Evaluation of type-B RR dimerization in poplar: A mechanism to preserve signaling specificity?

Plants possess specific signaling pathways, such as the MultiStep Phosphorelay (MSP), which is involved in cytokinin and ethylene sensing, and light, drought or osmotic stress sensing. These MSP comprise histidine-aspartate kinases (HKs) as receptors, histidine phosphotransfer (HPts) proteins acting as phosphorelay proteins, and response regulators (RRs), some of which act as transcription factors (type-B RRs). In previous studies, we identified partners of the poplar osmosensing signaling pathway, composed of two HKs, three main HPts, and six type-B RRs. To date, it is unresolved as to how cytokinin or osmotic stress signal specificity is achieved in the MSP in order to generate specific responses. Here, we present a large-scale interaction study of poplar type-B RR dimerization. Using the two-hybrid assay, we were able to show the homodimerization of type-B RRs, the heterodimerization of duplicated type-B RRs, and surprisingly, a lack of interaction between some type-B RRs belonging to different duplicates. The lack of interaction of the duplicates RR12-14 and RR18-19, which are involved in the osmosensing pathway has been confirmed by BiFC experiments. This study reveals, for the first time, an overview of type-B RR dimerization in poplar and makes way for the hypothesis that signal specificity for cytokinin or osmotic stress could be in part due to the fact that it is impossible for specific type-B RRs to heterodimerize.

Highlighting type A RRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus.

In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP.

[Yeasts from the CTG clade (Candida clade): Biology, impact in human health, and biotechnological applications]. / Les levures du clade CTG (clade Candida) : biologie, incidence en santé humaine et applications en biotechnologie.

Among the subdivision of Saccharomycotina (ascomycetes budding yeasts), the CTG clade (formerly the Candida clade) includes species that display a particular genetic code. In these yeasts, the CTG codon is predominantly translated as a serine instead of a leucine residue. It is now well-known that some CTG clade species have a major impact on human and its activities. Some of them are recognized as opportunistic agents of fungal infections termed candidiasis. In addition, another series of species belonging to the CTG clade draws the attention of some research groups because they exhibit a strong potential in various areas of biotechnology such as biological control, bioremediation, but also in the production of valuable biocompounds (biofuel, vitamins, sweeteners, industrial enzymes). Here we provide an overview of recent advances concerning the biology, clinical relevance, and currently tested biotechnological applications of species of the CTG clade. Future directions for scientific research on these particular yeasts are also discussed.

Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.

Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses.

Molecular cloning and characterisation of two calmodulin isoforms of the Madagascar periwinkle Catharanthus roseus.

Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.