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Background:. Few evidence-based suggestions are available to help applicants and mentors improve reapplication outcomes. We sought to provide program directors' (PDs) perspectives on actionable steps to improve reapplicants' chances for a match. Methods:. The PDs were asked to rank positions unmatched applicants can pursue, steps these applicants can take for the next application cycle, and reasons why reapplicants do not match. Results:. Responses from 66 of 123 PDs were received (53.6% response rate). Obtaining new recommendation letters and rotating with orthopaedics were the highest 20 ranked steps unmatched applicants can take. No curriculum vitae (CV) improvement, poor interview, and poor letters of recommendation were the most important reasons hindering applicants from matching when reapplying. Conclusions:. Steps reapplicants could prioritize include obtaining new recommendation letters, rotating in orthopaedics, and producing new research items. CV strengthening and improving interview skills address the 2 main reasons why unmatched applicants failed in subsequent attempts. Level of Evidence:. Level IV

Background:. Costs of healthcare in the US continue to rise at rates that are unsustainable. Prior studies, most of which come from non-surgical specialties, indicate that a variety of strategies to teach this material are utilized but without consensus on best practices. No studies exist regarding the teaching of cost-effective care in orthopaedic residency training programs. The goal of this study was to assess the landscape in this area from the perspective of program leadership. Methods:. A survey was developed that was sent to orthopaedic residency program leadership via email through their interaction with the COERG. Additional programs were included to enhance diversity of responding programs. The survey, based on those published from other areas of medicine, included questions about the experiences of the respondents in learning about cost-effective care, as well as how faculty and residents learned about this topic. Results:. Seventy one percent (30) of respondents noted that their faculty did not receive formal training in cost-effective care, and education in this area was likely to come from the department, especially review of practice data (12, 44%). Only 19% (8) of respondents agreed with the statement that “the majority of teaching faculty in our program consistently model cost-effective healthcare to residents”. Few of the programs (10, 24%) had formal curricula for residents regarding cost-effective care, and the primary mode of education in cost-effective care was through informal discussions with faculty (17, 43%). Few residents (3, 13%) were able to easily find the costs of tests or procedures. Discussion:. There is not consistent education in cost-effective care for orthopaedic surgery program leadership, faculty, or trainees. The results of this survey demonstrate a need for discussion of best practices, including increasing access to cost data at a local level, and engaging with the AOA, CORD, and the American Academy of Orthopaedic Surgeons more broadly in the development of standard education modules for faculty and residents, to improve the current and future delivery of cost-effective musculoskeletal care.

CONTEXT:. Near infrared spectroscopy (NIRS) is a noninvasive tool for assessing local oxygen balance. In circulatory shock, the microcirculatory environment as measured by NIRS during resuscitation may provide additional diagnostic tools of value to the critical care physician. HYPOTHESIS:. To assess whether a relative increase in peripheral NIRS was correlated with a clinically relevant increase in cardiac output following a fluid bolus in a swine model of shock. METHODS AND MODELS:. Nine healthy young adult swine with median weight 80 kg (interquartile range, 75–83 kg) were anesthetized and surgically instrumented. They underwent a controlled hemorrhage of 20% of their blood volume followed by partial or complete aortic occlusion to create a variable ischemia-reperfusion injury. Next, the animals underwent four 500-mL plasmalyte boluses over 9 minutes each followed by a 6-minute pause. The animal then underwent a 25% mixed auto/homologous blood transfusion followed by four more 500 mL plasmalyte boluses over 9 minutes. Finally, the animals underwent a 25% mixed auto/homologous blood transfusion followed by an additional four rounds of 500-mL plasmalyte boluses over 9 minutes. Left thoracic limb NIRS, descending thoracic aortic flow (dAF), arterial blood pressure (MAP), central venous pressure (CVP), and mixed central venous oxygen saturation (Svo2) were measured continuously for comparison. RESULTS:. The area under the receiver operating curve for an increase in dAF of 10% in response to a 500 mL bolus based on a percent increase in the proximal NIRS was 0.82 with 95% CI, 0.72–0.91; Svo2, 0.86 with 95% CI, 0.78–0.95; MAP, 0.75 with 95% CI, 0.65–0.85 and CVP, 0.64 with 95% CI, 0.53–0.76. INTERPRETATION AND CONCLUSIONS:. A dynamic relative increase in NIRS in response to a crystalloid challenge has moderate discriminatory power for cardiac output augmentation during shock in a swine model of ischemia-reperfusion injury. NIRS performed as well as invasive measurements (Svo2 and MAP) and better than CVP.

