Extracellular proteins of the tomato pathogen Cladosporium fulvum; role in pathogenicity and avirulence

The interaction between the biotrophic fungus Cladosporium fulvum (Cooke) and its only host, tomato ( Lycopersicon esculentum Miller) complies with the gene-for-gene model. Early genetic studies have shown that many different avirulence ( Avr ) and resistance ( R ) gene pairs exist between strains of the fungus and genotypes of the plant, respectively. Previous studies in our laboratory, as summarized in Chapter 1, led to the isolation and characterization of several proteins of fungal and plant origin that occur in the apoplast during infection. The search for proteinaceous fungal elicitors that could act as avirulence factors on resistant tomato genotypes, led to the isolation of two proteins, AVR4 and AVR9. The development of molecular tools to manipulate C. fulvum provided means to confirm that the two encoding genes, Avr 4 and Avr 9, are the avirulence genes that match the tomato resistance genes Cf-4 and Cf-9 , respectively. The genes encoding two additional fungal e xtra c ellular p roteins (ECPs), Ecp 1 and Ecp 2, have also been cloned. Based on their specific induction in planta , both genes were proposed to be putative pathogenicity genes.The object of the research presented in this thesis was to obtain a better understanding of the function of the ECPs in pathogenicity and virulence of C. fulvum on tomato and of their potential role as specific elicitors on particular tomato genotypes, which would possibly fit into additional Avr / R gene-for-gene pairs. Accordingly, most of the work presented in this thesis involves studies on the biological function of the ECP1 and ECP2 proteins and additionally characterized ECPs of C. fulvum .In Chapter 2 a review on our current knowledge on fungal avirulence genes is presented. Although only a few fungal Avr genes have been cloned so far, they encode very different proteins and are differently regulated. Their involvement in pathogenicity or virulence, the type of responses that their products trigger on plants carrying the matching resistance genes, and their potential application in molecular resistance breeding are discussed.Pathogenicity assays with transgenic strains of C. fulvum in which the Ecp 1 and/or Ecp 2 genes were deleted are presented in Chapter 3. Both genes are virulence factors, as their encoded products, ECP1 and ECP2, are required for full virulence. Nevertheless, when absent, the fungus is still able to cause disease on tomato. The studies also indicate that both genes might function as suppressors of plant defense responses. It is envisaged that pathogenicity of C. fulvum on tomato results from synergism between multiple virulence factors. That is, while each single virulence factor is partly involved in causing disease on tomato, the presence of a complete set of virulence factors ensures maximal parasitic ability.Both ECP1 and ECP2 proteins are secreted, have a low molecular weight and are cysteine-rich proteins. They are secreted early and abundantly in the intercellular space, in a similar manner as observed for the two race-specific elicitors, AVR4 and AVR9. In Chapter 4 a search for plant genotypes that could recognize these proteins as specific elicitors of HR is described. One genotype of tomato, which had been preselected for resistance to C. fulvum , showed specific HR upon exposure to ECP2. Significantly, resistance of this genotype was shown to rely solely on ECP2 recognition. The resistance character is conferred by a single dominant gene, designated Cf-ECP2 . Analogous to the Avr 4/ Cf-4 and Avr 9/ Cf-9 gene pairs, Ecp 2/ Cf-ECP2 represents a genuine gene-for-gene pair within the C. fulvum -tomato interaction.Since the Cf-ECP2 gene operates through recognition of a crucial virulence factor, it may, therefore, provide durable protection against C. fulvum in tomato crops. Thus, the same protein may have a role in virulence on all tomato plants and a role in avirulence on only particular tomato genotypes.In Chapter 5, we subsequently initiated an extensive screen for plants among breeding lines and wild accessions of Lycopersicon species that could respond specifically with HR to any of the five interaction-specific fungal ECPs that we had isolated. Indeed, for each of the five ECPs that were tested, we found at least one individual that responded with HR. This observation indicates that recognition specificities are scattered among populations of plants, suggesting that recognition of proteins of C. fulvum is a generic property of the Lycopersicon genus. To investigate whether this is also true for a non-host species of C. fulvum , wild accessions of Nicotiana spp. were screened for similar specific HR-associated recognition of proteins of C. fulvum . We identified a few N. paniculata plants that exhibited a specific HR towards ECP2. This suggests that generic recognition specificities of proteinaceous elicitors of a fungal pathogen exist in solanaceous plants outside its host range. Consequently, the plant system for recognition of foreign proteins seems to generate new specificities at random, rather than specificities against classes of proteins produced by the pathogens of a given host plant.Recently, the cloning of four tomato Cf genes, among which are the Cf-4 and the Cf-9 genes that match the avirulence genes Avr 4 and Avr 9 respectively, was reported. They all belong to the same gene family, which is predicted to encode membrane-anchored leucine-rich-repeat (LRR) proteins. As such, they might participate in the perception of the different fungal avirulence factors. The Cf genes are members of clusters containing several homologues.In Chapter 6 we tested whether some of the Cf-9 homologues, referred to as Hcr9-9 genes ( H omologue of C ladosporium fulvumR esistance gene Cf- 9 present at the Cf- 9 locus), could confer additional resistance against C. fulvum . Inoculation of fungal strains in which the Avr 9 gene was deleted onto Cf9 tomato genotypes, showed that additional partial resistance(s) are present on the L.pimpinellifolium genomic segment introgressed into L. esculentum . These additional resistance(s) are independent of the Avr 9/ Cf-9 gene pair and are most probably due to one or more of the Hcr9-9 genes. However, we could not identify any specific proteinaceous elicitor activity in apoplastic fluids of compatible C. fulvum /tomato interactions that could match the identified additional resistance(s). The suggestion that Hcr9-9 genes are responsible for the additional resistance(s) has been proven in an independent study in another research group. Therefore, the Lycopersicon genus is not only rich in recognition specificities for secreted C. fulvum proteins (AVRs and ECPs), also a single plant genotype appears to contain multiple recognition capacities towards a single fungal strain.The data obtained in this research are discussed in Chapter 7. They indicate that HR-associated recognition of extracellular, low-molecular weight, cysteine-rich proteins of C. fulvum , some of which are involved in pathogenicity, is a general phenomenon in the Lycopersicon genus. The finding that some of these proteins, that act as avirulence factors, are also important virulence factors of the fungus might help to develop a strategy for identification of putative durable resistance genes, as proposed for the Cf-ECP2 gene. The demonstration of partial resistance(s) conferred by the Cf-9 homologues, without detection of a HR-phenotype, could suggest that recognition capacities of these genes are not limited to proteins, or that gene-for-gene responses other than HR occur in the C. fulvum -tomato interaction. In addition, our data indicate that HR-recognition of C. fulvum proteins is not confined to its host genus, Lycopersicon , but also occurs in the non-host genus Nicotiana .In conclusion, it appears that the diversity of the HR-associated recognition of proteins by a variety of Cf genes in a population of tomato relatives, generates plants with a genuine surveillance system against invading pathogens. The importance of the recognized proteins in pathogenicity or virulence of the pathogen is likely to be positively correlated with the durability of the matching resistance gene. The wide occurrence of gene-for-gene systems in crop plants is most probably a result of human plant breeding activities, involving transfer and dissection of gene clusters that originate from wild species.</p

