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Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery.This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers.Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers.Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

SummaryShort read sequencing of two field-grown plants of red raspberry cv Glen Dee identified sequences derived from Rubus yellow net virus (RYNV). Surprisingly, PCR primers designed to target these sequences amplified RYNV-specific DNA fragments from some high health nuclear stock raspberry plants, previously tested as free from RYNV infection. The complete sequence of a Scottish isolate of RYNV (RYNV LG) was determined and a panel of primers covering the entire virus genome was designed to demonstrate that nuclear stock plants of cultivars Glen Dee, Glen Ericht, Glen Fyne and Glen Moy all contain partial fragments of the RYNV genome as integrated elements but do not contain the entire RYNV genome.The complete, circular genome of RYNV could be detected in RYNV LG-infected plants by rolling circle amplification but was not detected in Glen Dee plants. Grafting experiments showed that infectious RYNV could be transferred from RYNV LG plants but not from Glen Dee or Glen Moy plants, further demonstrating that these cultivars contain only partial, integrated RYNV elements. Grafting of RYNV LG to the certification-approved indexing speciesR. occidentalis(black raspberry) andR. macraei, showed that RYNV LG induced recognisable symptoms in both plants but thatR. macraeiwas more susceptible and produced more recognizable symptoms. Effective testing for RYNV in high health certified raspberry plants can be achieved by combining graft testing with multifragment PCR.

This work was funded by the Scottish Government Rural and Environment Science and Analytical Services Strategic Research Programme 2022–2027 (Project Ref. JHI-B1-1). Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected. Publisher PDF

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi) (1, 2). In plants, a small group of stem cells harbored within the shoot apical meristem (SAM) generates all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood (3, 4). Here we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

Background: Poor asthma control, medication adherence and knowledge of asthma have been identified as unmet needs of secondary school children with asthma. Barriers to self-management include embarrassment, fear of bullying, and a lack of peer awareness. Aims: To assess the impact of an interactive theatre play on the attitudes and awareness of teenagers without asthma in London secondary schools. We hypothesised that a theatre play delivered in school would improve peer awareness and attitudes about asthma. Methods: A play, developed by the research team and Greenwich and Lewisham Young People’s Theatre, is performed to children in schools. At the end, the main character stays in role and discusses her attitude towards her asthma with the audience. Pre- and post- questionnaire data are summarised using median and standard deviation and compared using Wilcoxon signed-rank analyses. Results: The play was delivered to 466 children (312 females; 154 males) aged 11 to 13 (mean = 11.8 y). Significant changes were seen in knowledge at baseline and post-theatre (M = 5.5, SD 2.4 vs M = 7.6, SD 2.5; Z = -14.5, p Conclusions: Theatre is an innovative way of raising awareness of asthma in schools. From this, children with asthma should feel empowered to self-manage their condition. This will form part of a larger randomised controlled trial in London schools.

Berry fruit crops are prone to infection by a wide range of viruses, with the list expanding every year, primarily because of the expansion of the crops to new geographic regions. Although some methods allow for virus detection in a nonspecific manner, the advent of cheap and effective nucleic acid sequencing technologies has allowed for the development of species-specific tests. This chapter describes methods for extraction of nucleic acids for molecular testing from a range of different berry fruit crops and lists oligonucleotide primers that have been developed for amplification of a large number of berry fruit viruses.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within β1 without preventing the membrane association or movement function of P4.

Publisher Summary The survey methods described in this chapter are questionnaires, rating scales, and structured interviews. Surveys are an established instrument for data gathering, and they are reported as being used with children as early as the 1890s. However, research and knowledge about the effectiveness of different child survey methods are scarce. Surveys involve questions and answers, and they typically fall into one of two categories: questionnaires or interviews. Three people are involved in surveys: the designer, the administrator, and the respondent. This chapter considers the different purposes of surveys and introduces the reader to the styles of questionnaires that are specifically for children and the general pitfalls and difficulties associated with the use of survey methods with children.

This chapter illustrates how some of the techniques are recruited and combined throughout the design of an interactive game. The case study provided here also illustrates how working with a small number of children, who participate in all evaluations, can be an efficient and effective way of involving children, although, at the end, testing with a different group of children could have been beneficial. The chapter shows how evaluation with children can be embedded in the interaction design process. It describes a series of evaluations that informed the design of Camelot, a mobile outdoor game intended to encourage social interaction among children aged 7 to 10. A single game concept named Camelot was formed as a synthesis and adaptation of the earlier game concepts. Its aim is to construct a toy castle before the opposing team constructs theirs. To build a castle, teams need to collect virtual resources spread around a large playing area, which could be the size of a playground.