Background:. It has been postulated that the process of—and stresses associated with—medical training may cause a loss of empathy among trainees. Because empathy is considered an important value for clinicians and may even be associated with better patient outcomes, we assessed the empathy of orthopaedic surgery trainees and identified factors associated with empathy. Methods:. Between June and September 2020, an anonymous survey was distributed electronically to trainees in 23 Accreditation Council for Graduate Medical Education-accredited orthopaedic surgery residency programs via the Collaborative Orthopaedic Educational Research Group. The survey comprised the validated Short-Form 8-Item Empathy Quotient (EQ-8) questionnaire—scored on a scale of 0, least empathetic, to 16, most empathetic—and single-item measure of emotional exhaustion and depersonalization derived from the Maslach Burnout Index—scored using a frequency scale. In total, 438 of 605 (72%) trainees completed the survey. The scores were compared via one-way analysis of variance, with Bonferroni correction and Tukey post-hoc testing, α = 0.05. Results:. The mean (±SD) EQ-8 score among respondents was 11.3 ± 3.3. Women scored significantly higher (mean, 12.2 ± 2.8) than men (mean, 11.2 ± 3.3) (p = 0.02). Mean scores were significantly higher for trainees planning on a career in academic medicine (12.0 ± 2.9) than those intending to pursue private practice (10.9 ± 3.3) or those with a military commitment (10.4 ± 3.4) (p = 0.01). An inverse relationship was found between EQ-8 scores and single-item Maslach Burnout Index measures in depersonalization and emotional exhaustion (both, p < 0.01). No significant differences were found in EQ-8 scores across postgraduate year, program location, primary training setting, intended fellowship, relationship status, or whether they reported having children. Conclusions:. We found no association between postgraduate year and EQ-8 score. Women and those intending to pursue a career in academic medicine had significantly higher levels of empathy. A significant inverse relationship was found between burnout and empathy. Respondents with higher levels of emotional exhaustion and depersonalization had lower levels of empathy.

