed necrotic lesions that appeared similar to lesions characteristic of the pathogen-induced hypersensitive reaction, and seen in the so-called lesion-mimic mutants. Plants expressing the antisense gene also had high endogenous salicylic acid levels, constitutive expression of the PR-1 gene, and were resistant to Peronospora parasitica, consistent with the activation of systemic acquired resistance (SAR). Treatment of wild-type plants with sublethal concentrations of herbicides that inhibit PPO also induced defence responses that conferred enhanced tolerance to P. parasitica. This effect was not observed in NahG and nim1 plants, which are compromised in their ability to activate SAR. These results demonstrate that genetic or chemical disruption of a metabolic pathway can lead to the induction of a set of defence responses including activation of SAR.

Pathogen resistance (R) genes of the NBS-LRR class (for nucleotide binding site and leucine-rich repeat) are found in many plant species and confer resistance to a diverse spectrum of pathogens. Little is known about the mechanisms that drive NBS-LRR gene evolution in the host-pathogen arms race. We cloned the RPP8 gene (for resistance to Peronospora parasitica) and compared the structure of alleles at this locus in resistant Landsberg erecta (Ler-0) and susceptible Columbia (Col-0) accessions. RPP8-Ler encodes an NBS-LRR protein with a putative N-terminal leucine zipper and is more closely related to previously cloned R genes that confer resistance to bacterial pathogens than it is to other known RPP genes. The RPP8 haplotype in Ler-0 contains the functional RPP8-Ler gene and a nonfunctional homolog, RPH8A. In contrast, the rpp8 locus in Col-0 contains a single chimeric gene, which was likely derived from unequal crossing over between RPP8-Ler and RPH8A ancestors within a Ler-like haplotype. Sequence divergence among RPP8 family members has been accelerated by positive selection on the putative ligand binding region in the LRRs. These observations indicate that NBS-LRR molecular evolution is driven by the same mechanisms that promote rapid sequence diversification among other genes involved in non-self-recognition.

Fungicide action is generally assumed to be dependent on an antibiotic effect on a target pathogen, although a role for plant defense mechanisms as mediators of fungicide action has not been excluded. Here, we demonstrate that in Arabidopsis, the innate plant defense mechanism contributes to the effectiveness of fungicides. In NahG and nim1 (for noninducible immunity) Arabidopsis plants, which normally exhibit increased susceptibility to pathogens, the fungicides metalaxyl, fosetyl, and Cu(OH)2 are much less active and fail to control Peronospora parasitica. In contrast, the effectiveness of these fungicides is not altered in Arabidopsis mutants defective in the ethylene or jasmonic acid signal transduction pathways. Application of the systemic acquired resistance activator benzothiadiazole (BTH) in combination with these fungicides results in a synergistic effect on pathogen resistance in wild-type plants and an additive effect in NahG and BTH-unresponsive nim1 plants. Interestingly, BTH treatment normally induces long-lasting pathogen protection; however, in NahG plants, the protection is transient. These observations suggest that BTH treatment can compensate only partially for an impaired signal transduction pathway and support the idea that pathogen defense mechanisms are under positive feedback control. These observations are strikingly reminiscent of the reduced efficacy of antifungal agents in immunocompromised animals.

Plant resistance (R) genes have evolved specific recognition capabilities in defense against pathogens. The evolution of R gene function and maintenance of R gene diversity within a plant species are therefore of great interest. In the Arabidopsis accession Wassilewskija, the RPP1 region on chromosome 3 contains four genetically linked recognition specificities, conditioning resistance to different isolates of the biotrophic oomycete Peronospora parasitica (downy mildew). We show that three of four tightly linked genes in this region, designated RPP1-WsA, RPP1-WsB, and RPP1-WsC, encode functional products of the NBS-LRR (nucleotide binding site-leucine-rich repeat) R protein class. They possess a TIR (Toll, interleukin-1, resistance) domain that is characteristic of certain other NBS-LRR-type R proteins, but in addition, they have unique hydrophilic or hydrophobic N termini. Together, the three RPP1 genes account for the spectrum of resistance previously assigned to the RPP1 region and thus comprise a complex R locus. The distinct but partially overlapping resistance capabilities conferred by these genes are best explained by the hypothesis that each recognizes a different pathogen avirulence determinant. We present evidence suggesting that the RPP genes at this locus are subject to the same selective forces that have been demonstrated for structurally different LRR-type R genes.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.

