Genetic architecture of Arabidopsis thaliana response to infection by Pseudomonas syringae.

Plant pathogens can severely reduce host yield and fitness. Thus, investigating the genetic basis of plant response to pathogens is important to further understand plant-pathogen coevolution and to improve crop production. The interaction between Arabidopsis thaliana and Pseudomonas syringae is an important model for studying the genetic basis of plant-pathogen interactions. Studies in this model have led to the discovery of many genes that differentiate a resistant from a susceptible plant. However, little is known about the genetic basis of quantitative variation in response to P. syringae. In this study, we investigate the genetic basis of three aspects of A. thaliana's response to P. syringae: symptom severity, bacterial population size and fruit production using a quantitative trait loci (QTL) analysis. We found two QTL for symptom severity and two for fruit production (possible candidate genes for observed QTL are discussed). We also found significant two-locus epistatic effect on symptom severity and fruit production. Although bacterial population size and symptom severity were strongly phenotypically correlated, we did not detect any QTL for bacterial population size. Despite the detected genetic variation observed for susceptibility, we found only a weak overall relationship between susceptibility traits and fitness, suggesting that these traits may not respond to selection.

Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms.

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.

Lethal and sublethal effects of bendiocarb, halofenozide, and imidacloprid on Harpalus pennsylvanicus (Coleoptera: Carabidae) following different modes of exposure in turfgrass.

Routes by which nontarget predatory insects can be exposed to turfgrass pesticides include topical, residual, and dietary exposure. We used each of these routes to evaluate potential lethal or sublethal effects of two novel turfgrass insecticides, imidacloprid and halofenozide, and a carbamate, bendiocarb, on survival, behavior, and fecundity of the ground beetle Harpalus pennsylvanicus DeGeer. Field-collected carabids were exposed to direct spray applications in turf plots, fed food contaminated by such applications, or exposed to irrigated or nonirrigated residues on turf cores. Halofenozide caused no apparent acute, adverse effects through topical, residual, or dietary exposure. Moreover, the viability of eggs laid by females fed halofenozide-treated food once, or continuously for 30 d, was not reduced. In contrast, topical or dietary exposure of carabids to bendiocarb inevitably was lethal. Exposure to imidacloprid by those routes caused high incidence of sublethal, neurotoxic effects including paralysis, impaired walking, and excessive grooming. Intoxicated beetles usually recovered within a few days in the laboratory, but in the field, they were shown to be highly vulnerable to predation by ants. One-time intoxication by imidacloprid did not reduce females' fecundity or viability of eggs. There was no apparent behavioral avoidance of insecticide residues, or of insecticide-treated food. Carabids exposed to dry residues on turfgrass cores suffered high mortality from bendiocarb, and some intoxication from imidacloprid, but these effects were greatly reduced by posttreatment irrigation. Implications for predicting hazards of insecticides to beneficial invertebrates in turfgrass are discussed.

The Pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells.

Several bacterial avr genes have been shown to contribute to virulence on susceptible plants lacking the corresponding resistance (R) gene. The mechanisms by which avr genes promote parasitism and disease, however, are not well understood. We investigated the role of the Pseudomonas syringae pv. tomato avrRpt2 gene in pathogenesis by studying the interaction of P. syringae pv. tomato strain PstDC3000 expressing avrRpt2 with several Arabidopsis thaliana lines lacking the corresponding R gene, RPS2. We found that PstDC3000 expressing avrRpt2 grew to significantly higher levels and often resulted in the formation of more severe disease symptoms in ecotype No-0 plants carrying a mutant RPS2 allele, as well as in two Col-0 mutant lines, cpr5 rps2 and coil rps2, that exhibit enhanced resistance. We also generated transgenic A. thaliana lines expressing avrRpt2 and demonstrated, by using several different assays, that expression of avrRpt2 within the plant also promotes virulence of PstDC3000. Thus, AvrRpt2 appears to promote pathogen virulence from within the plant cell.

A dsbA mutant of Pseudomonas syringae exhibits reduced virulence and partial impairment of type III secretion.