This chapter discusses the detailed design of an evaluation, introducing and explaining the need for pilot-testing, examining the design of tasks, and highlighting the need for organization of the evaluation in advance. The chapter assumes that the goals of the evaluation are already established, the style of the evaluation is already determined, and the location of, and possible participants in, the evaluation are already identified. The discussion begins with recruitment of participants and then considers the preparation of technology and evaluation instruments before considering order and activity planning and the communication of the plans to the people involved in the study. Preparing technology and evaluation instruments is discussed here, which explains that the initial evaluation plan will indicate what technology and what method of evaluation will be used. At the planning stage, the technology has to be checked and adapted to the location of the study, any work to be done by the children must be specified, and any evaluation instruments must be prepared.

This chapter describes the ethical issues that relate to evaluating products with children. The ethical principles for evaluation that are discussed in the chapter are respect for other people, beneficence and nonmaleficence, and justice. The chapter argues that ethical decisions are subjective, and even experienced evaluators are likely to disagree in some circumstances about whether a proposed action is ethical. One reason for some of the disagreement is that individuals will have different approaches to ethical issues involving children based on their own background, education, and views about children and childhood. The three different approaches are the utilitarian view, the virtue-based approach, and the rights-based approach. Most countries have laws that regulate some of the relevant decisions, such as health and safety, child protection, human rights, and data protection legislation.

Publisher Summary This chapter discusses some of the requirements for field evaluations and long-term evaluations, and it introduces the diary method and the parent evaluator method, which was developed as a way to evaluate home products for children who cannot yet read and write. The chapter outlines some of the key aspects of the diary method for evaluation. It describes how to design diaries, how to organize diary studies, and how to engage parents in the evaluation of interactive products. Diary methods appear to have considerable potential, especially when an evaluation needs to be carried out over an extended period of time, or when the evaluation is better carried out in a home situation. More research needs to be done to determine guidelines for the best design of diaries for various purposes. The parent evaluator method seems to be a good choice for evaluations with preschool children and may also be useful with babies and toddlers.

This chapter examines a range of technological solutions to the problem of recording interaction between children and products. Logging the activities of children is an attractive option for usability studies. These methods are unobtrusive and can often run alongside other evaluation activities. Some methods need special treatment when used with children but, in general, the use of these methods is relatively straightforward although care must be taken, especially when recording children, to ensure that the logging method does not interfere with the interaction of the child with the product. The technologies used for evaluation are normally used alongside or as part of the evaluation methods. The technologies covered include automated logging of interactions, video and audio recording, and gaze tracking. Automated logging involves recording the user's inputs to a system in a way that is not intrusive so that the user's actions can be examined or analyzed later. Video and audio technologies have obvious applications for recording users' reactions in observation studies, but they can also be used in interviews as an alternative to simultaneous note taking and in usability tests as a convenient method of recording task completion times. Gaze-tracking technologies can be used to record where users are looking as they perform tasks.

This chapter provides the preliminary steps for planning the evaluation study and the stage at which the study is crystallized and captured. It introduces terminology and concepts that will be useful when contemplating and discussing evaluation studies. It presents different approaches for, and purposes of, evaluations and outlines the different criteria that might be used to formulate evaluation goals. Further, it discusses how different approaches might be served by different evaluation methods and analysis procedures and demonstrates how any evaluation study represents a trade-off between control and the realism that a test setup can achieve. There are several purposes for the evaluation. The first step is deciding what the evaluation study is trying to achieve. Different purposes may require different approaches to evaluation, different data to be collected, and different analysis procedures. The evaluation purposes have several distinctions: diagnostic evaluations, formative and summative evaluations, and exploratory, measurement, and experimental studies.

This chapter is a practical guide to what should be done on the day of the evaluation. It discusses setting up the location, giving instructions, managing time, and dealing with difficulties. In an evaluation, much of the mood is set at the very beginning, when the children and the evaluators come together. What is said at this point by way of introductions and instructions can have a significant effect on the evaluation study as a whole. It is worth considering how the outsider feels at the start of an evaluation. The evaluation ends, and, depending on the sort of work that was done, there will be many outputs, probably including a great feeling of relief, a messy room, and a mound of paperwork or videotapes.