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has decimated kiwifruit orchards growing susceptible kiwifruit Actinidia chinensis varieties. Effector loss has occurred recently in Psa3 isolates from resistant kiwifruit germplasm, resulting in strains capable of partially overcoming resistance present in kiwiberry vines (Actinidia arguta, Actinidia polygama, and Actinidia melanandra). Diploid male A. melanandra recognises several effectors, sharing recognition of at least one avirulence effector (HopAW1a) with previously studied tetraploid kiwiberry vines. Sequencing and assembly of the A. melanandra genome enabled the characterisation of the transcriptomic response of this non-host to wild-type and genetic mutants of Psa3. A. melanandra appears to mount a classic effector-triggered immunity (ETI) response to wildtype Psa3 V-13, as expected. Surprisingly, the type III secretion (T3SS) system-lacking Psa3 V-13 ∆hrcC strain did not appear to trigger pattern-triggered immunity (PTI) despite lacking the ability to deliver immunity-suppressing effectors. Contrasting the A. melanandra responses to an effectorless Psa3 V-13 ∆33E strain and to Psa3 V-13 ∆hrcC suggested that PTI triggered by Psa3 V-13 was based on the recognition of the T3SS itself. The characterisation of both ETI and PTI branches of innate immunity responses within A. melanandra further enables breeding for durable resistance in future kiwifruit cultivars., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Testing effector knockout strains of the Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) for reduced in planta growth in their native kiwifruit host revealed a number of nonredundant effectors that contribute to Psa3 virulence. Conversely, complementation in the weak kiwifruit pathogen P. syringae pv. actinidifoliorum (Pfm) for increased growth identified redundant Psa3 effectors. Psa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus (ΔEEL; 10 effectors) were significant contributors to bacterial colonisation of the host and were additive in their effects on virulence. Four of the EEL effectors (HopD1a, AvrB2b, HopAW1a and HopD2a) redundantly contribute to virulence through suppression of pattern-triggered immunity (PTI). Important Psa3 effectors include several redundantly required effectors early in the infection process (HopZ5a, HopH1a, AvrPto1b, AvrRpm1a and HopF1e). These largely target the plant immunity hub, RIN4. This comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of ΦPsa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T = 9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage resistance is overcome by mutations in a tail fibre and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374-like phages that informs their use as antimicrobials against Psa., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Defence phytohormone pathways evolved to recognize and counter multiple stressors within the environment. Salicylic acid responsive pathways regulate the defence response to biotrophic pathogens whilst responses to necrotrophic pathogens, herbivory, and wounding are regulated via jasmonic acid pathways. Despite their contrasting roles in planta , the salicylic acid and jasmonic acid defence networks share a common architecture, progressing from stages of biosynthesis, to modification, regulation, and response. The unique structure, components, and regulation of each stage of the defence networks likely contributes, in part, to the speed, establishment, and longevity of the salicylic acid and jasmonic acid signaling pathways in response to hormone treatment and various biotic stressors. Recent advancements in the understanding of the Arabidopsis thaliana salicylic acid and jasmonic acid signaling pathways are reviewed here, with a focus on how the structure of the pathways may be influencing the temporal regulation of the defence responses, and how biotic stressors and the many roles of salicylic acid and jasmonic acid in planta may have shaped the evolution of the signaling networks., Competing Interests: JJ, MT, and ER are employed by The New Zealand Institute for Plant and Food Research Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stroud, Jayaraman, Templeton and Rikkerink.)

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors-AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a -suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars., Competing Interests: The authors have declared that no competing interests exist.

The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here, we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens , and a transient reporter eclipse assay for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the P. fluorescens and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l- and d-rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o-methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility., (© 2020 The Author. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization., Competing Interests: The authors have declared that no competing interests exist.

Key Message A resistant E. grandis genotype showed a constitutive overexpression of genes related to resistance to myrtle rust caused by A. psidii. Abstract Myrtle rust caused by Austropuccinia psidii is considered one of the most important fungal diseases affecting Eucalyptus spp. plantations in Brazil. Although the selection and planting of resistant eucalypt genotypes have been the major strategies to manage the disease in Brazil, the molecular mechanisms involved in resistance are still unclear. In this study, we evaluated the gene expression profile of two contrasting Eucalyptus grandis genotypes in resistance level to rust by RNA-Seq. The two genotypes showed a very different background gene expression level even without A. psidii infection. The resistant genotype had a constitutive overexpression of a large number of protein-coding genes compared to the susceptible genotype. These genes were mainly associated with signal transduction, photosynthesis, regulation and response to salicylic acid (SA), and protein kinase leucine-rich receptors (PK-LRR). PK-LRR and SA mediated disease resistance are well known to be effective against obligate biotroph pathogens, such as A. psidii. In addition, at 24 h after infection, the susceptible genotype was able to activate some response, however, several resistance-related proteins had their expression level reduced with A. psidii infection. Here, we present the first analysis of E. grandis genotypes transcriptomes infected by A. psidii and it reveals a constitutive overexpression of several resistance-related genes in the resistant genotype compared to the susceptible one. Our findings have the potential to be used as candidate molecular markers for resistance to myrtle rust.