Abstract To identify virulence genes of P. syringae pv. tomato strain DC3000 we screened for mutants with reduced virulence on its plant hosts, Arabidopsis thaliana and tomato. We isolated a Tn5-insertion mutant that exhibited reduced virulence on both hosts. Further characterization showed that this mutant carried a single Tn5 insertion in the dsbA gene, which encodes a periplasmic disulphide bond-forming protein. In addition to reduced virulence, the dsbA mutant exhibits mucoid colony morphology, loss of fluorescence, decreased motility, and a reduced growth rate in culture. The dsbA mutant is able to multiply in A. thaliana and tomato plants, trigger the hypersensitive response on tobacco and elicit Pto-mediated resistance in tomato, indicating that type III secretion occurs in this background. However, type III secretion appears to function with reduced efficiency in the dsbA mutant, as type III-dependent secretion of HrpZ and AvrRpt2 is impaired. These findings indicate that while the dsbA gene is required for multiple cellular functions in P. syringae, type III secretion in P. syringae is only partially dependent on dsbA.

Identification, characterization and comparative analysis of a novel chorismate mutase gene in Arabidopsis thaliana.

Phenylalanine, tyrosine, and tryptophan have a dual biosynthetic role in plants; they are required for protein synthesis and are also precursors to a number of aromatic secondary metabolites critical to normal development and stress responses. Whereas much has been learned in recent years about the genetic control of tryptophan biosynthesis in Arabidopsis and other plants, relatively little is known about the genetic regulation of phenylalanine and tyrosine synthesis. We have isolated, characterized and determined the expression of Arabidopsis thaliana genes encoding chorismate mutase, the enzyme catalyzing the first committed step in phenylalanine and tyrosine synthesis. Three independent Arabidopsis chorismate mutase cDNAs were isolated by functional complementation of a Saccharomyces cerevisiae mutation. Two of these cDNAs have been reported independently (Eberhard et al., 1993. FEBS 334, 233-236; Eberhard et al., 1996. Plant J. 10, 815-821), but the third (designated CM-3) represents a novel gene. The different organ-specific expression patterns of these cDNAs, their regulation in response to pathogen infiltration, as well as the different enzymatic characteristics of the proteins they encode are also described. Together, these data suggest that each isoform may play a distinct physiological role in coordinating chorismate mutase activity with developmental and environmental signals.

Diversity and molecular evolution of the RPS2 resistance gene in Arabidopsis thaliana.

The RPS2 gene in Arabidopsis thaliana governs resistance to strains of the bacterial pathogen, Pseudomonas syringae pv. tomato, that express the avrRpt2 gene. The two loci are involved in a gene-for-gene interaction. Seventeen accessions of A. thaliana were sequenced to explore the diversity present in the coding region of the RPS2 locus. An unusually high level of nucleotide polymorphisms was found (1.26%), with nearly half of the observed polymorphisms resulting in amino acid changes in the RPS2 protein. Seven haplotypes (alleles) were identified and their evolutionary relationships deduced. Several of the alleles conferring resistance were found to be closely related, whereas susceptibility to disease was conferred by widely divergent alleles. The possibility of selection at the RPS2 locus is discussed.

Translation of the mRNA for the sporulation gene spoIIID of Bacillus subtilis is dependent upon translation of a small upstream open reading frame.

We report the existence of a small open reading frame (usd) that is located between the promoter and coding sequence for the sporulation gene spoIIID in Bacillus subtilis. The mRNA from the usd-spoIIID operon contains an inverted repeat sequence that is predicted to form a stem-loop structure that would sequester the ribosome binding site for spoIIID. A mutation eliminating the ribosome binding site for the upstream open reading frame caused an oligosporogenous phenotype and interfered with the translation, but not the transcription, of the downstream gene spoIIID. We propose that efficient synthesis of SpoIIID requires that the putative stem-loop structure be disrupted by translation through the upstream open reading frame.

Traumatology--the Achilles heel in the rehabilitation of cardiovascular outpatients?