At least 10 Dm genes conferring resistance to the oomycete downy mildew fungus Bremia lactucae map to the major resistance cluster in lettuce. We investigated the structure of this cluster in the lettuce cultivar Diana, which contains Dm3. A deletion breakpoint map of the chromosomal region flanking Dm3 was saturated with a variety of molecular markers. Several of these markers are components of a family of resistance gene candidates (RGC2) that encode a nucleotide binding site and a leucine-rich repeat region. These motifs are characteristic of plant disease resistance genes. Bacterial artificial chromosome clones were identified by using duplicated restriction fragment length polymorphism markers from the region, including the nucleotide binding site-encoding region of RGC2. Twenty-two distinct members of the RGC2 family were characterized from the bacterial artificial chromosomes; at least two additional family members exist. The RGC2 family is highly divergent; the nucleotide identity was as low as 53% between the most distantly related copies. These RGC2 genes span at least 3.5 Mb. Eighteen members were mapped on the deletion breakpoint map. A comparison between the phylogenetic and physical relationships of these sequences demonstrated that closely related copies are physically separated from one another and indicated that complex rearrangements have shaped this region. Analysis of low-copy genomic sequences detected no genes, including RGC2, in the Dm3 region, other than sequences related to retrotransposons and transposable elements. The related but divergent family of RGC2 genes may act as a resource for the generation of new resistance phenotypes through infrequent recombination or unequal crossing over.

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.

In Arabidopsis, NPR1 mediates the salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR 6, that may function with NPR1 in regulating PR gene expression. The dominant CPR 6-1 mutant expresses the SA/NPR1-regulated PR genes (PR-1, BGL 2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr 6-1-induced PR gene expression is not suppressed in the cpr 6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr 6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr 6-1 npr1-1 double mutant, despite expression of PR-1, BGL 2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR 6 is an important regulator of multiple signal transduction pathways involved in plant defense.

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-delta 4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amide-linked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans had a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-delta 13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.

Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific corresponding avirulence genes. In Arabidopsis thaliana, resistance to both bacterial and fungal pathogens, mediated by several R gene products, requires the NDR1 gene. Positional cloning was used to isolate NDR1, which encodes a 660-base pair open reading frame. The predicted 219-amino acid sequence suggests that NDR1 may be associated with a membrane. NDR1 expression is induced in response to pathogen challenge and may function to integrate various pathogen recognition signals.

In many interactions of plants with pathogens, the primary host defense reaction is accompanied by plant cell death at the site of infection. The resulting lesions are correlated with the establishment of an inducible resistance in plants called systemic acquired resistance (SAR), for which salicylic acid (SA) accumulation is a critical signaling event in Arabidopsis and tobacco. In Arabidopsis, the lesions simulating disease (lsd) mutants spontaneously develop lesions in the absence of pathogen infection. Furthermore, lsd mutants express SAR marker genes when lesions are present and are resistant to the same spectrum of pathogens as plants activated for SAR by necrogenic pathogen infection. To assess the epistatic relationship between SA accumulation and cell death, transgenic Arabidopsis unable to accumulate SA due to the expression of the salicylate hydroxylase (nahG) gene were used in crosses with the dominant mutants lsd2 or lsd4. Progeny from the crosses were inhibited for SAR gene expression and disease resistance. However, these progeny retained the spontaneous cell death phenotype similar to siblings not expressing nahG. Because lesions form in the absence of SA accumulation for isd2 and lsd4, a model is suggested in which lesion formation in these two mutants is determined prior to SA accumulation in SAR signal transduction. By contrast, the loss of SAR gene expression and disease resistance in nahG-expressing lsd mutants indicates that these traits are dependent upon SA accumulation in the SAR signal transduction pathway.

