Genetic dissection of the tissue‐specific roles of type III effectors and phytotoxins in the pathogenicity of Pseudomonas syringae pv. syringae to cherry

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche‐specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644

Evolving strategies for enzyme engineering.

Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals. Introduction Enzymes are Nature's catalysts, tremendously accelerating the rates of a wide range of biochemical reactions, often with exquisite specificity. Harnessing enzymes for other purposes usually requires engineering them to improve their activity or stability. One approach to engineering enzymes is to make specific modifications, but this demands a detailed and frequently unattainable understanding of the relationship between sequence and function. Directed evolution bypasses this problem in much the same way as natural evolution, by combining mutation with selection or screening to identify improved variants. Because it is never possible to test more than an infinitesimal fraction of the vast number of possible protein sequences, it is essential to have a strategy for creating directed evolution sequence libraries that are rich in proteins with the desired enzymatic function. Such libraries can be designed by drawing on our knowledge of how proteins respond to mutation Directed evolution strategies Directed evolution works when the researcher can find at least one enzyme with improved properties in the sequence library. The most naïve strategy of creating a library of random protein sequences is not useful for most enzyme engineering goals. Although sequences with simple functions such as ATP binding Most directed evolution strategies involve making relatively small changes to existing enzymes. This takes advantage of the fact that enzymes often have a range of weak promiscuous activities that are quickly improved with just a few mutations Random mutagenesis The most straightforward strategy for library construction is to randomly mutate the full gene of an enzyme with a function close to the desired function. This approach requires no structural or mechanistic information, and can uncover unexpected beneficial mutations. Using sequential rounds of error-prone PCR to make an average of a few mutations per gene, followed by screening or selection for improved variants, is effective for a wide range of engineering goals. The creation of enantioselective catalysts from an enzyme whose structure is unknown is one such application. A single round of error-prone PCR produced several dozen cyclohexane monooxygenases with R or S selectivity Beneficial mutations found by random mutagenesis can be combined by DNA shuffling. A study with b-glucuronidase showed that beneficial mutations drive each other to extinction during recursive random mutagenesis, but that this problem can be eliminated by DNA shuffling Random mutagenesis can also uncover additional beneficial mutations in rationally designed enzymes. The Withers laboratory Targeted mutagenesis Some engineering goals, such as dramatically altering an enzyme's specificity or regioselectivity, may require mul- Random mutagenesis, targeted mutagenesis and recombination are three strategies for producing sequence libraries for directed evolution. (a) Random mutagenesis introduces amino acid substitutions throughout the protein and can uncover beneficial mutations distant from the active site. The red residues in the structure at top show four mutations uncovered by random mutagenesis that enhanced the activity of mammalian cytochrome P450 2B1 on several substrates Using a high-resolution crystal structure to target mutagenesis to three active site residues, Hill et al. [23] created a triple mutant of phosphotriesterase with a rate enhancement of three orders of magnitude for the degradation of organic triesters such as those used in chemical warfare agents. Crucially, two of the corresponding single mutants did not increase activity and so would not have been identified if they had been explored one at a time. The problem of inverting the enantioselectivity of a lipase offers an interesting comparison between full-gene random mutagenesis and targeted mutagenesis. Reetz and co-workers [24] used several rounds of full-gene random mutagenesis and DNA shuffling to invert the enantioselectivity of a lipase of unknown structure from S to R. Another lipase was engineered for the same goal by simultaneous mutation of four active site residues A variety of other enzymes have recently been engineered by targeted mutagenesis. Mutating three active site residues of penicillin acylase created six variants with improved activity, five of which were triple mutants [27]. Juillerat et al. [28] targeted four active site residues to engineer an O6-alkylguanine-DNA alkyltransferase for the efficient in vivo labeling of fusion proteins. They developed a selection system that allowed them to examine over 20,000 mutants and found that the best variants were triple mutants, suggesting the importance of simultaneously exploring multiple mutations. Novel DNA and RNA polymerases have also been engineered by targeted mutagenesis. Chelliserrykattil and Ellington [29] mutated four amino acids in RNA polymerase to engineer the enzyme to transcribe 2 0 -O-methyl RNA. Using a screen that selected variants that generated more RNA, they identified several mutants that incorporated nucleotides modified at the 2 0 position. Fa et al. [30] used targeted mutagenesis to engineer a DNA polymerase to specifically incorporate 2 0 -O-methyl ribonucleoside triphosphates by mutating six amino acids and selecting improved variants using phage display. Targeted mutagenesis of two active site residues was used to engineer a thioredoxin protein to replace the disulfide bond formation system in Escherichia coli Schultz and co-workers have created tRNA synthetases that charge orthogonal tRNAs with non-natural amino acids by targeting mutagenesis to five or six amino acids involved in substrate recognition. They then performed a positive selection for recognition of the non-natural amino acid and a negative selection against recognition of other amino acids The best mutants discovered by targeted mutagenesis almost always contain multiple mutations. These mutations are often beneficial as single mutants, but evidence is accumulating that at least some of them are beneficial only in combination Recombination Recombining structurally similar proteins can access larger degrees of sequence change than random mutagenesis The family shuffling protocol relies on regions of sequence identity to create crossovers that recombine the sequences of related proteins. This protocol is therefore limited to proteins with more than 70-75% identity, because libraries created from more diverged sequences tend to yield mostly parent sequences. A variety of methods have been developed to avoid this problem in the recombination of divergent sequences by using mismatched PCR primer pairs Although the studies described above demonstrate that recombining highly diverged but homologous sequences can produce libraries of diverse folded sequences, so far there has been little work to test whether it is also a useful method for discovering new functions. A tantalizing hint is that four out of fourteen chimeras of two cytochrome P450 proteins with 64% sequence identity show new product profiles Non-homologous recombination that combines fragments of unrelated proteins is another way to introduce large sequence changes. A new methodology was used to recombine the non-homologous chorismate mutase and fumarase proteins A striking application of non-homologous recombination is Ostermeier and co-workers' creation of a protein that combines the activity of a b-lactamase with the maltoseinduced conformational change of maltose-binding protein. In one experiment, they randomly inserted the lactamase sequence into the maltose-binding protein and screened for mutants with enhanced lactamase activity in the presence of maltose Conclusions Directed evolution is now an established method to engineer enzymes for a wide range of uses. Full-gene random mutagenesis continues to be a straightforward and powerful tool, and studies using this approach repeatedly illustrate that beneficial mutations can occur at unexpected sites. Targeted mutagenesis and recombination can extend directed evolution to the engineering of enzyme properties that require more than a few uncoupled changes in a protein's sequence (which are easily obtained by sequential rounds of random mutagenesis and screening). The increasing incorporation of structural and chemical knowledge will undoubtedly enhance the utility of these methods. The growing use of rational design in conjunction with directed evolution offers the exciting promise of generating libraries containing a high frequency of sequences with the desired functional properties. Update Recent work has emphasized the tendency of directed evolution to improve weak promiscuous functions by broadening specificity, as discussed i