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Ceratocystis fimbriata is an important plant pathogen known to cause Ceratocystis Wilt (CW), a prevalent fungal disease known to affect Eucalyptus spp. plantations in Brazil. To better understand the molecular mechanisms related to pathogenicity in eucalyptus, we generated a high-quality assembly and annotation of the Ce. fimbriata LPF1912 isolate (LPF1912) genome, as well as the first transcriptome of LPF1912 from 16 eucalyptus clones at three infection incubation periods (12, 18, and 24 h). The LPF1912 genome assembly contains 805 scaffolds, totaling 31.8 Mb, with 43% of the genome estimated to be coding sequence comprised of 7,390 protein-coding genes of which 626 (8.5%) were classified as secreted proteins, 120 ribosomal RNAs, and 532 transfer RNAs. Comparative genomic analysis among three eucalyptus fungal pathogens (Ce. fimbriata, Ce. eucalypticola, and Calonectria pseudoreteaudii), showed high similarity in the proteome (21.81%) and secretome (52.01%) of LPF1912 and Ce. eucalypticola. GO annotation of pathogenicity-related genes of LPF1912 and Ce. eucalypticola, revealed enrichment in cell wall degrading enzymes (CWDEs), and lipid/cutin metabolism for Ca. pseudoreteaudii. Additionally, a transcriptome analysis between resistant and susceptible eucalyptus clones to CW infection indicated that a majority (11) of LPF1912 differentially expressed genes had GO terms associated with enzymatic functions, such as the polygalacturonase gene family, confirming the crucial role of CWDEs for Ce. fimbriata pathogenicity. Finally, our genomic and transcriptomic analysis approach provides a better understanding of the mechanisms involved in Ce. fimbriata pathogenesis, as well as a framework for further studies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Bacterial canker is a destructive disease of kiwifruit caused by the Gram-negative bacterium Pseudomonas syringae pv. actinidiae (Psa). To understand the disease-causing mechanism of Psa, a kiwifruit yeast two-hybrid cDNA library was constructed to identify putative host targets of the Psa Type Three Secreted Effector AvrPto5., Results: In this study, we used the Mate & Plate™ yeast two-hybrid library method for constructing a kiwifruit cDNA library from messenger RNA of young leaves. The constructed library consisted of 2.15 × 10 6 independent clones with an average insert size of 1.52 kb. The screening of the kiwifruit yeast two-hybrid cDNA library with Psa AvrPto5 revealed the interaction of a V-type proton ATPase subunit-H, a proline rich-protein and heavy metal-associated isoprenylated plant protein 26. Among these, heavy metal-associated isoprenylated plant protein 26 showed a positive interaction with Psa AvrPto5 as both prey and bait.

Background: Pseudomonas syringae is a widespread bacterial species complex that includes a number of significant plant pathogens. Amongst these, P. syringae pv. actinidiae (Psa) initiated a worldwide pandemic in 2008 on cultivars of Actinidia chinensis var. chinensis. To gain information about the expression of genes involved in pathogenicity we have carried out transcriptome analysis of Psa during the early stages of kiwifruit infection., Results: Gene expression in Psa was investigated during the first five days after infection of kiwifruit plantlets, using RNA-seq. Principal component and heatmap analyses showed distinct phases of gene expression during the time course of infection. The first phase was an immediate transient peak of induction around three hours post inoculation (HPI) that included genes that code for a Type VI Secretion System and nutrient acquisition (particularly phosphate). This was followed by a significant commitment, between 3 and 24 HPI, to the induction of genes encoding the Type III Secretion System (T3SS) and Type III Secreted Effectors (T3SE). Expression of these genes collectively accounted for 6.3% of the bacterial transcriptome at this stage. There was considerable variation in the expression levels of individual T3SEs but all followed the same temporal expression pattern, with the exception of hopAS1, which peaked later in expression at 48 HPI. As infection progressed over the time course of five days, there was an increase in the expression of genes with roles in sugar, amino acid and sulfur transport and the production of alginate and colanic acid. These are both polymers that are major constituents of extracellular polysaccharide substances (EPS) and are involved in biofilm production. Reverse transcription-quantitative PCR (RT-qPCR) on an independent infection time course experiment showed that the expression profile of selected bacterial genes at each infection phase correlated well with the RNA-seq data., Conclusions: The results from this study indicate that there is a complex remodeling of the transcriptome during the early stages of infection, with at least three distinct phases of coordinated gene expression. These include genes induced during the immediate contact with the host, those involved in the initiation of infection, and finally those responsible for nutrient acquisition.