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Community-acquired pneumonia in children: cell-free plasma sequencing for diagnosis and management.

Community-acquired pneumonia (CAP) is a common cause of pediatric hospital admission. Empiric antibiotic therapy for hospitalized children with serious CAP now targets the most likely pathogen(s), including those that may demonstrate significant antibiotic resistance. Cell-free plasma next-generation sequencing (CFPNGS) was first made available for Pediatric Infectious Diseases physicians in June 1, 2017, to supplement standard-of-care diagnostic techniques. A retrospective chart review was performed for children hospitalized with CAP between June 1, 2017, and January 22, 2018, to evaluate the impact of CFPNGS. We identified 15 hospitalized children with CAP without other underlying medical conditions for whom CFPNGS was performed. CFPNGS identified a pathogen in 13 of 15 (86%) children compared with 47% for those using standard culture and PCR-based methods alone. Changes in antibiotic management were made in 7 of 15 (47%) of children as a result of CFPNGS

Bactericidal Kinetics of Marine-Derived Napyradiomycins against Contemporary Methicillin-Resistant Staphylococcus aureus

There is an urgent need for new antibiotics to treat hospital- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections. Previous work has indicated that both terrestrial and marine-derived members of the napyradiomycin class possess potential anti-staphylococcal activities. These compounds are unique meroterpenoids with unusual levels of halogenation. In this paper we report the evaluation of two previously described napyradiomycin derivatives, A80915A (1) and A80915B (2) produced by the marine-derived actinomycete, Streptomyces sp. strain CNQ-525, for their specific activities against contemporary and clinically relevant MRSA. Reported are studies of the in vitro kinetics of these chemical scaffolds in time-kill MRSA assays. Both napyradiomycin derivatives demonstrate potent and rapid bactericidal activity against contemporary MRSA strains. These data may help guide future development and design of analogs of the napyradiomycins that could potentially serve as useful anti-MRSA therapeutics

Characteristics of children with hip displacement in cerebral palsy

<p>Abstract</p> <p>Background</p> <p>Hip dislocation in children with cerebral palsy (CP) is a common and severe problem. The dislocation can be avoided, by screening and preventive treatment of children with hips at risk. The aim of this study was to analyse the characteristics of children with CP who develop hip displacement, in order to optimise a hip surveillance programme.</p> <p>Methods</p> <p>In a total population of children with CP a standardised clinical and radiological follow-up of the hips was carried out as a part of a hip prevention programme. The present study is based on 212 children followed until 9–16 years of age.</p> <p>Results</p> <p>Of the 212 children, 38 (18%) developed displacement with Migration Percentage (MP) >40% and further 19 (9%) MP between 33 and 39%. Mean age at first registration of hip displacement was 4 years, but some hips showed MP > 40% already at two years of age. The passive range of hip motion at the time of first registration of hip displacement did not differ significantly from the findings in hips without displacement.</p> <p>The risk of hip displacement varied according to CP-subtype, from 0% in children with pure ataxia to 79% in children with spastic tetraplegia. The risk of displacement (MP > 40%) was directly related to the level of gross motor function, classified according to the gross motor function classification system, GMFCS, from 0% in children in GMFCS level I to 64% in GMFCS level V.</p> <p>Conclusion</p> <p>Hip displacement in CP often occurs already at 2–3 years of age. Range of motion is a poor indicator of hips at risk. Thus early identification and early radiographic examination of children at risk is of great importance. The risk of hip displacement varies according to both CP-subtype and GMFCS. It is sometimes not possible to determine subtype before 4 years of age, and at present several definitions and classification systems are used. GMFCS is valid and reliable from 2 years of age, and it is internationally accepted.</p> <p>We recommend a hip surveillance programme for children with CP with radiographic examinations based on the child's age and GMFCS level.</p

Development of lower limb range of motion from early childhood to adolescence in cerebral palsy: a population-based study

<p>Abstract</p> <p>Background</p> <p>The decreasing range of joint motion caused by insufficient muscle length is a common problem in children with cerebral palsy (CP), often worsening with age. In 1994 a CP register and health care programme for children with CP was initiated in southern Sweden. The aim of this study was to analyse the development of the passive range of motion (ROM) in the lower limbs during all the growth periods in relation to gross motor function and CP subtype in the total population of children with CP.</p> <p>Methods</p> <p>In total, 359 children with CP born during 1990-1999, living in the southernmost part of Sweden in the year during which they reached their third birthday and still living in the area in the year of their seventh birthday were analysed. The programme includes a continuous standardized follow-up with goniometric measurements of ROM in the lower limbs. The assessments are made by each child's local physiotherapist twice a year until 6 years of age, then once a year. In total, 5075 assessments from the CPUP database from 1994 to 1 January 2007 were analysed.</p> <p>Results</p> <p>The study showed a decreasing mean range of motion over the period 2-14 years of age in all joints or muscles measured. The development of ROM varied according to GMFCS level and CP subtype.</p> <p>Conclusion</p> <p>We found a decreasing ROM in children with CP from 2-14 years of age. This information is important for both the treatment and follow-up planning of the individual child as well as for the planning of health care programmes for all children with CP.</p