The Human Disease Ontology 2022 update.

The Human Disease Ontology (DO) (www.disease-ontology.org) database, has significantly expanded the disease content and enhanced our userbase and website since the DO\u27s 2018 Nucleic Acids Research DATABASE issue paper. Conservatively, based on available resource statistics, terms from the DO have been annotated to over 1.5 million biomedical data elements and citations, a 10× increase in the past 5 years. The DO, funded as a NHGRI Genomic Resource, plays a key role in disease knowledge organization, representation, and standardization, serving as a reference framework for multiscale biomedical data integration and analysis across thousands of clinical, biomedical and computational research projects and genomic resources around the world. This update reports on the addition of 1,793 new disease terms, a 14% increase of textual definitions and the integration of 22 137 new SubClassOf axioms defining disease to disease connections representing the DO\u27s complex disease classification. The DO\u27s updated website provides multifaceted etiology searching, enhanced documentation and educational resources

Identifying resistance in wild and ornamental cherry towards bacterial canker caused by Pseudomonas syringae

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry ( ) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental species and sweet cherry × ornamental cherry hybrids with the canker pathogens, pvs , races 1 and 2, and . Several accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 . strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of . . Progeny resulting from a cross of a resistant ornamental species with susceptible sweet cherry ( . ) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm. [Abstract copyright: © 2021 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.

