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Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.

Common bean (Phaseolus vulgaris L.) is the most important legume for human consumption worldwide and an important source of vegetable protein, minerals, antioxidants, and bioactive compounds. The N2-fixation capacity of this crop reduces its demand for synthetic N fertilizer application to increase yield and quality. Fertilization, yield, and quality of common bean may be optimised by several other agronomic practices such as irrigation, rhizobia application, sowing density, etc. Taking this into consideration, a systematic review integrated with a bibliometric analysis of several agronomic practices that increase common bean yield and quality was conducted, based on the literature published during 1971–2021. A total of 250 publications were found dealing with breeding (n = 61), sowing density and season (n = 14), irrigation (n = 36), fertilization (n = 27), intercropping (n = 12), soilless culture (n = 5), tillage (n = 7), rhizobia application (n = 36), biostimulant/biofertilizer application (n = 21), disease management (n = 15), pest management (n = 2) and weed management (n = 14). The leading research production sites were Asia and South America, whereas from the Australian continent, only four papers were identified as relevant. The keyword co-occurrence network analyses revealed that the main topics addressed in relation to common bean yield in the scientific literature related to that of “pod”, “grain”, “growth”, “cultivar” and “genotype”, followed by “soil”, “nitrogen”, “inoculation”, “rhizobia”, “environment”, and “irrigation”. Limited international collaboration among scientists was found, and most reported research was from Brazil. Moreover, there is a complete lack in interdisciplinary interactions. Breeding for increased yield and selection of genotypes adapted to semi-arid environmental conditions combined with the suitable sowing densities are important agronomic practices affecting productivity of common bean. Application of fertilizers and irrigation practices adjusted to the needs of the plants according to the developmental stage and selection of the appropriate tillage system are also of high importance to increase common bean yield and yield qualities. Reducing N-fertilization via improved N-fixation through rhizobia inoculation and/or biostimulants application appeared as a main consideration to optimise crop performance and sustainable management of this crop. Disease and weed management practices appear neglected areas of research attention, including integrated pest management.

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by \"click\" chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor.

Background Agriculture is facing an unprecedented challenge in having to reduce its environmental footprint whilst ensuring food security to an ever-growing global population. Towards this end, several strategies have been investigated and implemented to help maintain or improve crop yield under reduced water and/or nutrient provision for key commercial commodities such as tomatoes. Despite the high commercial, nutritional, and food-cultural value, there is no synthesis of evidence regarding yield maintenance of tomato (as a model crop) under resource-deficit. This systematic map therefore provides an overview of the evidence that exists on the effectiveness of techniques and management approaches aimed at improving the productivity of field-grown tomatoes under conditions of water-, nitrogen- (N) and/or phosphorus (P)-deficit. Methods Following the published map protocol, systematic searches of peer reviewed- and grey-literature were conducted using research publication databases, and specialist websites. A total of 14,377 unique articles were identified as potentially relevant to our research question, of which 927 were screened at the full-text level. Of that subset, 291 articles met all the pre-defined eligibility criteria. Basic information and meta-data on the interventions reported were recorded for these articles and a systematic map was compiled with the extracted data. Results The articles included in the systematic map database were used to identify several significant points including: (1) from the year 2000, the number of articles investigating strategies to improve field-grown tomato yield under conditions of water and/or nutrient deficit follows an upward trend; (2) large evidence bases (> 50%) originated from the United States, India, and Italy; (3) most studies addressed water alone as a resource (49%), with only 18% of studies focussing on N and 4% on P alone. Only 4% of records assessed all three resources simultaneously; (4) most evidence (77%) aims to improve resource use-efficiency via either irrigation, fertilisation, or crop and soil management strategies; and (5) different geographical regions appear to focus on different groups of interventions. Conclusions This systematic map identifies a range of interventions that have been successfully implemented in fields to improve the yield of commercial tomatoes under conditions of water, N and/or P deficit. However, only half of the relevant literature reported evidence on more than one intervention, which highlights the need for more integrated approaches to assess multiple interventions to adapt to deficits of key-resources simultaneously. In addition, the use of ‘techno-chemical’, ‘breeding and genetic’ and ‘computational’ interventions are only reported in a small number of records (< 8% of the gathered evidence). Hence, these interventions may also be considered as subjects to prioritise in future funding strategies.

The common bean (Phaseolous vulgaris L.) is a grain legume functionally characterized by its capacity for symbiotic of biological nitrogen fixation. As such, it does not demand the application of synthetic nitrogenous fertilizer and can offer environmental benefits as part of holistic cropping systems. While common bean commodities are highly nutritious, commercial cultivation of this crop is declining in already-industrialized countries. However, recent interest of consumers towards diets that benefit environmental and personal health has rekindled commercial interest in legumes, including the common bean. The aim of this protocol is to identify agronomic practices that are capable of increasing the yield and quality of the common bean for use as food. To address this research question, published literature will be screened for inclusion on the basis of defined eligibility criteria to ensure data sources are selected in an objective and consistent manner. Consistency checks will be carried out for the title, abstract and full texts of the literature collated. The output is expected to be a summary of the knowledge available to maximize the productivity and quality of the common bean as food. This anticipated synthesis will be of utility for a wide range of value-chain stakeholders from farmers and consumers, to research scientists and policy makers.

Background There is an urgent need to ensure that food production is maintained in response to either a reduction in use or lack of availability of natural resources. To this end, several strategies have been investigated to determine which agronomic approaches may improve crop yields under conditions of reduced water and/or nutrients provision, with special attention upon nitrogen (N) and phosphorus (P). New technologies and practices have been developed for key commercial crops, such as tomatoes. However, few of these are widely adopted in the field and evidence of their value in this production setting is limited. Methods This protocol sets out a systematic map methodology that aims to provide a coherent synthesis of the available evidence among the literature on the techniques and management approaches that may ensure the productivity of field-grown tomatoes under conditions of water-, N- and/or P-deficits, either as single or combined stresses. To conduct the literature search, a search strategy was produced to define the scope of the systematic map and to allow reproducibility of the approach. A list of published and unpublished sources of literature were selected and a preliminary trial identified best-fit-for-purpose search-terms and -strings. A literature screening process was set with consistency checks amongst reviewers at the title, abstract and full text screening stages. A series of eligibility criteria were defined to ensure objectivity and consistency in the selection of studies that are best suited to address the research question of the systematic map. In addition, a coding strategy was designed to set the means for meta-data extraction out from the literature for review. A drafted structured questionnaire will serve as the base for collating the meta-data to produce a database where variables will be queried for the evidence synthesis. This work is expected to inform stakeholders, researchers and policy makers regarding the extent and nature of the existing evidence base, and so serve as a basis by-which specific approaches may be highlighted as potential focal-areas in future.

The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as a protein target identification method, the Y3H technique has not yet been widely adopted, in part due to the challenges associated with the synthesis of the complex chemical inducers of dimerisation (CIDs). The development of a modular approach using potentially “off the shelf” synthetic components was achieved and allowed the synthesis of a family of four triazole-containing CIDs, MTX-Cmpd2.2-2.5. These CIDs were then compared using the Y3H approach with three of them giving a strong positive interaction with a known target of compound 2, TgCDPK1. These results showed that the modular nature of our synthetic strategy may help to overcome the challenges currently encountered with CID synthesis and should contribute to the Y3H approach reaching its full potential as an unbiased target identification strategy.

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by \"click\" chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor.