Wheat stripe rust resistance locus YR63 is a hot spot for evolution of defence genes - a pangenome discovery.

BACKGROUND: Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a threat to global wheat production. Deployment of widely effective resistance genes underpins management of this ongoing threat. This study focused on the mapping of stripe rust resistance gene YR63 from a Portuguese hexaploid wheat landrace AUS27955 of the Watkins Collection. RESULTS: YR63 exhibits resistance to a broad spectrum of Pst races from Australia, Africa, Asia, Europe, Middle East and South America. It was mapped to the short arm of chromosome 7B, between two single nucleotide polymorphic (SNP) markers sunCS_YR63 and sunCS_67, positioned at 0.8 and 3.7 Mb, respectively, in the Chinese Spring genome assembly v2.1. We characterised YR63 locus using an integrated approach engaging targeted genotyping-by-sequencing (tGBS), mutagenesis, resistance gene enrichment and sequencing (MutRenSeq), RNA sequencing (RNASeq) and comparative genomic analysis with tetraploid (Zavitan and Svevo) and hexaploid (Chinese Spring) wheat genome references and 10+ hexaploid wheat genomes. YR63 is positioned at a hot spot enriched with multiple nucleotide-binding and leucine rich repeat (NLR) and kinase domain encoding genes, known widely for defence against pests and diseases in plants and animals. Detection of YR63 within these gene clusters is not possible through short-read sequencing due to high homology between members. However, using the sequence of a NLR member we were successful in detecting a closely linked SNP marker for YR63 and validated on a panel of Australian bread wheat, durum and triticale cultivars. CONCLUSIONS: This study highlights YR63 as a valuable source for resistance against Pst in Australia and elsewhere. The closely linked SNP marker will facilitate rapid introgression of YR63 into elite cultivars through marker-assisted selection. The bottleneck of this study reinforces the necessity for a long-read sequencing such as PacBio or Oxford Nanopore based techniques for accurate detection of the underlying resistance gene when it is part of a large gene cluster.

Sr65: a widely effective gene for stem rust resistance in wheat.

KEY MESSAGE: Sr65 in chromosome 1A of Indian wheat landrace Hango-2 is a potentially useful all-stage resistance gene that currently protects wheat from stem rust in Australia, India, Africa and Europe. Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), threatened global wheat production with the appearance of widely virulent races that included TTKSK and TTRTF. Indian landrace Hango-2 showed resistance to Pgt races in India and Australia. Screening of a Hango-2/Avocet 'S' (AvS) recombinant inbred line population identified two stem rust resistance genes, a novel gene (temporarily named as SrH2) from Hango-2 and Sr26 from AvS. A mapping population segregating for SrH2 alone was developed from two recombinant lines. SrH2 was mapped on the short arm of chromosome 1A, where it was flanked by KASP markers KASP_7944 (proximal) and KASP_12147 (distal). SrH2 was delimited to an interval of 1.8-2.3 Mb on chromosome arm 1AS. The failure to detect candidate genes through MutRenSeq and comparative genomic analysis with the pan-genome dataset indicated the necessity to generate a Hango-2 specific assembly for detecting the gene sequence linked with SrH2 resistance. MutRenSeq however enabled identification of SrH2-linked KASP marker sunCS_265. Markers KASP_12147 and sunCS_265 showed 92% and 85% polymorphism among an Australian cereal cultivar diversity panel and can be used for marker-assisted selection of SrH2 in breeding programs. The effectiveness of SrH2 against Pgt races from Europe, Africa, India, and Australia makes it a valuable resource for breeding stem rust-resistant wheat cultivars. Since no wheat-derived gene was previously located in chromosome arm 1AS, SrH2 represents a new locus and named as SR65.

User-operated audiometry - an evaluation of expert vs. non-expert headphone placement.

OBJECTIVE: User-operated audiometry faces multiple barriers. One of these is the concern of audiologists that patients (non-experts) placing headphones by themselves results in invalid hearing thresholds due to greater placement variability. DESIGN: Comparative study. Participants took the AMTAS pure-tone air-conduction audiometry under two different conditions, expert and non-expert circumaural headphone placement for five frequencies within the range 250-8000 Hz. Questionnaires were also used to gain insight into the usability of the user-operated audiometry system - as well as the participants' perceived handling of the audiometry headphones. STUDY SAMPLE: Thirty participants (mean age 67.5 years). RESULTS: No statistically significant mean differences in hearing thresholds between the expert and non-expert conditions were found. The mean system usability scale score was 84.5. Handling the headphones was also rated as being easy (30%) or very easy (60%) by most non-experts. CONCLUSION: The conclusion of the study is that non-experts can be trusted to properly equip a pair of circumaural audiometry headphones for the correct conduction of pure-tone audiometry with only a few digital instructions.