This chapter deals with a case study that describes how the Wizard of Oz (WOz) method was employed in the design of computer vision-based action game that are controlled with body movements. The game does not require keyboards or traditional game controllers, and it uses a web camera and a microphone to “see and hear” the player. The main challenge in the interaction design of perceptual action games is to ensure the usability and playability of the game controls. These qualities are, on the other hand, highly dependent on the intuitiveness and physical appropriateness of the movements used to control the game character. This case study also illustrates how the selection of the evaluation method is bound to the features of the interactive product, context of use, and target user groups.

Publisher Summary This chapter explores the inspection methods, where an expert or a team of experts analyzes the designed interaction with the goal of creating a list of design improvements. An expert in this context is someone who is trained in the use of the evaluation method. Inspection methods are quite well studied in the literature, where they are referred to by a variety of names, such as expert evaluations, reviews, and analytical evaluation methods. The term inspection implies that the evaluation is not empirical, so it does not require that children use a product or prototype, and the evaluation requires the judgment of an expert rather than calculating quality metrics or running simulations. Inspection methods are a good option when it is difficult to involve children in the evaluation process directly. The nature of inspection methods is such that some detailed decisions must be in place regarding the interaction design, and the evaluation requires some level of detail in the prototypes to be evaluated.

Publisher Summary This chapter covers a range of techniques that encourage, prompt, or require children to talk about the interaction with the product. The terms verbalization data, verbal protocol, and verbal data are used interchangeably to refer to a record of things. Verbalization techniques are a valuable complement to observation during testing. They help in understanding why children behave the way they do, whether or not the system is clear to them, and whether or not it is fun or boring. Verbalization can be obtained from a structured monologue, through a structural dialogue with the tester, and between children. The chapter also discusses constructive interaction, an established technique that is adapted for child testers, and peer tutoring, a technique developed especially for obtaining verbal data from children.

This chapter provides an overview of child development theories and highlights the diverse nature of children, their lack of homogeneity, and their widely differing talents and motivations. It provides a sense of children and childhood and also outlines some basic child psychology. As a population children change over time in similar ways: They grow bigger, get stronger, and learn more. However, across a similar population, individual children can also have many differences. Child development is the study of these differences and similarities. An understanding of child development is necessary for individuals who want to carry out evaluation studies with and for children. This understanding will help prevent errors in judgment, minimize poor design of evaluation studies, and provide more believable results. The chapter also discusses the stages of child development that include physical development, socioemotional development, and cognitive development. These developments are intended to explain the differences and similarities between children that change with age.

This chapter discusses several issues, including the selection of participants, the selection of metrics, and the difficulties in gathering opinions from children, especially where novel technology is involved. The study that is described is intended to determine the relative usefulness of four different input technologies for children. It compares four input methods—a mouse, a keyboard, speech recognition, and handwriting recognition—and includes children between ages 6 and 10. This case study's most important characteristics are the organization of the study and the method used to ensure that the findings had reasonable reliability. This case study demonstrates some key aspects of using small groups of children in observational and behavioral studies. The use of survey methods is explored in the study, and interesting aspects about children's views of input technologies are gathered.

There are some activities that are supposed to be completed after evaluation. This process involves some reflection and action and a more analytical mindset. This chapter examines the functions, beginning when the evaluation has concluded and ending when the findings are reported. The products of the evaluation, in their untreated form, constitute raw data. Raw data can exist in a variety of forms, including questionnaires, interview notes, observation notes, videotapes, or electronic logs. Raw data generally need some attention before they can be interpreted and used. The data extracted are analyzed and coded. Analysis is the process of taking data and discovering what story they tell. Generally, the process of coding and analyzing data is different for quantitative and qualitative data. It is important to safeguard the data gathered during an evaluation and the media on which they are stored.

Publisher Summary The term observation refers to an examination of a phenomenon that results in a description suitable for the purposes of a research or evaluation study. Observations can be direct or indirect. Direct observations rely on the senses of the observer, who might watch or listen to how children use the product evaluated. Indirect observations are those where records of use are created through technology, such as logs of user interactions, logs of eye gaze, and so forth. Observation is invaluable when it comes to evaluating products for children. Observing children who test a prototype or product can provide new insights to designers and can provide solid evidence to support design recommendations or other conclusions based on evaluation. This chapter discusses approaches to observation that are applicable for evaluating products with children.