Identification and Characterization of the SnTox6-Snn6 Interaction in the Parastagonospora nodorum-Wheat Pathosystem.

Parastagonospora nodorum is a necrotrophic fungal pathogen that causes Septoria nodorum blotch (SNB) (formerly Stagonospora nodorum blotch) on wheat. P. nodorum produces necrotrophic effectors (NE) that are recognized by dominant host sensitivity gene products resulting in disease development. The NE-host interaction is critical to inducing NE-triggered susceptibility (NETS). To date, seven NE-host sensitivity gene interactions, following an inverse gene-for-gene model, have been identified in the P. nodorum-wheat pathosystem. Here, we used a wheat mapping population that segregated for sensitivity to two previously characterized interactions (SnTox1-Snn1 and SnTox3-Snn3-B1) to identify and characterize a new interaction involving the NE designated SnTox6 and the host sensitivity gene designated Snn6. SnTox6 is a small secreted protein that induces necrosis on wheat lines harboring Snn6. Sensitivity to SnTox6, conferred by Snn6, was light-dependent and was shown to underlie a major disease susceptibility quantitative trait locus (QTL). No other QTL were identified, even though the P. nodorum isolate used in this study harbored both the SnTox1 and SnTox3 genes. Reverse transcription-polymerase chain reaction showed that the expression of SnTox1 was not detectable, whereas SnTox3 was expressed and, yet, did not play a significant role in disease development. This work expands our knowledge of the wheat-P. nodorum interaction and further establishes this system as a model for necrotrophic specialist pathosystems.

The usefulness of fungicide mixtures and alternation for delaying the selection for resistance in populations of Mycosphaerella graminicola on winter wheat: a modeling analysis.

A fungicide resistance model (reported and tested previously) was amended to describe the development of resistance in Mycosphaerella graminicola populations in winter wheat (Triticum aestivum) crops in two sets of fields, connected by spore dispersal. The model was used to evaluate the usefulness of concurrent, alternating, or mixture use of two high-resistance-risk fungicides as resistance management strategies. We determined the effect on the usefulness of each strategy of (i) fitness costs of resistance, (ii) partial resistance to fungicides, (iii) differences in the dose-response curves and decay rates between fungicides, and (iv) different frequencies of the double-resistant strain at the start of a treatment strategy. Parameter values for the quinine outside inhibitor pyraclostrobin were used to represent two fungicides with differing modes of action. The effectiveness of each strategy was quantified as the maximum number of growing seasons that disease was effectively controlled in both sets of fields. For all scenarios, the maximum effective lives achieved by the use of the strategies were in the order mixtures ≥ alternation ≥ concurrent use. Mixtures were of particular benefit where the pathogen strain resistant to both modes of action incurred a fitness penalty or was present at a low initial frequency.

Emergence of tan spot disease caused by toxigenic Pyrenophora tritici-repentis in Australia is not associated with increased deployment of toxin-sensitive cultivars.

The wheat disease tan (or yellow leaf) spot, caused by Pyrenophora tritici-repentis, was first described in the period 1934 to 1941 in Canada, India, and the United States. It was first noted in Australia in 1953 and only became a serious disease in the 1970s. The emergence of this disease has recently been linked to the acquisition by P. tritici-repentis of the ToxA gene from the wheat leaf and glume blotch pathogen, Stagonospora nodorum. ToxA encodes a host-specific toxin that interacts with the product of the wheat gene Tsn1. Interaction of ToxA with the dominant allele of Tsn1 causes host necrosis. P. tritici-repentis races lacking ToxA give minor indistinct lesions on wheat lines, whereas wheat lines expressing the recessive tsn1 are significantly less susceptible to the disease. Although the emergence and spread of tan spot had been attributed to the adoption of minimum tillage practices, we wished to test the alternative idea that the planting of Tsn1 wheat lines may have contributed to the establishment of the pathogen in Australia. To do this, wheat cultivars released in Australia from 1911 to 1986 were tested for their sensitivity to ToxA. Prior to 1941, 16% of wheat cultivars were ToxA-insensitive and hence, all other factors being equal, would be more resistant to the disease. Surprisingly, only one of the cultivars released since 1940 was ToxA insensitive, and the area planted to ToxA-insensitive cultivars varied from 0 to a maximum of only 14% in New South Wales. Thus, the majority of the cultivars were ToxA-sensitive both before and during the period of emergence and spread of the disease. We therefore conclude that the spread of P. tritici-repentis in Australia cannot be causally linked to the deployment of ToxA-sensitive cultivars.