Interactions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurring Pseudomonas syringae isolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae (Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenic Psa and commensal P. syringae PG3a isolated from the same leaf was tested using reciprocal invasion from rare assays in vitro and in planta. P. syringae G33C (PG3a) inhibited Psa NZ54, while the presence of Psa NZ54 enhanced the growth of P. syringae G33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system of Psa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

Type-III secreted effectors (T3Es) play critical roles during bacterial pathogenesis in plants. Plant recognition of certain T3Es can trigger defence, often accompanied by macroscopic cell death, termed the hypersensitive response (HR). Economically important species of kiwifruit are susceptible to Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker. Although Psa is non-pathogenic in Arabidopsis thaliana, we observed that a T3E, HopZ5 that is unique to a global outbreak clade of Psa, triggers HR and defence in Arabidopsis accession Ct-1. Ws-2 and Col-0 accessions are unable to produce an HR in response to Pseudomonas-delivered HopZ5. While Ws-2 is susceptible to virulent bacterial strain Pseudomonas syringae pv. tomato DC3000 carrying HopZ5, Col-0 is resistant despite the lack of an HR. We show that HopZ5, like other members of the YopJ superfamily of acetyltransferases that it belongs to, autoacetylates lysine residues. Through comparisons to other family members, we identified an acetyltransferase catalytic activity and demonstrate its requirement for triggering defence in Arabidopsis and Nicotiana species. Collectively, data herein indicate that HopZ5 is a plasma membrane-localized acetyltransferase with autoacetylation activity required for avirulence.

Background: Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity., Results: Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes., Conclusions: Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

Horizontal gene transfer can precipitate rapid evolutionary change. In 2010 the global pandemic of kiwifruit canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) reached New Zealand. At the time of introduction, the single clone responsible for the outbreak was sensitive to copper, however, analysis of a sample of isolates taken in 2015 and 2016 showed that a quarter were copper resistant. Genome sequences of seven strains showed that copper resistance - comprising czc/cusABC and copABCD systems - along with resistance to arsenic and cadmium, was acquired via uptake of integrative conjugative elements (ICEs), but also plasmids. Comparative analysis showed ICEs to have a mosaic structure, with one being a tripartite arrangement of two different ICEs and a plasmid that were isolated in 1921 (USA), 1968 (NZ) and 1988 (Japan), from P. syringae pathogens of millet, wheat and kiwifruit respectively. Two of the Psa ICEs were nearly identical to two ICEs isolated from kiwifruit leaf colonists prior to the introduction of Psa into NZ. Additionally, we show ICE transfer in vitro and in planta, analyze fitness consequences of ICE carriage, capture the de novo formation of novel recombinant ICEs, and explore ICE host-range., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Pseudomonas syringae pv. actinidiae is the causal agent of bacterial canker of kiwifruit, a disease that has rapidly spread worldwide. We have fully sequenced and assembled the chromosomal and plasmid DNA from P. syringae pv. actinidiae ICMP 18884 using the PacBio RS II platform., (Copyright © 2015 Templeton et al.)

