Boosting the full potential of PyMOL with structural biology plugins

Over the past few decades, the number of available structural bioinformatics pipelines, libraries, plugins, web resources and software has increased exponentially and become accessible to the broad realm of life scientists. This expansion has shaped the field as a tangled network of methods, algorithms and user interfaces. In recent years PyMOL, widely used software for biomolecules visualization and analysis, has started to play a key role in providing an open platform for the successful implementation of expert knowledge into an easy-to-use molecular graphics tool. This review outlines the plugins and features that make PyMOL an eligible environment for supporting structural bioinformatics analyses

Effect of zinc and protein content in different barley cultivars: use of controlled release matrices

Barley is one of the most consumed cereals, with many different cultivars available worldwide. Like other crops, its yield has been affected by climate change and soil degradation. This work proposes controlled-release protein-based matrices with incorporated zinc to improve barley seed germination and zinc content in the plant. Thus, the main objective of this study was to investigate the use of controlled-release protein-based matrices for massive crops, such as barley. Different barley cultivars of barley were studied: Barke, Golden Promise, Morex, WB-200, WB379, and WB-446. The seeds of each cultivar were also analyzed in order to explain the behavior of plants observed during the growth. To this end, the physico-chemical (FT-IR, Raman spectroscopy, and Zn concentration) and microstructural (SEM) properties of the different seeds were firstly evaluated to establish differences between the studied cultivars. In addition, the use of controlled-release soybean protein-based matrices without zinc (M) or with zinc incorporated (MZ) was evaluated as fertilizers in the different barley cultivars. In this sense, the use of these matrices as a zinc carrier improved seed germination and zinc content in the plants, indicating that the use of matrices improves the amount of zinc assimilated by the crops (up to 30 and 50% with M and MZ, respectively) and allows the proper root growth of all cultivars of barley. In conclusion, this article shows the potential of controlled-release protein-based matrices as substitutes for conventional fertilization

The archaeal elongation factor EF-2 induces the release of aIF6 from 50S ribosomal subunit

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release

Cloning the barley nec3 disease lesion mimic mutant using complementation by sequencing

Disease lesion mimic (DLM) or necrotic mutants display necrotic lesions in the absence of pathogen infections. They can show improved resistance to some pathogens and their molecular dissection can contribute to revealing components of plant defense pathways. Although forward-genetics strategies to find genes causal to mutant phenotypes are available in crops, these strategies require the production of experimental cross populations, mutagenesis, or gene editing and are time- and resource-consuming or may have to deal with regulated plant materials. In this study, we described a collection of 34 DLM mutants in barley (Hordeum vulgare L.) and applied a novel method called complementation by sequencing (CBS), which enables the identification of the gene responsible for a mutant phenotype given the availability of two or more chemically mutagenized individuals showing the same phenotype. Complementation by sequencing relies on the feasibility to obtain all induced mutations present in chemical mutants and on the low probability that different individuals share the same mutated genes. By CBS, we identified a cytochrome P450 CYP71P1 gene as responsible for orange blotch DLM mutants, including the historical barley nec3 locus. By comparative phylogenetic analysis we showed that CYP71P1 gene family emerged early in angiosperm evolution but has been recurrently lost in some lineages including Arabidopsis thaliana (L.) Heynh. Complementation by sequencing is a straightforward cost-effective approach to clone genes controlling phenotypes in a chemically mutagenized collection. The TILLMore (TM) collection will be instrumental for understanding the molecular basis of DLM phenotypes and to contribute knowledge about mechanisms of host-pathogen interaction

A lineage-specific Exo70 is required for receptor kinase–mediated immunity in barley

In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity but lack causal association. Here, we show that Rps8-mediated resistance in barley to the pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: Pur1 and Exo70FX12, which are together necessary and sufficient. Pur1 encodes a leucine-rich repeat receptor kinase and is the ortholog of rice Xa21, and Exo70FX12 belongs to the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Pur1-mediated immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling

Root angle is controlled by EGT1in cereal crops employing anantigravitropic mechanism

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus anti-gravitropic offset (AGO) mechanisms. Here we report a new root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss of function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes a F-box and Tubby domain containing protein which is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wildtype. Transcript profiling revealed Hvegt1 roots deregulate ROS homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shown that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic Force Microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wildtype. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery’s known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing a novel anti-gravitropic mechanism

Characterization and cloning of chemically-induced mutants in barley (Hordeum vulgare L.)