Targeting Fungicide Inputs According to Need.

Fungicides should be used to the extent required to minimize economic costs of disease in a given field in a given season. The maximum number of treatments and maximum dose per treatment are set by fungicide manufacturers and regulators at a level that provides effective control under high disease pressure. Lower doses are economically optimal under low or moderate disease pressure, or where other control measures such as resistant cultivars constrain epidemics. Farmers in many countries often apply reduced doses, although they may still apply higher doses than the optimum to insure against losses in high disease seasons. Evidence supports reducing the number of treatments and reducing the applied dose to slow the evolution of fungicide resistance. The continuing research challenge is to improve prediction of future disease damage and account for the combined effect of integrated control measures to estimate the optimum number of treatments and the optimum dose needed to minimize economic costs. The theory for optimizing dose is well developed but requires translation into decision tools because the current basis for farmers' dose decisions is unclear.

Action spectra for ultraviolet light-induced transformation of human cells to anchorage-independent growth.

We have determined action spectra for transformation of human embryonic skin and muscle fibroblasts to anchorage-independent growth. Tests under our experimental conditions indicate that reciprocity holds for photon rate and exposure time and that all the dose-effect curves in the wavelength range of 248 to 297 nm are smaller. These data can be used to construct action spectra with a maximum at about 265 nm, which do not implicate moieties other than nucleic acids as absorbers in the transformation process.

Ultraviolet light-induced transformation of human cells to anchorage-independent growth.

We have developed a system for ultraviolet light (UV) transformation of human embryonic cells to anchorage-independent growth. The procedure involves multiple UV irradiations, post irradiation growth, and plating in soft agar. Transformants are obtained at frequencies from 1 to 80 per 10(5) cells at UV exposures to 25 J/sq m. The resulting transformants can be subcultured on solid surfaces. The cells show crisscrossing and piling up; they reach 2- to 5-fold higher saturation densities than the parental cells. Some subcultures show increased plating efficiency in soft agar and increased life span. The susceptibility of the UV transformation process to apparent photoenzymatic reversal implies that purimidine dimers play a role in its induction.

The automatic implantable cardioverter-defibrillator in young patients.

An international survey identified 40 patients less than 20 years old who underwent surgical implantation of an automatic implantable cardioverter-defibrillator (AICD). There was a history of aborted sudden cardiac death or sustained ventricular tachycardia in 92.5% of these patients. Twenty-two patients (55%) had structural heart disease; dilated and hypertrophic cardiomyopathy were the most common diagnoses. Eighteen patients (45%) had primary electrical abnormalities including seven with the congenital long QT syndrome. There were no perioperative deaths associated with device implantation. Concomitant drug therapy was administered to 75% of the patients. Defibrillator discharge occurred in 70% of the patients, with 17 patients (42.5%) receiving at least one appropriate shock. There were two sudden and two nonsudden deaths at 28.2 months' median follow-up. Sudden death-free survival rates by life table analysis at 12 and 33 months were 0.94 and 0.88, respectively. Total survival rates at 12 and 33 months were 0.94 and 0.82, respectively. The AICD represents an effective treatment approach for young patients with life-threatening ventricular tachyarrhythmias.

Optimal electrode position for transvenous defibrillation: a prospective randomized study.