In outpatient rehabilitation involving cardiac patients apprehension of the rare cardiovascular complications are crucial determinants of the sports activities whereas prophylactics of injuries is thought to be less important. Retrospectively, questionnaires to specify injuries were answered by 903 patients (61.7 +/- 8 years, 753 men, 150 women from 116 coronary care groups, approximately 270,000 patient exercise hours [PEH]). Independently of the patient's age and frequency of participation 123 traumas (1 per 2,200 PEH) occurred. Ball games accounted for the majority of the injuries (101/123 = 82.1%). Overstrains and distorsions were most frequent (53.7% approximately equal to 1 per 4,100 PEH) followed by bruises/ hematomas (15.4% approximately equal to 1 per 14,200 PEH), ruptured muscles, tendons and ligaments (12.2% approximately equal to 1 per 18,000 PEH), bone fractures (11.4% approximately 1 per 19,300 PEH), and abrasions or slashes (6.5% approximately 1 per 33,750 PEH). Two of the slashes and the loss of eyesight in one patient were caused by broken spectacles. Strains and distorsions were primarily located in the upper (25/27 = 43.8%) or lower limbs (22/57 = 38.6%). All ruptured muscles, tendons and ligaments were of the lower extremities. The 14 bone fractures were localized over the body. In sports with cardiac patients physicians must focus on the cardiovascular system and stress the prophylaxis of injuries by teaching motor and technical skills as well as tactics. Non-breakable spectacles are mandatory.

Preliminary results achieved by a computer-assisted system for controlled balloon dilatation of coronary and peripheral arteries.

In percutaneous balloon angioplasty the extent of trauma to the vessel as determined by slope of balloon inflation, peak pressure, and inflation time is crucial to the success of the intervention. These parameters are still not standardized and hence open to the operator. To elucidate this problem, a computer-assisted PTCA system (CAPS) was developed. CAPS is composed of a motor driven unit, a central processing and power unit, and a notebook. A syringe is clamped onto the motor unit and connected to a pressure gauge. CAPS may be linked to all types of balloon catheters. The notebook allows for preselection of peak pressure, slope of pressure increase, and inflation time. During balloon inflation, adjustments are made in a closed-loop system. On a screen, the inflation process is supervised in digital numbers and analogous curves. After the procedure, patient data and inflation curves may be recalled for analysis. In conclusion, CAPS by controlled inflation theoretically may reduce the mechanical trauma to the arteries. Further refinements should aim at gaining information on the lesions' characteristics and on the dilatation process itself.

Analysis of adhesion molecules in myocardial biopsies of cardiac allografts and coronary artery disease with CABG.

Interactions of leukocytes with vascular endothelium are important components of inflammation tissue reactions and have been implicated in cardiac transplant rejection and demonstrated to be mediated by cell adhesion molecules (CAM's). The expression of ICAM-1, VCAM-1 and E-selectin in human myocardium is variable and little is known about the expression of LFA-1 and Mac-1 during allograft rejection. This study investigated these CAM's in myocardial biopsies of transplanted hearts (HTX) and of coronary artery disease eligible for coronary artery bypass grafting (CABG) as non-inflammatory controls and explicitly examines vascular endothelium, cardiomyocytes and infiltrating cells. Immunohistochemistry was performed using the APAAP-method and directing specific mouse anti-human monoclonal antibodies against ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), alpha-LFA-1 (CD11a), alpha-Mac-1 (CD11b), alpha-p150/95 (CD11c) and the beta2-integrin chain (CD18). CD18, LFA-1 (CD11a), Mac-1 (CD11b) and p150/95 (CD11c) were markedly expressed on infiltrating immunocytes in HTX compared to CABG where no expression of beta2-integrins was observed. Cardiac allografts demonstrated a strong expression of ICAM-1 on vascular endothelium and on infiltrating cells. ICAM-1 was not detected on cardiomyocytes. In CABG a weak expression of ICAM-1 was observed on endothelial cells but not on myocytes. VCAM-1 was expressed on vascular endothelium and perivascular infiltrating cells in HTX but not in CABG. VCAM-1 was not found to be expressed on myocytes. There was no evidence for the presence of E-selectin in any of our biopsy specimens. Our study shows that the study of cell adhesion molecules adds to the pathophysiological understanding of inflammation after transplantation in cardiac disease. This offers a potential for the development of diagnostic tools and new therapeutic strategies.

[Traumatological risk in ambulatory cardiological rehabilitation]. / Traumatologische Risiken der ambulanten kardiologischen Rehabilitation.