Arabidopsis thaliana (At) RPM1-interacting protein 4 (RIN4), targeted by many defence-suppressing bacterial type III effectors and monitored by several resistance proteins, regulates plant immune responses to pathogen-associated molecular patterns and type III effectors. Little is known about the overall protein structure of AtRIN4, especially in its unbound form, and the relevance of structure to its diverse biological functions. AtRIN4 contains two nitrate-induced (NOI) domains and is a member of the NOI family. Using experimental and bioinformatic approaches, we demonstrate that the unbound AtRIN4 is intrinsically disordered under physiological conditions. The intrinsically disordered polypeptide chain of AtRIN4 is interspersed with molecular recognition features (MoRFs) and anchor-identified long-binding regions, potentially allowing it to undergo disorder-to-order transitions upon binding to partner(s). A poly-l-proline II structure, often responsible for protein recognition, is also identified in AtRIN4. By performing bioinformatics analyses on RIN4 homologues from different plant species and the NOI proteins from Arabidopsis, we infer the conservation of intrinsic disorder, MoRFs and long-binding regions of AtRIN4 in other plant species and the NOI family. Intrinsic disorder and MoRFs could provide RIN4 proteins with the binding promiscuity and plasticity required to act as hubs in a pivotal position within plant defence signalling cascades., (© 2014 FEBS.)

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries--even millennia--ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 (cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix-loop-helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A (15)N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain-domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain-domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in α-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a "beads-on-a-string" organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Unlabelled: The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis., Taxonomy: Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes., (© 2010 The New Zealand Institute for Plant & Food Research Ltd. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd.)

The identification of clones expressing high levels of recombinant protein in Pichia pastoris is usually dependant upon SDS-PAGE, Western blotting, or bioactivity-based assays that are labour and time-consuming. We describe a rapid method that images green fluorescence protein (GFP) of individual P. pastoris clones transformed with vectors that express the proteins as GFP C- terminal fusion. In this report we have used the system to monitor expression of three proteins from Venturia inaequalis. Culture plates containing individual colonies were imaged on a Fuji LAS-3000 system and the intensity of fluorescence of GFP [Mean Gray Value (MGV)] of each colony recorded. Two common variables, the time course of expression and induction temperature were also optimised using this method. The results show that colonies with high levels of GFP fluorescence can be successfully used to identify, at an early stage, colonies expressing high levels of recombinant proteins. This correlation can be used to monitor the conditions for optimization of the expression and accumulation of extracellular recombinant protein in medium and to identify fractions containing GFP-tagged recombinant proteins during protein purification.

The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel V. inaequalis gene, cin1, is strongly up-regulated in the early stages of infection. This gene codes for a 523 residue secreted protein, containing eight imperfect repeats of approximately 60 amino acids. Cin1 was expressed in the methanolytic yeast Pichia pastoris using the pPICZ vector system. A protein of 57 kDa was secreted by these transformants and peptide fingerprinting indicated that it was the Cin1 protein product. Multiple angle laser light scattering confirmed the predicted mass of Cin1, showing it was not glycosylated by Pichia and was monomeric in solution. Through measurements of the hydrodynamic properties of Cin1, the experimental Stokes radius of Cin1 was calculated and corresponded to the theoretical value for a natively folded globular protein of size 57 kDa. The mobility of recombinant Cin1 on native PAGE was also consistent with that of a folded protein. To simplify future structural analyses, a two-domain truncated version, Cin1-2D, consisting of domains one and two, was also expressed using the same vector system. Both proteins were purified to homogeneity. Conditions for maximal (>98%) incorporation of 13C and 15N were determined. A mouse polyclonal antibody and three monoclonal antibodies (MAbs) were raised against the full-length version of Cin1. Analysis of the three MAbs using surface plasmon resonance indicated binding to distinct epitopes on the Cin1 protein. Western blots confirmed the different specificities of each MAb.