OBJECTIVES: This study was performed to determine the optimal position for the proximal electrode in a two-electrode transvenous defibrillation system. BACKGROUND: Minimizing the energy required to defibrillate the heart has several potential advantages. Despite the increased use of two-electrode transvenous defibrillation systems, the optimal position for the proximal electrode has not been systematically evaluated. METHODS: Defibrillation thresholds were determined twice in random sequence in 16 patients undergoing implantation of a two-lead transvenous defibrillation system; once with the proximal electrode at the right atrial-superior vena cava junction (superior vena cava position) and once with the proximal electrode in the left subclavian-innominate vein (innominate vein position). RESULTS: The mean (+/- SD) defibrillation threshold with the proximal electrode in the innominate vein position was significantly lower than with the electrode in the superior vena cava position (13.4 +/- 5.7 J vs. 16.3 +/- 6.6 J, p = 0.04). Defibrillation threshold with the proximal electrode in the innominate vein position was lower or equal to that achieved in the superior vena cava position in 75% of patients. In patients with normal heart size (cardiothoracic ratio < or = 0.55), the improvement in defibrillation threshold with the proximal electrode in the innominate vein position was more significant than in patients with an enlarged heart (innominate vein 13.0 +/- 6.5 J vs. superior vena cava 17.9 +/- 5.1 J, p < 0.01). In patients with an enlarged heart, no difference between the two sites was observed (innominate vein 13.9 +/- 4.5 J vs. superior vena cava 13.6 +/- 8.3 J, p = NS). CONCLUSIONS: During implantation of a two-lead transvenous defibrillation system, positioning the proximal defibrillation electrode in the subclavian-innominate vein will lower defibrillation energy requirements in the majority of patients.

Starvation-induced genes of the tomato pathogen Cladosporium fulvum are also induced during growth in planta.

The pathogenicity of fungal pathogens is presumably dependent on genes that are expressed during infection. In order to isolate such genes from the tomato pathogen Cladosporium fulvum, and to test the hypothesis that starvation-induced genes are also plant induced, a cDNA library was prepared from mycelia grown in a defined medium and then transferred to a starvation medium. The library was then screened with cDNA prepared from starved and replete fungal mycelium. Five unique, differentially expressed cDNAs were isolated from 1,000 clones screened. Northern (RNA) hybridization confirmed that all five were starvation induced. Interestingly, all five were also found to be plant induced. The identity of two of the clones was indicated by partial DNA sequencing as alcohol and aldehyde dehydrogenase. The observed correlation between starvation induction and plant induction in discussed.

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable.

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.

Complementation of the Magnaporthe grisea deltacpkA mutation by the Blumeria graminis PKA-c gene: functional genetic analysis of an obligate plant pathogen.

Obligate plant-pathogenic fungi have proved extremely difficult to characterize with molecular genetics because they cannot be cultured away from host plants and only can be manipulated experimentally in limited circumstances. Previously, in order to characterize signal transduction processes during infection-related development of the powdery mildew fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei, we described a gene similar to the catalytic subunit of cyclic AMP-dependent protein kinase A (here renamed Bka1). Functional characterization of this gene has been achieved by expression in a deltacpkA mutant of the nonobligate pathogen Magnaporthe grisea. This nonpathogenic M. grisea deltacpkA mutant displays delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal transduction processes that control the maturation of infection cells. Transformation of the deltacpkA mutant with the mildew Bka1 open reading frame, controlled by the M. grisea MPG1 promoter, restored pathogenicity and appressorium maturation kinetics. The results provide, to our knowledge, the first functional genetic analysis of pathogenicity in an obligate pathogen and highlight the remarkable conservation of signaling components regulating infection-related development in pathogenic fungi.

Expression of reverse transcriptase genes in Fulvia fulva.

