Joël Crèche

Cytokinins and ethylene stimulate indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus by two distinct mechanisms

Abstract The interactions between cytokinins and ethylene on alkaloid accumulation in a periwinkle cell line have been examined. It was found that (a) either exogenously-applied cytokinins or ethylene (the latter through ethephon degradation) greatly enhanced ajmalicine accumulation in cells subcultured in a 2,4-dichlorophenoxyacetic acid-free medium; (b) the enhancing effect of cytokinin was not mediated by enhancement of endogenous ethylene production contrary to what is found in some plant models; (c) the responses to exogenous cytokinin and ethylene were additive and showed a different pattern of expression. It may be concluded that cytokinin and ethylene can up-regulate the alkaloid production in a periwinkle cells through independent pathways when added exogenously to the cultures.

Candida guilliermondii: biotechnological applications, perspectives for biological control, emerging clinical importance and recent advances in genetics

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last 40 years due to its biotechnological interest, biological control potential and clinical importance. Such a wide range of applications in various areas of fundamental and applied scientific research has progressively made C. guilliermondii an attractive model for exploring the potential of yeast metabolic engineering as well as for elucidating new molecular events supporting pathogenicity and antifungal resistance. All these research fields now take advantage of the establishment of a useful molecular toolbox specifically dedicated to C. guilliermondii genetics including the construction of recipient strains, the development of selectable markers and reporter genes and optimization of transformation protocols. This area of study is further supported by the availability of the complete genome sequence of the reference strain ATCC 6260 and the creation of numerous databases dedicated to gene ontology annotation (metabolic pathways, virulence, and morphogenesis). These genetic tools and genomic resources represent essential prerequisites for further successful development of C. guilliermondii research in medical mycology and in biological control by facilitating the identification of the multiple factors that contribute to its pathogenic potential. These genetic and genomic advances should also expedite future practical uses of C. guilliermondii strains of biotechnological interest by opening a window into a better understanding of the biosynthetic pathways of valuable metabolites.

Histidine-containing phosphotransfer domain extinction by RNA interference turns off a cytokinin signalling circuitry in Catharanthus roseus suspension cells

We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures. Here, we characterized the CrHPt1 gene that encodes a histidine‐containing phosphotransfer domain. CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway. In transgenic lines stably transformed with an intron‐spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription. These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators. They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry.

Effect of cytokinin on alkaloid accumulation in periwinkle callus cultures transformed with a light-inducible ipt gene

The effect of cytokinins on accumulation of indole alkaloids in periwinkle callus cultures was investigated. Firstly, it was found that exogenously-applied cytokinin increased the ajmalicine and serpentine content of untransformed callus culture obtained from cotyledons. Secondly, periwinkle cotyledons were transformed with the isopentenyl transferase (ipt) gene under the control of a light-inducible promoter and two transformed callus lines were used in order to investigate whether endogenously-produced cytokinin could also increase the alkaloid production. We found that the ipt-transgenic tissues accumulated higher levels of isopentenyl transferase transcripts as well as zeatin riboside, even under non-inductive condition, but lower concentration of alkaloids compared to that of untransformed tissues. A 28 kDa polypeptide whose accumulation was previously found to be associated with alkaloid production in a periwinkle cell suspension was also present in the non-transformed tissue and its level was increased in parallel to the CK-enhanced alkaloid production. Neither light induction condition, nor exogenous cytokinin treatment led to the increase of the 28 kDa polypeptide accumulation in the transformed tissues. All these data show that endogenously-produced cytokinin does not mimic the effect of exogenously-applied cytokinin on the alkaloid production in periwinkle calli.

Drug‐resistant cassettes for the efficient transformation of Candida guilliermondii wild‐type strains

Candida guilliermondii is an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains an interesting biotechnological model for the industrial production of value-added metabolites. The recent whole-genome sequencing of the C. guilliermondii ATCC 6260 reference strain provides an interesting resource for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering. In the present study, we designed an efficient transformation system for C. guilliermondii wild-type strains using both nourseothricin- and hygromycin B-resistant markers. To demonstrate the potential of these drug-resistant cassettes, we carried out the disruption and the complementation of the C. guilliermondii FCY1 gene (which encodes cytosine deaminase) known to be associated with flucytosine sensitivity in yeast. These two new dominant selectable markers represent powerful tools to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.

Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH#), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondiiku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.

Fluorescent protein fusions in Candida guilliermondii

Candida guilliermondii is an emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains a promising biotechnological model for the industrial production of value-added metabolites. In the present study, we developed a recipient strain as well as a set of plasmids for construction of fluorescent protein (FP) fusions in this species. We demonstrated that C. guilliermondii phosphoglycerate kinase transcription-regulating sequences allow a constitutive expression of codon-optimized green, cyan, yellow and mCherry FP genes in C. guilliermondii cells and the fluorescence signal could be directly observed at the colony and blastospore level by epifluorescence microcopy. To illustrate differential targeting of the FPs into specified cellular compartments, we studied and validated the expected subcellular localization of various C. guilliermondii predicted proteins fused to FPs. Furthermore, co-expression experiments of various couples of FP-tagged C. guilliermondii predicted proteins in the same cell showed that the fluorescence of each FP could be detected independently, providing firm evidences that YFP/CFP and GFP/mCherry pairs can be used for dual labeling in C. guilliermondii cells. This technical advance will facilitate future studies of protein co-expression and co-localization in C. guilliermondii and will give precious help for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering.

Deciphering the Evolution, Cell Biology and Regulation of Monoterpene Indole Alkaloids

Monoterpene indole alkaloids (MIAs) constitute a large group of specialised metabolites with many potent pharmaceutical properties, including the antitumoral vinblastine and hypotensive ajmalicine. Hence a large body of phytochemical investigation delineates the distribution and diversity of various MIA structural classes in Gentianales families. The biosynthetic pathway of these secondary metabolites involves several specific branches, including indole and monoterpenoid formations, secoiridoid assembly, central MIA biosynthesis and branch-specific reactions, as well as supply of primary metabolite precursors by the methylerythritol phosphate and shikimate pathways. Several genes and enzymatic activities involved in these pathways have been characterised, allowing detailed analysis of the molecular biology of this system in model plants such as Catharanthus roseus and Rauvolfia serpentina. With the prospects of improving production of MIAs in plant and cell culture, regulations of biosynthetic capacities have been thoroughly investigated. This pathway also presents a high degree of spatial organisation at the organ, cellular and subcellular levels. This chapter presents an overview of the structural diversity, the complexity of MIA biosynthesis, and regulation with an evolutionary perspective.

Changes in the accumulation of cytosolic cyclophilin transcripts in cultured periwinkle cells following hormonal and stress treatments

Summary Cyelophilins (CyPs) are a family of highly conserved proteins that catalyze cis-trans isomerization of peptidylprolyl bonds and also act as chaperones. In spite of the biological importance of these enzymes, the regulation of the CyP genes remains poorly known. We found in periwinkle cell cultures that zeatin, abscisic acid, NaCl, LaCl 3 , high sucrose concentration and cold stress caused an increase in the accumulation of transcripts from a cytosolic CyP gene. By contrast, the expression of the CyP gene was neither heat shock-nor gibberellic acid-responsive, whereas hypoxia or presence of phosphates in the medium led to a rapid and severe decrease in the CyP messages. We also examined the pattern of expression of the CyP gene in young periwinkle plants. CyP transcripts accumulated in all organs studied, but in lower amounts in flower buds than in leaves and roots. From our data, we conclud that: (i) CyP expression is often but not always associated with stress responses; (ii) CyP transcript accumulation is independent of cell mitotic activity; and (iii) there is no correlation between the alkaloid biosynthesis and the content of CyP messages in the periwinkle cells.

Variability in tissue cultures of Choisya ternata: alkaloid accumulation in protoclones and aggregate clones obtained from established strains

Protoclones and aggregate clones have been prepared from 5 callus strains of C. ternata chosen for their dihydrofuroquinoline-accumulating capacities in a population of well-established strains. The results show that it is possible to obtain cell lines which accumulate higher alkaloid contents than the highest alkaloid-producing strain; the probability of obtaining a high-producing clone is greater if a high-producing strain is chosen as the parent strain for cloning. Compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in greater amounts in some clones, but another alkaloid (balfourodinium) was always found in lesser quantities, even in the clones which accumulated most alkaloids. Aggregate clones were easier to obtain than protoclones and alkaloid production was rather unstable in all the clones. The protoplast-cloning procedure was not more efficient than the aggregate-cloning procedure for the selection of high-alkaloid producing lines.