Evaluation of Spray and Point Inoculation Methods for the Phenotyping of Puccinia striiformis on Wheat.

The fungus Puccinia striiformis causes yellow (stripe) rust on wheat worldwide. In the present article, new methods utilizing an engineered fluid (Novec 7100) as a carrier of urediniospores were compared with commonly used inoculation methods. In general, Novec 7100 facilitated a faster and more flexible application procedure for spray inoculation and it gave highly reproducible results for virulence phenotyping. Six point inoculation methods were compared to find the most suitable for assessment of pathogen aggressiveness. The use of Novec 7100 and dry dilution with Lycopodium spores gave an inoculation success rate of 100% in two independent trials, which was significantly higher and more consistent than for spore suspension in Soltrol 170, water, water + Tween 20, and Noble agar + Tween 20. Both Soltrol 170 and Novec 7100 allowed precise quantification of inoculum, which is important for the assessment of quantitative epidemiological parameters. New protocols for spray and point inoculation of P. striiformis on wheat are presented, along with the prospect for applying these in rust research and resistance breeding activities.

Sexual structures and recombination of the wheat rust fungus Puccinia striiformis on Berberis vulgaris.

An isolate of the basidiomycete Puccinia striiformis, which causes yellow (stripe) rust on wheat, was selfed on the newly discovered alternate host, Berberis vulgaris. This allowed a study of the segregation of molecular markers and virulence in the progeny isolates, and of the development of fungal sexual structures and spore forms. Pycnia and aecia were obtained after inoculation of B. vulgaris with basidiospores resulting from germinating teliospores from infected wheat leaves. Subsequent inoculation of wheat with aeciospores from bulked aecia resulted in 16 progeny isolates of the S1 generation. Genotyping with 42 simple sequence repeat (SSR) markers confirmed a parental origin of progeny isolates. Of the 42 analyzed loci, 15 were heterozygous in the parental isolate and 14 revealed segregation in the progenies. This resulted in 11 new multilocus genotypes (MLGs), which confirmed segregation following sexual reproduction. Additionally, parental and progeny isolates were phenotyped using a genetic stock of wheat genotypes representing 21 resistance genes. All S1 progeny isolates had virulence for 14 out of 15 loci where the parental isolate was virulent. This was consistent with the hypothesis that virulence in plant pathogens is often recessive to avirulence, i.e., only expressed in a homozygous state. Furthermore, no segregation was observed for five out of six loci, for which the parental isolate had an avirulent phenotype. The results for one of the two segregating virulence/avirulence loci suggested that the parental isolate was heterozygous with Avr alleles resulting in different but clearly avirulent phenotypes. The other locus indicated that additional genes modifying the phenotypic expression of avirulence were involved.

New Races of Puccinia striiformis Found in Europe Reveal Race Specificity of Long-Term Effective Adult Plant Resistance in Wheat.

Resistance to Puccinia striiformis was examined in nine wheat recombinant inbred lines (RILs) from a cross between 'Camp Rémy' (resistant parent) and 'Récital' (susceptible parent) using an isolate of a strain common to the northwestern European population before 2011 (old) and two additional isolates, one representing an aggressive and high-temperature-adapted strain (PstS2) and another representing a virulence phenotype new to Europe since 2011 (new). The RILs carried different combinations of quantitative trait loci (QTL) for resistance to P. striiformis. Under greenhouse conditions, the three isolates gave highly contrasting results for infection type, latent period, lesion length, and diseased leaf area. The PstS2 isolate revealed Yr genes and QTL which conferred complete resistance in adult plants. Six QTL had additive effects against the old isolate whereas the effects of these QTL were significantly lower for the new isolate. Furthermore, the new isolate revealed previously undetected resistance in the susceptible parent. Disease severity under field conditions agreed with greenhouse results, except for Camp Rémy being fully resistant to the new isolate and for two RILs being susceptible in the field. These results stress the need of maintaining high genetic diversity for disease resistance in wheat and of using pathogen isolates of diverse origin in studies of host resistance genetics.

Eye-Tracking on Touch Screen - Evaluating User Interaction.

This paper suggests a setup for using remote eye-tracking on a touchscreen tablet to evaluate user interaction for older adults interacting with a user-driven hearing test. By using video recordings to support the eye-tracking data, it was possible to evaluate quantitative usability metrics that could be compared to other research findings. The video recordings revealed useful information to distinguish between reasons for gaps in data and missing data and to inform future similar studies of human-computer interaction on a touch screen. Using only portable equipment allows researchers to move to the location of the user and investigate the user interaction of devices in real-world scenarios.

AMTASTM and user-operated smartphone research application audiometry-An evaluation study.

OBJECTIVE: To evaluate two user-operated audiometry methods, the AMTASTM PC-based audiometry and a low-cost smartphone audiometry research application (R-App). DESIGN: A repeated-measures within-subject study design was used to compare both user-operated methods to traditional manual audiometry and to evaluate test-retest reliability of each method. STUDY SAMPLE: 58 subjects were recruited in the study of which 83 ears had normal hearing thresholds and 33 ears had hearing loss (pure-tone average > 25 dB HL). Average age of participants was 44.8 years, with an age range of 11-85. RESULTS: Standard deviation of absolute differences ranged between 3.9-6.9 dB on AMTASTM and 4.5-6.8 dB on the R-App. The highest variability was found at the 8000 Hz frequency (R-App and AMTASTM test) and 3000 Hz frequency (AMTASTM retest). Evaluation of test-retest reliability of AMTASTM and R-App showed SD of absolute differences ranging between 3.5-5.8 dB and 3.1-5.0 dB, respectively. The mean threshold difference between test and retest was within ±1.5 dB on AMTASTM and ±1 dB on the R-App. CONCLUSION: Accuracy of AMTASTM and the R-App was within acceptable limits for audiometry and comparable to traditional manual audiometry on all tested frequencies (250-8000 Hz). Evaluation of test-retest reliability showed acceptable variation on both AMTASTM and R-App. Both user-operated methods could be reliably performed in a quiet non-soundproofed environment.

Ambient Noise in Candidate Rooms for User-Operated Audiometry.

Hearing loss is a widespread problem while treatment is not always accessible, mainly because of the limited availability of hearing care professionals and clinics. In this work, part of the User-Operated Audiometry project, we investigate the acoustic environment of inexpensive non-sound-treated rooms that could be used for unsupervised audiometric testing. Measurements of 10 min of ambient noise were taken from 20 non-sound-treated rooms in libraries and private and public clinics, nine of which were measured twice. Ambient noise was compared against two traditional audiometric sound-treated rooms and Maximum Permissible Ambient noise levels by ISO 8231-1, while factoring for the attenuation by the DD450 circumaural headphones provided. In most non-sound-treated rooms, MPAs were violated only by transient sounds, while the floor-noise level was below MPAs. Non-sound-treated rooms' ambient noise levels presented with much larger fluctuations compared to sound-treated rooms. Almost all violations occurred at low to mid-low frequencies. Our results suggest that large-scale implementation of user-operated audiometry outside traditional audiometric rooms is possible, at least under some realizable conditions. Circumaural headphones' attenuation is probably a necessary condition for all cases. Depending on the room, an online system making decisions based on ambient noise might also be included in combination with active attenuation.

Comparison of hearing aid fitting effectiveness with audiograms from either user-operated or traditional audiometry in a clinical setting: a study protocol for a blinded non-inferiority randomised controlled trial.

INTRODUCTION: There is a worldwide need to enhance the capacity of audiometry testing. The objective of this study is to compare the User-operated Audiometry (UAud) system with traditional audiometry in a clinical setting, by investigating if hearing aid effectiveness based on UAud is non-inferior to hearing aid effectiveness based on traditional audiometry, and whether thresholds obtained with the user-operated version of the Audible Contrast Threshold (ACT) test correlates to traditional measures of speech intelligibility. METHODS AND ANALYSIS: The design will be a blinded non-inferiority randomised controlled trial. 250 adults referred for hearing aid treatment will be enrolled in the study. Study participants will be tested using both traditional audiometry as well as the UAud system and they will answer the questionnaire Speech, Spatial and Qualities of Hearing Scale (SSQ12) at baseline. Participants will be randomly divided to receive hearing aids fitted based on either UAud or traditional audiometry. Three months after participants have started using their hearing aids, they will undergo a hearing in noise test with hearing aids to measure their speech-in-noise performance and answer the following questionnaires: SSQ12, the Abbreviated Profile of Hearing Aid Benefit and the International Outcome Inventory for Hearing Aids. The primary outcome is a comparison of the change in SSQ12 scores from baseline to follow-up between the two groups. Participants will undergo the user-operated ACT test of spectro-temporal modulation sensitivity as part of the UAud system. The ACT results will be compared with measures of speech intelligibility from the traditional audiometry session and follow-up measurements. ETHICS AND DISSEMINATION: The project was evaluated by the Research Ethics Committee of Southern Denmark and judged not to need approval. The findings will be submitted to an international peer-reviewed journal and presented at national and international conferences. TRIAL REGISTRATION NUMBER: NCT05043207.