Induced mutagenesis has been exploited for crop improvement and for investigating gene function and regulation. To unravel molecular mechanisms of stress resilience, we applied state-of-the-art genomics-based gene cloning methods to barley mutant lines showing altered root and shoot architecture and disease lesion mimic phenotypes. With a novel method that we named complementation by sequencing, we cloned NEC3, the causal gene for an orange-spotted disease lesion mimic phenotype. NEC3 belongs to the CYP71P1 gene family and it is involved in serotonin biosynthesis. By comparative phylogenetic analysis we showed that CYP71P1 emerged early in angiosperm evolution but was lost in some lineages including Arabidopsis thaliana. By BSA-Seq, we cloned the gene whose mutation increased leaf width, and we showed that the gene corresponded to the previously cloned BROADLEAF1. By BSA coupled to WGS sequencing, we cloned EGT1 and EGT2, two genes that regulate root gravitropic set point angle. EGT1 encodes a Tubby-like F-box protein and EGT2 encodes a Sterile Alpha Motive protein; EGT2 is phylogenetically related to AtSAM5 in Arabidopsis and to WEEP in peach where it regulates branch angle. Both EGT1 and EGT2 are conserved in wheat. We hypothesized that both participate to an anti-gravitropic offset mechanism since their disruption causes mutant roots to grow along the gravity vector. By the MutMap+ method, we cloned the causal gene of a short and semi-rigid root mutant and found that it encodes for an endoglucanase and is the ortholog of OsGLU3 in rice whose mutant has the same phenotype, suggesting that the gene is conserved in barley and rice. The mutants and the corresponding genes which were cloned in this work are involved in the response to stress and can potentially contribute to crop adaptation

Advances in molecular breeding techniques for barley: targeted induced local lesions in genomes (TILLING)

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics method enabling the identification of individuals carrying chemically induced allelic variants at a gene of interest, within a mutagenized population. TILLING was first described in Arabidopsis (McCallum et al., 2000) and Drosophila (Bentley et al., 2000), and then extended to several plant species including maize (Till et al., 2004b), wheat (Slade et al., 2005), rice (Suzuki et al., 2008; Till et al., 2007), and others. It can be implemented in any species where a population of mutant individuals can be generated starting from a homogeneous line. Additionally, a version of TILLING has been described to help in discovery of polymorphisms in natural populations or in cultivars collections (EcoTILLING; Comai et al., 2004). Commonly, TILLING a gene implies three consecutive steps, namely (i) production or availability of a mutagenized population, (ii) PCR amplification of the target gene in a pool of individual genomic DNA samples, and (iii) detection of mutant alleles and individuals carrying them by means of a biochemical essay suitable for nucleotide polymorphism (e.g. SNP) identification. Most TILLING resources have been developed using chemical mutagenesis which mostly induces single-point mutations. This provides the investigator with a very informative range of allelic effects, from effective protein knockouts produced by stop-gain mutations to hypomorphic alleles caused by missense (i.e. amino acid substitution) mutations. Such different functional alleles are not usually available within populations generated with other types of mutational agents such as insertional mutagenesis (e.g. T-DNA, transposons) or irradiation (e.g. gamma-ray, fast-neutrons), all of which mostly generate knockouts. One additional positive aspect of TILLING is that it does not require any prior genomic knowledge (except for the target gene sequence, which must be known)

Design and development of a versatile graphical user interface for assisting consensus docking analyses on therapeutic targets for cancer treatment.