The hemi-biotrophic fungus Venturia inaequalis infects members of the Maloideae, causing the economically important apple disease, scab. The plant-pathogen interaction of Malus and V. inaequalis follows the gene-for-gene model. cDNA libraries were constructed, and bioinformatic analysis of the resulting expressed sequence tags (ESTs) was used to characterize potential effector genes. Effectors are small proteins, secreted in planta, that are assumed to facilitate infection. Therefore, a cDNA library was constructed from a compatible interaction. To distinguish pathogen from plant sequences, the library was probed with genomic DNA from V. inaequalis to enrich for pathogen genes, and cDNA libraries were constructed from in vitro-grown material. A suppression subtractive hybridization library enriched for cellophane-induced genes was included, as growth on cellophane may mimic that in planta, with the differentiation of structures resembling those formed during plant colonization. Clustering of ESTs from the in planta and in vitro libraries indicated a fungal origin of the resulting non-redundant sequence. A total of 937 ESTs was classified as putatively fungal, which could be assembled into 633 non-redundant sequences. Sixteen new candidate effector genes were identified from V. inaequalis based on features common to characterized effector genes from filamentous fungi, i.e. they encode a small, novel, cysteine-rich protein, with a putative signal peptide. Three of the 16 candidates, in particular, conformed to most of the protein structural characteristics expected of fungal effectors and showed significant levels of transcriptional up-regulation during in planta growth. In addition to candidate effector genes, this collection of ESTs represents a valuable genomic resource for V. inaequalis.

Class I hydrophobins are fungal proteins that self-assemble into robust amphipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the amphipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.

Hydrophobins are a remarkable class of small cysteine-rich proteins found exclusively in fungi. They self-assemble to form robust polymeric monolayers that are highly amphipathic and play numerous roles in fungal biology, such as in the formation and dispersal of aerial spores and in pathogenic and mutualistic interactions. The polymeric form can be reversibly disassembled and is able to reverse the wettability of a surface, leading to many proposals for nanotechnological applications over recent years. The surprising properties of hydrophobins and their potential for commercialization have led to substantial efforts to delineate their morphology and molecular structure. In this review, we summarize the progress that has been made using a variety of spectroscopic and microscopic approaches towards understanding the molecular mechanisms underlying hydrophobin structure.

Venturia inaequalis is a hemibiotrophic ascomycete that causes apple scab. Germ tubes, from conidia or ascospores, penetrate the leaf or fruit surface directly via appressoria-like swellings; subsequently the hyphae divide laterally to form a stroma between the cuticle and the outer wall of the epidermal cells. This morphological switch can be mimicked by growing the fungus in vitro on cellophane discs. The aim of this work was to identify genes upregulated in planta using growth on cellophane as a model. Four cDNA clones were found to be induced by growth on cellophane, and qRT-PCR showed two of these genes were up-regulated over a thousand fold in infected apple leaves compared to liquid culture. The predicted proteins for both genes possess putative signal peptides for secretion but have no similarity to sequences in publicly available databases. Both genes encode proteins with novel, imperfect repeat domain structures, the number of which vary in an isolate-specific fashion. Cin1 has seven or eight repeats of about 60 amino acids with four conserved cysteine residues per repeat, while Cin3 has four or five repeats of 32 amino acids with no cysteines. Both proteins appear to have evolved through internal duplication. Cin3, in particular, shows considerable between-strain variation in domain structure, indicating a high degree of recombination at this locus and revealing that the repeat structure has most likely arisen by unequal crossing-over. Results of this study support the hypothesis that cellophane-grown V. inaequalis mimics aspects of biotrophic infection and provide the first insights into novel fungal genes expressed during apple scab infection and their mechanisms of evolution.

