Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.

Comparative mutant analyses reveal a novel mechanism of ARF regulation in land plants.

A major challenge in plant biology is to understand how the plant hormone auxin regulates diverse transcriptional responses throughout development, in different environments, and in different species. The answer may lie in the specific complement of auxin signaling components in each cell. The balance between activators (class-A AUXIN RESPONSE FACTORS) and repressors (class-B ARFs) is particularly important. It is unclear how this balance is achieved. Through comparative analysis of novel, dominant mutants in maize and the moss Physcomitrium patens , we have discovered a ∼500-million-year-old mechanism of class-B ARF protein level regulation, important in determining cell fate decisions across land plants. Thus, our results add a key piece to the puzzle of how auxin regulates plant development.

Boundary domain genes were recruited to suppress bract growth and promote branching in maize.

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

The power of classic maize mutants: Driving forward our fundamental understanding of plants.

Since Mendel, maize has been a powerhouse of fundamental genetics research. From testing the Mendelian laws of inheritance, to the first genetic and cytogenetic maps, to the use of whole-genome sequencing data for crop improvement, maize is at the forefront of genetics advances. Underpinning much of this revolutionary work are the classic morphological mutants; the "freaks" that stood out in the field to even the untrained eye. Here we review some of these classic developmental mutants and their importance in the history of genetics, as well as their key role in our fundamental understanding of plant development.

Plant Development: Coordinating across Space and Time.

Organ development requires coordination between gene expression patterns and cellular processes across developmental axes to generate consistent shapes. A new study shows that, in plants, this coordination may be in part mediated by precise spatial hormone synthesis, regulated by a conserved family of genes.

A heat-shock inducible system for flexible gene expression in cereals.

BACKGROUND: Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana, they have not yet been implemented in grasses. RESULTS: Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). CONCLUSIONS: This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

Drawing a Line: Grasses and Boundaries.

Delineation between distinct populations of cells is essential for organ development. Boundary formation is necessary for the maintenance of pluripotent meristematic cells in the shoot apical meristem (SAM) and differentiation of developing organs. Boundaries form between the meristem and organs, as well as between organs and within organs. Much of the research into the boundary gene regulatory network (GRN) has been carried out in the eudicot model Arabidopsis thaliana. This work has identified a dynamic network of hormone and gene interactions. Comparisons with other eudicot models, like tomato and pea, have shown key conserved nodes in the GRN and species-specific alterations, including the recruitment of the boundary GRN in leaf margin development. How boundaries are defined in monocots, and in particular the grass family which contains many of the world's staple food crops, is not clear. In this study, we review knowledge of the grass boundary GRN during vegetative development. We particularly focus on the development of a grass-specific within-organ boundary, the ligule, which directly impacts leaf architecture. We also consider how genome engineering and the use of natural diversity could be leveraged to influence key agronomic traits relative to leaf and plant architecture in the future, which is guided by knowledge of boundary GRNs.

Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity.

Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth.

The Plant Membrane-Associated REMORIN1.3 Accumulates in Discrete Perihaustorial Domains and Enhances Susceptibility to Phytophthora infestans.

Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized host-derived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3 revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility to an oomycete pathogen.