Molecular Docking, and its application in high-throughput virtual screening (HTVS), has arisen over the years as one of the most used tools in Drug Discovery, which is important to eliminate much of the effort and to decrease costs of experimental assays. A comprehensive understanding of the system that is subject to docking and of its underlying structural principles, may need a parallel usage of Molecular Graphics Visualization Software to visualize, at first bio-molecules, but also, interactions and search spaces. The steps that must be carried on to complete a docking process, from the preparation of input files to the analyses of the results, usually take advantage of more than one platform. Furthermore, although docking algorithms are usually implemented for a command-line usage only, just for some of them is currently available a graphical user interface (GUI) that may facilitate their usage. Nevertheless, the already developed GUIs are limited to the implementation of a single docking algorithm but, it is a general opinion, that in some cases it is not enough to rely on a single one to reach accurate results, making it necessary the usage of more docking algorithms in parallel. To overcome these drawbacks, I’ve developed a versatile, user-friendly and cross-platform GUI for assisting the process in all its steps, which works as a plugin of the molecular visualization system PyMOL. The plugin’s peculiar feature, which is not found in any other GUI currently developed, is to be suited for the implementation and usage of more docking algorithms in parallel. Therefore, in order to truly reflect its purposes, we have both tested and implemented a new scoring scheme, which is based own on this feature and which is termed as “Consensus Scoring”. This approach re-scores the results combining the output of different algorithms and it is primary useful to have a much more accurate ranking of the real bio-active molecules. Indeed it becomes necessary when dealing with Virtual Screening that, too often, lead to False Positives (FP) results. The biological interest was focused on Aurora-A protein, a Serine-Threonine Kinase involved in different Cell-Cycle related (CCR) processes, for which have been reported several mis-regulations that lead to the appearance of a neoplastic phenotype. By now, diverse types of compounds able to bind to the active site, impeding its catalytic activity, have been developed but, when talking about kinases, off-target events and cell toxicity may arise; meaning that it is very challenging the identification of selective and potent inhibitors. Therefore, it is needed the development of new methods to reach this aim. On these bases I’ve decided to evaluate the accuracy of “Consensus Scoring” method on Aurora-A, exploiting the information coming from crystallographic experiments about the real bio-active conformation adopted by different types of ligands in the interaction with this kinase. When applied to Aurora-A, this method revealed both an improvement of accuracy and some scoring issues in the docking algorithms used. Thus, concluding, we have identified the combination of algorithms and protocols to be used to reach considerable accurate predictions on Aurora-A system

QTL mapping identifies novel major loci for kernel row number-associated ear fasciation, ear prolificacy and tillering in maize (Zea mays L.)

Maize ear fasciation originates from excessive or abnormal proliferation of the ear meristem and usually manifests as flattened multiple-tipped ear and/or disordered kernel arrangement. Ear prolificacy expresses as multiple ears per plant or per node. Both ear fasciation and prolificacy can affect grain yield. The genetic control of the two traits was studied using two recombinant inbred line populations (B73 x Lo1016 and Lo964 x Lo1016) with Lo1016 and Lo964 as donors of ear fasciation and prolificacy, respectively. Ear fasciation-related traits, number of kernel rows (KRN), ear prolificacy and number of tillers were phenotyped in multi-year field experiments. Ear fasciation traits and KRN showed relatively high heritability (h(2) > 0.5) except ratio of ear diameters. For all ear fasciation-related traits, fasciation level positively correlated with KRN (0.30 <= r <= 0.68). Prolificacy and tillering were not correlated and their h(2) ranged from 0.41 to 0.78. QTL mapping identified four QTLs for ear fasciation, on chromosomes 1 (two QTLs), 5 and 7, the latter two overlapping with QTLs for number of kernel rows. Notably, at these QTLs, the Lo1016 alleles increased both ear fasciation and KRN across populations, thus showing potential breeding applicability. Four and five non-overlapping QTLs were mapped for ear prolificacy and tillering, respectively. Two ear fasciation QTLs, qFas1.2 and qFas7, overlapped with fasciation QTLs mapped in other studies and spanned compact plant2 and ramosa1 candidate genes. Our study identified novel ear fasciation loci and alleles positively affecting grain yield components, and ear prolificacy and tillering loci which are unexpectedly still segregating in elite maize materials, contributing useful information for genomics-assisted breeding programs