BACKGROUND: In Israel in 1956 Gottheiner introduced outpatient rehabilitation programs in patients who had survived a myocardial infarction. In Germany one decade later these WHO phase III activities were established as well. At present any patient with cardiovascular disease is included unless suffering from acute illnesses or presenting with symptoms at rest. Gymnastics, stretching and the "historic" volleyball are completed by jogging, soccer, basketball and anaerobic exercise. Thus, a notable trauma rate would be expected especially in the elderly and those who are on anticoagulation. METHODS: A retrospective analysis evaluated questionnaires of 903 patients in 116 outpatient coronary care groups covering approximately equal to 270,000 patient exercise hours. RESULTS: 101 of 123 injuries (approximately equal to 1/2 200 patient exercise hours) occurred during games the majority of which having been distortions (53.7%), hematomas, or bruises (15.4%). Severe traumas included bone fractures (11.4%) and ruptured muscles, tendons, or ligaments (12.2%). One patient lost the sight of one eye due to a broken spectacle frame. Patients who had felt exercise as too exhausting (p < 0.001) or complained of cardiovascular symptoms (p < 0.01) showed a significantly elevated trauma rate. CONCLUSIONS: Thus, in outpatient cardiac rehabilitation the trauma rate is within the range of healthy sports men. To prevent injuries sports should be limited to the patient's level of activity. Improved skills in techniques and strategies as well as unbreakable glasses are recommended.

A useful weed put to work: genetic analysis of disease resistance in Arabidopsis thaliana.

Plant resistance to disease caused by phytopathogenic organisms is often triggered by the ability of the plant to specifically recognize the invading pathogen. One of the most fascinating areas in plant biology research focusses on understanding the mechanisms governing this process. Several recent breakthroughs in this area have come from the genetic analyses of disease resistance in Arabidopsis thaliana.

[The late cardiac sequelae after mantle-field irradiation. The results in Erlangen's patient caseload]. / Kardiale Spätfolgen nach Mantelfeldbestrahlung. Ergebnisse im Erlanger Patientenkollektiv.

PURPOSE: We have retrospectively evaluated the cardiac function in patients after mantle-irradiation for Hodgkin's disease. PATIENTS AND METHODS: Forty-three patients, mean age 39.8 +/- 13 years, who had been treated with mantle-irradiation from 1979 through 1984 at the University of Erlangen-Nürnberg, were examined. All of them were in first remission at 5 to 11 years (mean 8.1 years) after radiotherapy (n = 24) or combined modality treatment (n = 19). Mantle-irradiation had been administered through equally weighted anterioposterior-posterioanterior portals with 5 fractions of 2 Gy per week up to a total mediastinal dose of 41.8 +/- 7 Gy (including boost). The examination program included anamnesis, physical examination, Doppler echocardiography and ergometry. Hundred and twenty-two non-irradiated volunteers and cardiological patients were used as control group. RESULTS: None of the patients suffered from clinical symptoms. Pericardial thickening was present in 26%. The left ventricular end-systolic diameter (31.3 +/- 5.5 mm, normal value 26 to 42 mm) and the thickness of the left ventricular posterior wall (8.1 +/- 1.8 mm, normal value 6 to 11 mm) were within the normal range. The left ventricular ejection fraction was also normal (67.1 +/- 8% in patients versus 67.0 +/- 8% in control subjects). Evaluation of diastolic parameters, however, revealed significant changes. The isovolumic relaxation time was significantly decreased as compared to the control group (38.8 +/- 17 ms versus 50.8 +/- 21 ms, p < 0.05). The same we found for the shortening fraction (33.6 +/- 6% versus 38.1 +/- 6%, p < 0.05). The most significant changes were found after combined modality therapy, especially after radiotherapy and anthracycline-based chemotherapy. 30% of the patients were tachycardic (pulse > 100/min) at rest. At ergometry, the frequency of signs of ischemia (5%), conduction disturbances (2%), and rhythm disturbances (7%) was not elevated. CONCLUSIONS: In this retrospective investigation, patients after mantle irradiation with modern techniques showed only minimal cardiologic abnormalities within the first 10 years after treatment. The most sensitive parameters were the isovolumic relaxation times. The clinical relevance of such findings remains to be defined. Pathophysiologically, our findings (decreased relaxation time plus tachycardia) support the theory that myocardial damage after radiotherapy may result in subsequent increased beta-receptor density as proposed by Schultz-Hector et al. [21] on the basis of experimental data.

RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes.

Plant disease resistance genes function is highly specific pathogen recognition pathways. PRS2 is a resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.