Background: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs)., Results: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified., Conclusion: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Homologs of the gene encoding the hydrophobin EAS from Neurospora crassa have been identified both in the other conidial species of Neurospora (N. discreta, N. intermedia, N. sitophila, and N. tetrasperma) and selected aconidial species (N. africana, N. dodgei, N. lineolata, N. pannonica, and N. terricola). Southern blot analysis indicated the presence of a single gene in all species examined. EAS-like proteins were purified from the conidial species and each was shown to be the proteolytically processed gene-product of the corresponding eas homolog. While EAS-like proteins were not detected in the aconidial species, putative eas transcripts were detected in some isolates following RT-PCR and the aerial hyphae of these species were hydrophobic. DNA sequences of the coding region of the eas homologs were amplified by PCR and cloned and sequenced from all species except N. pannonica. Phylogenetic analysis of these sequences produced two clusters, the first comprising the conidiating species N. crassa, N. intermedia, N. sitophila, and N. tetrasperma forming a closely related group with N. discreta more distant, and the second comprising the aconidial species N. africana, N. dodgei, N. lineolata forming another closely related group with N. terricola more distant.

Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a beta-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar beta-structures that continue to be discovered in the protein world.

Background: We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes., Results: We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs., Conclusion: In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).

ABSTRACT DNA sequence polymorphisms in the putative two-component histidine protein kinase encoded by the Daf1 gene have been identified within a sample of 5 sensitive and 27 dicarboximide-resistant field strains of Botryotinia fuckeliana (anamorph Botrytis cinerea). The gene of 3948 bp is predicted to encode a 1315-amino acid protein comprising an N-terminal region, an amino acid repeat region, which has been hypothesized to be the binding site for dicarboximide fungicide, and a C-terminal region encompassing kinase and response regulator domains. Two amino acid variants were distinguished among the sensitive strains characterized by alanine (group 1), or threonine (group 2), at position 1259 in the C-terminal region. All resistant strains could be classified into either group 1 or group 2 but, in addition, all showed changes in the second amino acid repeat region. On the basis of the differences in this repeat region, four classes of resistant strains were recognized; class 1 characterized by an isoleucine to serine mutation, class 2 by an isoleucine to asparagine mutation, class 3 by an isoleucine to arginine mutation (all at position 365), and class 4 by an isoleucine to serine mutation (position 365) as well as a glutamine to proline mutation (position 369). All classes showed similar low levels of resistance to iprodione and to vinclozolin, except for class 3 and class 4 strains, which show low resistance to iprodione but moderate (class 3) or high (class 4) resistance to vinclozolin. The classes as a group did not differ from sensitive strains in osmotic sensitivity measured as mycelial growth response, but some class 1 strains showed an abnormal morphology on osmotically amended medium. The evolution of the amino acid differences is discussed in relation to field observations. It is proposed that class 1 and class 2 strains arose by single mutations within the sensitive population, whereas classes 3 and 4 arose by single mutations within a resistant population.

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.

The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.

Background: Fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. These monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. The molecular details underlying the formation of these monolayers, however, have been elusive. We have studied EAS, the hydrophobin from the ascomycete Neurospora crassa, in an effort to understand the structural aspects of hydrophobin polymerization., Results: We have purified both wild-type and uniformly 15N-labeled EAS from N. crassa conidia, and used a range of physical methods including multidimensional NMR spectroscopy to provide the first high resolution structural information on a member of the hydrophobin family. We have found that EAS is monomeric but mostly unstructured in solution, except for a small region of antiparallel beta sheet that is probably stabilized by four intramolecular disulfide bonds. Polymerised EAS appears to contain substantially higher amounts of beta sheet structure, and shares many properties with amyloid fibers, including a characteristic gold-green birefringence under polarized light in the presence of the dye Congo Red., Conclusions: EAS joins an increasing number of proteins that undergo a disorder-->order transition in carrying out their normal function. This report is one of the few examples where an amyloid-like state represents the wild-type functional form. Thus the mechanism of amyloid formation, now thought to be a general property of polypeptide chains, has actually been applied in nature to form these remarkable structures.