3-D imaging of temporal and spatial development of Puccinia striiformis haustoria in wheat.

Differentiation of haustoria on primary infection hyphae of the fungal pathogen Puccinia striiformis was studied in wheat seedlings with two-photon microscopy in combination with a classical staining technique. Our results showed a significant increase in the average haustorium size 22, 44, 68, 92 and 116 h after inoculation (hai). After 116 hai no significant change was observed until 336 hai. Haustorium morphology also changed significantly during the time of infection. Initially small spherical haustoria were seen, but as they grew the haustoria gradually became apically branched. At 22 hai all observed haustoria were spherical, but at 44 hai most haustoria had an irregular structure, and at 92 hai all observed haustoria appeared branched. Along with the changes of the haustorial body the haustorial neck changed from narrow and slender to having an expanded appearance with a rough and invaginated structure. The structural changes were similar in two susceptible wheat varieties, 514W and Cartago, although the mean haustorium size was larger in 514W than in Cartago at all intervals.

User-Operated Audiometry Project (UAud) - Introducing an Automated User-Operated System for Audiometric Testing Into Everyday Clinic Practice.

Hearing loss is the third leading cause of years lived with disability. It is estimated that 430 million people worldwide are affected, and the number of cases is expected to increase in the future. There is therefore increased pressure on hearing health systems around the world to improve efficiency and reduce costs to ensure increased access to quality hearing health care. Here, we describe the User-Operated Audiometry project, the goal of which is to introduce an automated system for user-operated audiometric testing into everyday clinic practice as a means to relieve part of this pressure. The alternative to the existing referral route is presented in which examination is executed via the user-operated system. This route is conceptualized as an interaction between the patient, the system, and the hearing care professional (HCP). Technological requirements of the system and challenges that are related to the interaction between patients, the user-operated system, and the HCPs within the specific medical setting are discussed. Lastly, a strategy for the development and implementation of user-operated audiometry is presented, which includes initial investigations, a validation study, and implementation in a real-life clinical situation.

A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat.

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness.

Distinct Life Histories Impact Dikaryotic Genome Evolution in the Rust Fungus Puccinia striiformis Causing Stripe Rust in Wheat.

Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f.sp. tritici, is a major threat to wheat production worldwide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole-genome comparative genomics of two P. striiformis f.sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural, and allele levels. At the whole-genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates, whereas 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall, this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f.sp. tritici is the first step in understanding the evolution of dikaryotic rust fungi at a whole-genome level.

Plant nonhost resistance: paradigms and new environments.

Nonhost resistance (NHR) protects plants from a large and diverse array of potential phytopathogens. Each phytopathogen can parasitise some plant species, but most plant species are nonhosts that are innately immune due to a series of physical, chemical and inducible defenses these nonadapted pathogens cannot overcome. New evidence supports the NHR paradigm that posits the inability of potential pathogens to colonise nonhost plants is frequently due to molecular incompatibility between pathogen virulence factors and plant cellular targets. While NHR is durable, it is not insurmountable. Environmental changes can facilitate pathogen host jumps or alternatively result in new encounters between previously isolated plant species and pathogens. Climate change is predicted to substantially alter the current distribution of plants and their pathogens which could result in parasitism of new plant species.

Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family.

Yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease threatening much of global wheat production. Race-specific resistance (R)-genes are used to control rust diseases, but the rapid emergence of virulent Pst races has prompted the search for a more durable resistance. Here, we report the cloning of Yr15, a broad-spectrum R-gene derived from wild emmer wheat, which encodes a putative kinase-pseudokinase protein, designated as wheat tandem kinase 1, comprising a unique R-gene structure in wheat. The existence of a similar gene architecture in 92 putative proteins across the plant kingdom, including the barley RPG1 and a candidate for Ug8, suggests that they are members of a distinct family of plant proteins, termed here tandem kinase-pseudokinases (TKPs). The presence of kinase-pseudokinase structure in both plant TKPs and the animal Janus kinases sheds light on the molecular evolution of immune responses across these two kingdoms.