Solar Occultation Satellite Data and Derived Meteorological Products: Sampling Issues and Comparisons with Aura MLS

Derived Meteorological Products (DMPs, including potential temperature (theta), potential vorticity, equivalent latitude (EqL), horizontal winds and tropopause locations) have been produced for the locations and times of measurements by several solar occultation (SO) instruments and the Aura Microwave Limb Sounder (MLS). DMPs are calculated from several meteorological analyses for the Atmospheric Chemistry Experiment-Fourier Transform Spectrometer, Stratospheric Aerosol and Gas Experiment II and III, Halogen Occultation Experiment, and Polar Ozone and Aerosol Measurement II and III SO instruments and MLS. Time-series comparisons of MLS version 1.5 and SO data using DMPs show good qualitative agreement in time evolution of O3, N2O, H20, CO, HNO3, HCl and temperature; quantitative agreement is good in most cases. EqL-coordinate comparisons of MLS version 2.2 and SO data show good quantitative agreement throughout the stratosphere for most of these species, with significant biases for a few species in localized regions. Comparisons in EqL coordinates of MLS and SO data, and of SO data with geographically coincident MLS data provide insight into where and how sampling effects are important in interpretation of the sparse SO data, thus assisting in fully utilizing the SO data in scientific studies and comparisons with other sparse datasets. The DMPs are valuable for scientific studies and to facilitate validation of non-coincident measurements

Merging paleobiology with conservation biology to guide the future of terrestrial ecosystems

Conservation of species and ecosystems is increasingly difficult because anthropogenic impacts are pervasive and accelerating. Under this rapid global change, maximizing conservation success requires a paradigm shift from maintaining ecosystems in idealized past states toward facilitating their adaptive and functional capacities, even as species ebb and flow individually. Developing effective strategies under this new paradigm will require deeper understanding of the long-term dynamics that govern ecosystem persistence and reconciliation of conflicts among approaches to conserving historical versus novel ecosystems. Integrating emerging information from conservation biology, paleobiology, and the Earth sciences is an important step forward on the path to success. Maintaining nature in all its aspects will also entail immediately addressing the overarching threats of growing human population, overconsumption, pollution, and climate change.Peer reviewe

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Genome-wide meta-analysis of 158,000 individuals of European ancestry identifies three loci associated with chronic back pain

Back pain is the #1 cause of years lived with disability worldwide, yet surprisingly little is known regarding the biology underlying this symptom. We conducted a genome-wide association study (GWAS) meta-analysis of ch

Quantifying neutralising antibody responses against SARS-CoV-2 in dried blood spots (DBS) and paired sera

The ongoing SARS-CoV-2 pandemic was initially managed by non-pharmaceutical interventions such as diagnostic testing, isolation of positive cases, physical distancing and lockdowns. The advent of vaccines has provided crucial protection against SARS-CoV-2. Neutralising antibody (nAb) responses are a key correlate of protection, and therefore measuring nAb responses is essential for monitoring vaccine efficacy. Fingerstick dried blood spots (DBS) are ideal for use in large-scale sero-surveillance because they are inexpensive, offer the option of self-collection and can be transported and stored at ambient temperatures. Such advantages also make DBS appealing to use in resource-limited settings and in potential future pandemics. In this study, nAb responses in sera, venous blood and fingerstick blood stored on filter paper were measured. Samples were collected from SARS-CoV-2 acutely infected individuals, SARS-CoV-2 convalescent individuals and SARS-CoV-2 vaccinated individuals. Good agreement was observed between the nAb responses measured in eluted DBS and paired sera. Stability of nAb responses was also observed in sera stored on filter paper at room temperature for 28 days. Overall, this study provides support for the use of filter paper as a viable sample collection method to study nAb responses.</p