Characterisation of intracellular membrane structures derived from a massive expansion of ER membrane due to synthetic ER-membrane-resident polyproteins

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER-proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation organised smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterised and this hampers their potential development for plant synthetic biology. Here we characterise a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the final compartment morphology. We show that a cytosol-facing oligomerisation domain is an essential component for compartment formation. Using FRAP, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions

The β‐1,3‐glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium‐mediated plant infection

[EN] The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+, which carry a putative carbohydrate-binding module, and the GH72− Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.SIWe acknowledge BBSRC grant BB/J008923/1

Hormone-regulated expansins : expression, localization, and cell wall biomechanics in Arabidopsis root growth

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young’s modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN: XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fouriertransform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.peer-reviewe

Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway

GTPases of the Rab1 subclass are essential for membrane traffic between the endoplasmic reticulum (ER) and Golgi complex in animals, fungi and plants. Rab1-related proteins in higher plants are unusual because sequence comparisons divide them into two putative subclasses, Rab-D1 and Rab-D2, that are conserved in monocots and dicots. We tested the hypothesis that the Rab-D1 and Rab-D2 proteins of Arabidopsis represent functionally distinct groups. RAB-D1 and RAB-D2a each targeted fluorescent proteins to the same punctate structures associated with the Golgi stacks and trans-Golgi-network. Dominant-inhibitory N121I mutants of each protein inhibited traffic of diverse cargo proteins at the ER but they appeared to act via distinct biochemical pathways as biosynthetic traffic in cells expressing either of the N121I mutants could be restored by coexpressing the wild-type form of the same subclass but not the other subclass. The same interaction was observed in transgenic seedlings expressing RAB-D1 [N121I]. Insertional mutants confirmed that the three Arabidopsis Rab-D2 genes were extensively redundant and collectively performed an essential function that could not be provided by RAB-D1, which was non-essential. However, plants lacking RAB-D1, RAB-D2b and RAB-D2c were short and bushy with low fertility, indicating that the Rab-D1 and Rab-D2 subclasses have overlapping functions

The molecular core of transcriptome responses to abiotic stress in plants : a machine learning-driven meta-analysis

Understanding how plants adapt their physiology to overcome severe stress conditions is vital in light of the current climate crisis. This remains a challenge given the complex nature of the underlying molecular mechanisms. To provide a full picture of stress mitigation mechanisms, an exhaustive analysis of publicly available stress-related transcriptomic data was conducted. We combined a meta-analysis with an unsupervised machine learning algorithm to identify a core of stress-related genes. To ensure robustness and biological significance of the output, often lacking in meta-analyses, a three-layered biovalidation was incorporated. Our results present a ‘stress gene core’, a set of key genes involved in plant tolerance to a multitude of adverse environmental conditions rather than specific ones. In addition, we provide a biologically validated database to assist in design of multi-stress resilience. Taken together, our results pave the way towards future proof sustainable agriculture

Characterisation of intracellular membrane structures derived from a massive expansion of ER membrane due to synthetic ER membrane-resident polyproteins

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring.  Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER-proliferation: interaction of the proteins with each other alters the local  structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes  leading to the formation organised smooth endoplasmic reticulum (OSER). However, these membrane  structures formed by ER proliferation are poorly characterised and this hampers their potential development for plant synthetic biology. Here we characterise a range of ER-derived membranous  compartments in tobacco and show how the nature of the polyproteins introduced into the ER  membrane affect the final compartment morphology. We show that a cytosol-facing oligomerisation  domain is an essential component for compartment formation. Using FRAP, we demonstrate that  although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER  and the cytosol associated with the compartment. Using quantitative image analysis, we also show  that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we  demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment and  for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing  the compartment-forming polyproteins grew and developed normally under standard conditions

The New GPI-Anchored Protein, SwgA, Is Involved in Nitrogen Metabolism in the Pathogenic Filamentous Fungus Aspergillus fumigatus

International audienceGPI-anchored proteins display very diverse biological (biochemical and immunological) functions. An in silico analysis has revealed that the genome of Aspergillus fumigatus contains 86 genes coding for putative GPI-anchored proteins (GPI-APs). Past research has demonstrated the involvement of GPI-APs in cell wall remodeling, virulence, and adhesion. We analyzed a new GPI-anchored protein called SwgA. We showed that this protein is mainly present in the Clavati of Aspergillus and is absent from yeasts and other molds. The protein, localized in the membrane of A. fumigatus, is involved in germination, growth, and morphogenesis, and is associated with nitrogen metabolism and thermosensitivity. swgA is controlled by the nitrogen regulator AreA. This current study indicates that GPI-APs have more general functions in fungal metabolism than cell wall biosynthesis

Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete's foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete's foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host's immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.National Human Genome Research InstituteNational Human Genome Research InstituteNIHNIH [R21-AI081235

Transactivated and chemically inducible gene expression in plants.

Several vector systems are available for tissue-specific transactivation or chemical induction of transgene expression in plants. The choice facing researchers is which promoter system to commit to as this determines the range and characteristics of the expression resources available. The decision will not be the same for all species or applications. We present some general discussion on the use of these technologies and review in detail the properties in various (mainly angiosperm) species of the most promising: mGal4:VP16/UAS and pOp/LhG4 for transactivation, and the alc-switch, GVE/VGE, GVG, pOp6/LhGR, and XVE systems for chemical induction