Assessment of Aggressiveness of Puccinia striiformis on Wheat.

A simple point-inoculation method using Novec™ 7100, a volatile engineered fluid, is presented for the assessment of aggressiveness of Puccinia striiformis isolates on seedlings of wheat. The method allows for quantification of the applied inoculum with a minimal risk of cross-contamination of rust from leaves grown side by side. The method is also applicable for the assessment of qualitative differences inferred by compatible and incompatible host-pathogen interactions, and it can be adjusted to other cereal rust and powdery mildew fungi on other host species, and other plant growth stages as appropriate.

Temporal and Spatial Variability of Fungal Structures and Host Responses in an Incompatible Rust-Wheat Interaction.

Information about temporal and spatial variability of fungal structures and host responses is scarce in comparison to the vast amount of genetic, biochemical, and physiological studies of host-pathogen interactions. In this study, we used avirulent wild type and virulent mutant isolates of Puccinia striiformis to characterize the interactions in wheat carrying yellow rust Yr2 resistance. Both conventional and advanced microscopic techniques were used for a detailed study of morphology and growth of fungal colonies and associated host cell responses. The growth of the wild type isolates was highly restricted due to hypersensitive response (HR, plant cell death) indicated by autofluorescence and change in the shape of the affected plant cells. The host response appeared post-haustorial, but large variation in the time and stage of arrest was observed for individual fungal colonies, probably due to a delay between detection and response. Some colonies were stopped right after the formation of the primary infection hyphae whereas others formed highly branched mycelia. HR was first observed in host cells in direct contact with fungal structures, after which the defense responses spread to adjacent host cells, and eventually led to encasement of the fungal colony. Several cells with HR contained haustoria, which were small and underdeveloped, but some cells contained normal sized haustoria without signs of hypersensitivity. The growth of the virulent mutants in the resistant plants was similar to the growth in plants without Yr2 resistance, which is a strong indication that the incompatible phenotype was associated with Yr2. The interaction between P. striiformis and wheat with Yr2 resistance was highly variable in time and space, which demonstrate that histological studies are important for a deeper understanding of host-pathogen interactions and plant defense mechanisms in general.

Race Typing of Puccinia striiformis on Wheat.

A procedure for virulence phenotyping of isolates of yellow (stripe) rust using spray inoculation of wheat seedlings by spores suspended in an engineered fluid, Novec™ 7100, is presented. Differential sets consisting of near-isogenic Avocet lines, selected lines from the "World" and "European" sets, and additional varieties showing race-specificity facilitate a robust assessment of race, irrespectively of geographical and evolutionary origin of isolates. A simple procedure for purification of samples consisting of multiple races is also presented.

Genetic diversity within and among aecia of the wheat rust fungus Puccinia striiformis on the alternate host Berberis vulgaris.

An isolate of the fungus Puccinia striiformis, causing yellow (stripe) rust on cereals and grasses, was selfed on the alternate (sexual) host, Berberis vulgaris. This enabled us to investigate genetic variability of progeny isolates within and among aecia. Nine aecial clusters each consisting of an aecium (single aecial cup) and nine clusters containing multiple aecial cups were selected from 18 B. vulgaris leaves. Aeciospores from each cluster were inoculated on susceptible wheat seedlings and 64 progeny isolates were recovered. Molecular genotyping using 37 simple sequence repeat markers confirmed the parental origin of all progeny isolates. Thirteen molecular markers, which were heterozygous in the parental isolate, were used to analyse genetic diversity within and among aecial cups. The 64 progeny isolates resulted in 22 unique recombinant multilocus genotypes and none of them were resampled in different aecial clusters. Isolates derived from a single cup were always of the same genotype whereas isolates originating from clusters containing up to nine aecial cups revealed one to three genotypes per cluster. These results implied that each aecium was the result of a successful fertilization in a corresponding pycnium and that an aecium consisted of genetically identical aeciospores probably multiplied via repetitive mitotic divisions. Furthermore, the results suggested that aecia within a cluster were the result of independent fertilization events often involving genetically different pycniospores. The application of molecular markers represented a major advance in comparison to previous studies depending on phenotypic responses on host plants. The study allowed significant conclusions about fundamental aspects of the biology and genetics of an important cereal rust fungus.