Antibodies raised against intercellular fluid antigens isolated from diseased tomato leaves have revealed that the fungal pathogen Fulvia fulva expresses genes for a fungal reverse transcriptase (RNA-dependent DNA polymerase). This enzyme is required for the replication of retroviruses and retroviral-like transposable elements and could provide a mechanism for increasing the mutation rate of fungal pathogens, perhaps explaining their ability to evolve new races rapidly. We report here the DNA sequence of a 225-bp clone from a lambda gt11 genomic library of F. fulva. This clone, designated P5, exhibits a high degree of sequence homology with the reverse transcriptase (pol) gene of the Drosophila melanogaster copia-like retrotransposon 17.6. Southern blot analysis of genomic DNA of F. fulva showed that P5-related sequences are moderately reiterated with 30-100 copies, some of which exhibit restriction fragment length polymorphism in different races of the pathogen. Western blot analysis of extracts from F. fulva with antibodies raised to purified reverse transcriptase (from human immunodeficiency virus-1) revealed immunoreactive proteins. Reverse transcriptase previously has been detected in a variety of organisms including yeast, insects, protozoa, and mammals, but to our knowledge, this is the first report of its occurrence in filamentous fungi.

Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp. hordei.

Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew, is an obligate biotroph. On arrival on the host, a primary germ tube (PGT) emerges from the conidium. An appressorial germ tube (AGT) then appears, forms an appressorium, and effects host penetration. Such developmental precision may be due to multiple, plant-derived signals and to endogenous tactile and chemical signals. The transduction mechanism remains obscure. The isolation of an expressed sequence tag (EST) homologue of the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) enabled the corresponding gene to be characterized and the transcript to be identified in conidia and in PGT and AGT stage spores. cAMP-dependent PKA activity was detected in ungerminated conidia. These data suggest that PKA and cAMP are involved in conidial development. To substantiate this we exploited the responses of developing conidia to various surfaces, including exposure to the host leaf (fully inductive to AGT formation), cellulose membrane (semi-inductive), and glass (non-inductive). Assessment of fungal development, following application of exogenous cAMP or cAMP analogues, revealed that, at different concentrations and on different surfaces, cAMP either promoted or inhibited conidial differentiation. Various PKA inhibitors were tested for their effect on PKA activity and conidial development. A negative correlation was established between PKA inhibition in vitro and fungal development in vivo. Taken collectively, these data suggest that PKA and cAMP play a role in conidial differentiation in this obligate, plant-pathogenic fungus.

Heterologous expression of Septoria lycopersici tomatinase in Cladosporium fulvum: effects on compatible and incompatible interactions with tomato seedlings.

The anti-fungal, steroidal, glycoalkaloid saponin, alpha-tomatine, is present in uninfected tomato plants in substantial concentrations, and may contribute to the protection of tomato plants against attack by phytopathogenic fungi. In general, successful fungal pathogens of tomato are more resistant to alpha-tomatine in vitro than fungi that do not infect this plant. For a number of tomato pathogens, this resistance has been associated with the ability to detoxify alpha-tomatine through the action of enzymes known as tomatinases. In contrast, the biotrophic tomato pathogen Cladosporium fulvum is sensitive to alpha-tomatine and is unable to detoxify this saponin. This paper describes the effects of heterologous expression of the cDNA encoding tomatinase from the necrotroph Septoria lycopersici in two different physiological races of C. fulvum. Tomatinase-producing C. fulvum transformants showed increased sporulation on cotyledons of susceptible tomato lines. They also caused more extensive infection of seedlings of resistant tomato lines. Thus, alpha-tomatine may contribute to the ability of tomato to restrict the growth of C. fulvum in both compatible and incompatible interactions.

Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens.

In previous work, UEA1 and UCSC1, two geographically distinct, powdery mildew isolates, were recognized for their ability to infect Arabidopsis thaliana. We have clarified the identity of these isolates by determining their host ranges, reexamining their morphology, and comparing their DNA sequences for the 5.8S ribosomal RNA and two flanking internal transcribed spacer sequences. These experiments confirm that UEA1 is a member of Erysiphe cruciferarum and that UCSC1 belongs to E. cichoracearum. Interactions of the two Erysiphe isolates with 360 A. thaliana accessions were examined to provide a comprehensive profile of naturally occurring powdery mildew resistance in this weedy species. The majority of A. thaliana accessions (213) were susceptible to both isolates. Among the accessions exhibiting some degree of resistance, most (84) responded differentially to UEA1 and UCSC1 and the remainder were resistant to both isolates. Notably, resistance to UCSC1 cosegregated with RPW7, a locus previously demonstrated to confer resistance to UEA1 in Ms-0 x Landsberg (erecta) crosses. With this large collection of resistant accessions, questions about species specificity, genetic diversity and the evolution of resistance to powdery mildews can be addressed.