Advantages of the Ilizarov external fixation in the management of intra-articular fractures of the distal tibia

<p>Abstract</p> <p>Background</p> <p>Treatment of distal tibial intra-articular fractures is challenging due to the difficulties in achieving anatomical reduction of the articular surface and the instability which may occur due to ligamentous and soft tissue injury. The purpose of this study is to present an algorithm in the application of external fixation in the management of intra-articular fractures of the distal tibia either from axial compression or from torsional forces.</p> <p>Materials and methods</p> <p>Thirty two patients with intra-articular fractures of the distal tibia have been studied. Based on the mechanism of injury they were divided into two groups. Group I includes 17 fractures due to axial compression and group II 15 fractures due to torsional force. An Ilizarov external fixation was used in 15 patients (11 of group I and 4 of group II). In 17 cases (6 of group I and 11 of group II) a unilateral hinged external fixator was used. In 7 out of 17 fractures of group I an additional fixation of the fibula was performed.</p> <p>Results</p> <p>All fractures were healed. The mean time of removal of the external fixator was 11 weeks for group I and 10 weeks for group II. In group I, 5 patients had radiological osteoarthritic lesions (grade III and IV) but only 2 were symptomatic. Delayed union occurred in 3 patients of group I with fixed fibula. Other complications included one patient of group II with subluxation of the ankle joint after removal of the hinged external fixator, in 2 patients reduction found to be insufficient during the postoperative follow up and were revised and 6 patients had a residual pain. The range of ankle joint motion was larger in group II.</p> <p>Conclusion</p> <p>Intra-articular fractures of the distal tibia due to axial compression are usually complicated with cartilaginous problems and are requiring anatomical reduction of the articular surface. Fractures due to torsional forces are complicated with ankle instability and reduction should be augmented with ligament repair, in order to restore normal movement of talus against the mortise. Both Ilizarov and hinged external fixators are unable to restore ligamentous stability. External fixation is recommended only for fractures of the ankle joint caused by axial compression because it is biomechanically superior and has a lower complication rate.</p

Alignment, orientation, and Coulomb explosion of difluoroiodobenzene studied with the pixel imaging mass spectrometry (PImMS) camera

Citation: Amini, K., Boll, R., Lauer, A., Burt, M., Lee, J. W. L., Christensen, L., . . . Rolles, D. (2017). Alignment, orientation, and Coulomb explosion of difluoroiodobenzene studied with the pixel imaging mass spectrometry (PImMS) camera. Journal of Chemical Physics, 147(1). doi:10.1063/1.4982220Laser-induced adiabatic alignment and mixed-field orientation of 2,6-difluoroiodobenzene (C6H3F2I) molecules are probed by Coulomb explosion imaging following either near-infrared strong-field ionization or extreme-ultraviolet multi-photon inner-shell ionization using free-electron laser pulses. The resulting photoelectrons and fragment ions are captured by a double-sided velocity map imaging spectrometer and projected onto two position-sensitive detectors. The ion side of the spectrometer is equipped with a pixel imaging mass spectrometry camera, a time-stamping pixelated detector that can record the hit positions and arrival times of up to four ions per pixel per acquisition cycle. Thus, the time-of-flight trace and ion momentum distributions for all fragments can be recorded simultaneously. We show that we can obtain a high degree of one-and three-dimensional alignment and mixed-field orientation and compare the Coulomb explosion process induced at both wavelengths. © 2017 Author(s)