Amphibians appear to be declining worldwide. One cause of their decline may be used crankcase oil which leaks from motor vehicles and washes into ponds. Once in ponds, the oil may either be directly toxic to amphibians, or may indirectly affect them by disrupting food chains. The effects of oil may also be compounded by naturally occurring materials in the water column such as silt. Silt may interfere with respiration across gill surfaces. This study examined the effects of oil and silt on the growth and metamorphosis of larval mole salamanders, Ambystoma opacum and A. tigrinum tigrinum. In Experiment One it examined ponds with and without silty water and oil pollution to determine their suitability as habitats for salamander larvae. In Experiment Two it studied the effects of low levels of oil combined with silt on animals raised in the laboratory and fed prey items not raised in oil. In Experiment Three, it explored the effects of oil at an ecosystem level by raising the salamanders in the field in plastic micromesocosms that mimicked small ponds. Finally, in Experiment Four, in the laboratory, it examined the short-term survival of salamanders in high concentrations of oil. This study found that ponds containing oil and silt produce salamanders of reduced size and weight. Furthermore, while salamanders are relatively robust to the short term effects of large concentrations of used motor oil, oil has deleterious effects on the community and therefore exerts an indirect negative effect on salamanders. In the mi- cro-mesocosms containing oil, salamanders were smaller and weighed less than animals not raised in oil. Furthermore, silt results in reduced growth, earlier metamorphosis, and increased susceptibility to the water mold Saprolegnia parasitica.

Little is known about the signal transduction events that lead to the establishment of the broad-spectrum, inducible plant immunity called systemic acquired resistance (SAR). Salicylic acid (SA) accumulation has been shown to be essential for the expression of SAR and plays a key role in SAR signaling. Hydrogen peroxide has been proposed to serve as a second messenger of SA. However, our results do not support such a role in the establishment of SAR. Further elucidation of SAR signal transduction has been facilitated by the identification and characterization of mutants. The lesions simulating disease (lsd). resistance response mutant class exhibits spontaneous lesions similar to those that occur during the hypersensitive response. Interestingly, some lsd mutants lose their lesioned phenotype when SA accumulation is prevented by expression of the nahG gene (encoding salicylate hydroxylase), thereby providing evidence for a feedback loop in SAR signal transduction. Characterization of a mutant non-responsive to SAR activator treatments has provided additional evidence for common signaling components between SAR and gene-for-gene resistance.

Lagenidium giganteum, a facultative parasite of mosquito larvae, cannot synthesize sterols, and requires an exogenous source of these lipids in order to enter its reproductive cycle. This parasite grows vegetatively in the absence of sterols, but requires cholesterol or structurally related compounds to produce motile zoospores, which are the only stage capable of infecting mosquitoes. Sterols structurally related to cholesterol and some steryl esters inhibited the activity of L. giganteum phospholipase A2 (PLA2), an enzyme that hydrolyzes fatty acids from the sn-2 position of glycerophospholipids. Sterols that induce reproduction inhibited L. giganteum PLA2 activity, while sterols and steroids that do not support sporulation had minimal effect. Most steryl esters had no effect on enzyme activity, but cholesteryl arachidonate (CA) was a potent inhibitor of parasite PLA2. Not all enzymes partly purified using a DEAE-Sephacel column were affected by these lipids, demonstrating selective inhibition of specific enzymes. Potency was enhanced by up to several orders of magnitude if epoxy fatty acids were esterified to the cholesterol nucleus. The steryl ester pool was dynamic during morphogenesis, and the fatty acid composition of the steryl esters did not mimic total cell or membrane (glycerophospholipid) fatty acid composition as L. giganteum proceeded through its growth cycle. Synthesis of CA and monoepoxy CA by the parasite was confirmed using electrospray mass spectrometry and collision-induced dissociation. Steryl derivatives selectively inhibited PLA2 enzymes from bovine pancreas, snake venom, and human cytoplasmic 85-kDa PLA2.