[Extended reversible global left ventricular contraction dysfunction with symptomless coronary system as effect of cytostatic therapy with 5-fluorouracil]. / Ausgedehnte reversible globale linksventrikuläre Kontraktionsstörung als Folge einer zytostatischen Therapie mit 5-Fluorouracil bei unauffälligem Koronarsystem.

CASE REPORT: We report on a young female patient suffering from anal-cancer who received simultaneous radiochemotherapy and developed a "hibernating myocardium" during continuous infusion of 5-FU. CONCLUSION: In literature the incidence of cardiac complications caused by 5-FU-therapy is found to be between 1% and 10%. Patients with coronary heart disease have a four-fold higher risk. Possible pathogenic mechanisms are spasm of coronary artery, direct cardiotoxicity, immunological reactions and disturbance of the coagulation system.

[Angiography follow-up after transluminal angioplasty of coronary bypass grafts]. / Angiographische Verlaufskontrollen nach transluminaler Angioplastie von koronaren Bypass-Grafts.

Between January 1979 and October 1991, percutaneous transluminal angioplasty of stenosed or occluded coronary bypass grafts was attempted 180 times in 146 patients (180 lesions in 157 bypass grafts); 6/157 grafts were internal mammary grafts. The procedure was successful in 129/157 grafts (82%) and in 151/180 lesions (84%). Failures occurred almost exclusively in recanalization attempts. Cardiac complications occurred in 4/146 patients (2.7%). Three patients developed an acute myocardial infarction, another patient died after acute occlusion of a native vessel dilated during the same procedure. In successful attempts the severity of stenosis was reduced from 87 +/- 10% to 33 +/- 15%. 113/129 successfully dilated grafts had at least one (mean 2.7) control angiogram. 54/113 (48%) showed recurrence after a mean follow up of 6 months. An additional 15 grafts showed late restenosis in a second control angiogram (mean follow-up 23 months). The total restenosis rate was 61%. Restenoses were dilated again one to six times (mean 1.9) with comparable success and recurrence rate. Two patients died during the sixth angioplasty. Finally, 32/129 (25%) grafts were occluded or presumably occluded, and 97/129 (75%) were angiographically confirmed open without restenosis. Thus, angioplasty of bypass grafts is an alternative to a repeat revascularization surgery. The acute results are comparable to the results of angioplasty in native coronary arteries. The restenosis rate is high. One has to be aware of late restenosis. Restenosis can be dilated repeatedly with a comparable success rate and with no significant increase in restenosis rate.

Nested PCR for specific detection and rapid identification of human picornaviruses.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.

PTCA in elderly patients: acute results and long-term follow-up.

Acute results and long-term follow-up of percutaneous transluminal coronary angioplasty (PTCA) in 125 patients aged 75 to 90 years (79 men; 46 female; mean age 78 +/- 3 years), with mainly unstable angina (102 patients) are reported. Successful PTCA was achieved in 96 out of 108 patients (89%); occlusions could be reopened in eight out of 17 patients (47%). Thirty-eight procedures were multiple vessel or multiple lesion PTCA, so that the lesion-related success rate of PTCA was higher (91%). Major complications occurred in seven patients (5.6%) including one procedure-related death. In-hospital mortality was 3% and concerned exclusively patients with unstable angina and unsuccessful procedure. At a mean follow-up of 27 +/- 16 months (2 to 82 months) 13 additional patients had died: 10 from cardiovascular causes, three from cancer. In the meantime, three patients underwent non-fatal myocardial infarction and three elective bypass surgery; 26 patients had repeat PTCA. Eighty-seven of the 93 surviving patients with successful primary procedure and reintervention if necessary, were in anginal class I or II; only six were in class III. We conclude that the success rate of PTCA in elderly patients is comparable to that in younger patients, and that a successful procedure leads to continued relief of symptoms.

[Recurrent myxoma]. / Rezidivmyxom.

In a now 53-year-old woman, who suffered from recurrent syncope and dizziness, a biatrial myxoma originating from the interatrial septum was resected nearly 4 years ago, and the resulting septal defect was closed by a Dacron patch. In the symptomless patient the recurrent myxoma was revealed by echocardiography. The left atrial myxoma was surgically removed without repeat heart catheterization. This case underlines the importance of regular, echocardiographic follow-up examinations in patients in whom a myxoma has been excised. In case of suspicious findings, an additional transesophageal examination or nuclear magnetic resonance imaging should be performed.