Cloning and characterisation of telomeric DNA from Cladosporium fulvum.

The molecular cloning of DNA from the telomere of one chromosome of Cladosporium fulvum, a fungal pathogen of tomato, is described. The telomeric DNA exhibits tandem repeats of the sequence TTAGGG running 5' to 3' toward the chromosome end. At least 16 tracts of TTAGGG repeats are present in the C. fulvum genome. All such tracts are telomeric, and all chromosome-sized DNAs separated by pulsed-field gel electrophoresis exhibit the repeats. It is probable that tracts of these repeats are present at all chromosome termini. The cloned telomeric DNA exhibits 19 copies of the TTAGGG hexanucleotide motif, and evidence is presented indicating that all tracts of TTAGGG repeats are quite short. Telomere-linked restriction-fragment length polymorphisms between races of C. fulvum have been detected, and groupings based on these polymorphisms are consistent with those determined previously. Sub-telomeric DNA, centromere proximal to the cloned telomeric DNA, contains sequences reiterated many times in the genome; some of these repeats are at non-terminal locations. Partial sequence analysis indicates an absence of homology with the sub-telomeric DNA of other organisms.

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli.

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.

Use of implantable cardioverter-defibrillators in the congenital long QT syndrome.

We surveyed the use of implantable cardioverter-defibrillators in patients with congenital long QT syndrome. The implantable cardioverter-defibrillator was used primarily in high-risk persons and appeared safe and effective over a mean 31-month follow-up.

Ribosomal DNA variation and population differentiation in Salicornia L.

Analysis of nuclear rDNA variation within and between populations of Salicornia from the salt marsh at Stiffkey, Norfolk has detected new patterns of genetic differentiation. Individual plants may have alternative 10.5 and 12 kb repeat units. A strong association between the frequency of particular genetic variants and the environmental gradient of tidal inundation was evident. Salicornia dolichostachya Moss from sandy flats on the lowest parts of the marsh, near to the seaward edge, and S. fragilis Ball & Tutin from deep creeks were both monomorphic for the 12 kb variant; in contrast, S. pusilla J. Woods, characteristic of the highest parts of the tidal range, was monomorphic for the 10-5 kb variant. The more phenotypically variable S. europaea L. and S. ramosissima J. Woods populations that are found in large areas of lower and upper marsh, respectively, were heterogeneous for rDNA variant type. Nevertheless, the frequency of the 12 kb variant was significantly higher in plants from the lower marsh than in those from upper marsh, and in plants from low-lying pans than in those from raised interfluves; the 10.5 kb variant had the converse distribution. Variation in rDNA was not obviously associated with variation in morphology, or with variations in isozyme frequency established previously. Comparison of these results with those for populations from an extensive study, ranging from Anglesey (N. Wales) to S.W. Spain and the Gulf coast of Saudi Arabia, suggested that the rDNA variation and its association with environmental variation are complex and site-dependent. Plants from Anglesey had 10.5 and 11.5 kb variants, whereas only a 10-75 kb variant was detected in material from Spain and Saudi Arabia. rDNA variants were used as genetic markers in order to test the hypothesis that Salicornia is predominantly an inbreeder; conventional breeding experiments have been hampered by its highly specialized morphology and this represents the first direct, sensitive test of an idea that has been suggested mainly on morphological and phenological grounds. Analysis of rDNA in 38 maternal plants from Stiffkey and 2112 of their progeny provided no evidence for out-crossing.

Transformation of the nematode-trapping fungus Arthrobotrys oligospora.

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.