Receptor–ligand and parasite protein–protein interactions in Plasmodium vivax: Analysing rhoptry neck proteins 2 and 4

Elucidating receptor–ligand and protein–protein interactions represents an attractive alternative for designing effective Plasmodium vivax control methods. This article describes the ability of P. vivax rhoptry neck proteins 2 and 4 (RON2 and RON4) to bind to human reticulocytes. Biochemical and cellular studies have shown that two PvRON2‐ and PvRON4‐derived conserved regions specifically interact with protein receptors on reticulocytes marked by the CD71 surface transferrin receptor. Mapping each protein fragment's binding region led to defining the specific participation of two 20 amino acid‐long regions selectively competing for PvRON2 and PvRON4 binding to reticulocytes. Binary interactions between PvRON2 (ligand) and other parasite proteins, such as PvRON4, PvRON5, and apical membrane antigen 1 (AMA1), were evaluated and characterised by surface plasmon resonance. The results revealed that both PvRON2 cysteine‐rich regions strongly interact with PvAMA1 Domains II and III (equilibrium constants in the nanomolar range) and at a lower extent with the complete PvAMA1 ectodomain and Domains I and II. These results strongly support that these proteins participate in P. vivax's complex invasion process, thus providing new pertinent targets for blocking P. vivax merozoites' specific entry to their target cells.     

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), β-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits.

Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads     

Antifungal Activity of <Emphasis Type="Italic">Pv</Emphasis>D1 Defensin Involves Plasma Membrane Permeabilization,Inhibition of Medium Acidification,and Induction of ROS in Fungi Cells

In recent years, studies have demonstrated the function of many antimicrobial peptides against an extensive number of microorganisms that have been isolated from different plant species and that have been used as models for the study of various cellular processes linked to these peptides’ activities. Recently, a new defensin from Phaseolus vulgaris (L.) seeds, named PvD_1, was isolated and characterized. PvD₁ was purified through anion exchange and phase-reverse chromatography. PvD₁’s antifungal activity was tested. A SYTOX Green uptake assay revealed that the defensin PvD₁ is capable of causing membrane permeabilization in the filamentous fungi Fusarium oxysporum, Fusarium solani, and Fusarium laterithium and in yeast strains Candida parapsilosis, Pichia membranifaciens, Candida tropicalis, Candida albicans, Kluyveromyces marxiannus, and Saccharomyces cerevisiae at a concentration of 100 μg/ml. Ultrastructural analysis of C. albicans and C. guilliermondii cells treated with this defensin revealed disorganization of both cytoplasmic content and the plasma membrane. PvD₁ is also able to inhibit glucose-stimulated acidification of the medium by yeast cells and filamentous fungi, as well as to induce the production of reactive oxygen species and nitric oxide in C. albicans and F. oxysporum cells.     

Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana: ENZYMES CATALYZING UP TO 10 REARRANGEMENT STEPS YIELDING FRIEDELIN AND OTHER TRITERPENOIDS*

The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C₃₀H₅₀O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761–779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.     

ISOLATION OF ALGAL GENES BY FUNCTIONAL COMPLEMENTATION OF YEAST1

Algal cDNAs were isolated and characterized by functional complementation of yeast auxotrophs. Two cDNA libraries, one derived from the diatom Phaeodactylum tricornutum Bohlin and the other from the dinoflagellate Crypthecodinium cohnii Biecheler, were constructed using the Saccharomyces cerevisiae expression vector pFL‐61. These libraries were used for functional complementation of auxotrophic markers in two yeast strains. Yeast tryptophan auxotrophs, complemented by the P. tricornutum library, contained a plasmid that encoded a two‐domain protein associated with tryptophan synthesis, indole‐3‐glycerol phosphate synthase‐N‐(5′‐phosphoribosyl) anthranilate isomerase. Another cDNA originating from the C. cohnii library rescued S. cervisiae from a defect in adenine biosynthesis. This cDNA encoded a fusion of phosphoribosylamidoimidazole‐succinocarboxamide synthetase and phosphoribosylaminoimidazole carboxylase, which correspond to the yeast ade1 and ade2 genes, respectively. These results demonstrate that heterologous functional complementation can be used to identify algal genes and may provide advantages over other gene discovery methods.     

Identification of marneral synthase,which is critical for growth and development in Arabidopsis

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3‐oxidosqualene (OS), which is cyclized by 2,3‐oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock‐out mutant displaying round‐shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.     

Isolation and characterization of a new repetitive DNA family recently amplified in the Mesoamerican gene pool of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L., Fabaceae)

The common bean (Phaseolus vulgaris) is one of the most important crop plants. About 50% of its genome is composed of repetitive sequences, but only a little fraction was isolated and characterized so far. In this paper, a new repetitive DNA family from the species, named PvMeso, was isolated and characterized in both gene pools of P. vulgaris (Andean and Mesoamerican) and related species. Two fragments, 1.7 and 2.3 kb long, were cloned from BAC 255F18, which has previously shown a repetitive pattern. The subclone PvMeso-31 showed a terminal block in chromosome 7. This subclone contains a 1,705 bp long, AT-rich repeat with small internal repeats and shares a 1.2 kb region with PvMeso-47, derived from the 2.3 kb fragment. The presence of this repetitive block was restricted to Mesoamerican accessions of the common bean. In P. acutifolius, P. leptostachyus and Andean P. vulgaris, only a faint, 2.3 kb fragment was visualized in Southern experiments. Moreover, in Mesoamerican accessions, two other fragments (1.7 kb and 3.4 kb) were strongly labelled as well. Taken together, our results indicate that PvMeso is a recently emerged, repeat family initially duplicated in chromosome 11, on ancestral Mesoamerican accession, and later amplified in chromosome 7, after the split of the two major gene pools of the common bean.     

Divergence of defensive cucurbitacins in independent Cucurbita pepo domestication events leads to differences in specialist herbivore preference

Crop domestication and improvement often concurrently affect plant resistance to pests and production of secondary metabolites, creating challenges for isolating the ecological implications of selection for specific metabolites. Cucurbitacins are bitter triterpenoids with extreme phenotypic differences between Cucurbitaceae lineages, yet we lack integrated models of herbivore preference, cucurbitacin accumulation, and underlying genetic mechanisms. In Cucurbita pepo, we dissected the effect of cotyledon cucurbitacins on preference of a specialist insect pest (Acalymma vittatum) for multiple tissues, assessed genetic loci underlying cucurbitacin accumulation in diverse germplasm and a biparental F₂ population (from a cross between two independent domesticates), and characterized quantitative associations between gene expression and metabolites during seedling development. Acalymma vittatum affinity for cotyledons is mediated by cucurbitacins, but other traits contribute to whole-plant resistance. Cotyledon cucurbitacin accumulation was associated with population structure, and our genetic mapping identified a single locus, Bi-4, containing genes relevant to transport and regulation – not biosynthesis – that diverged between lineages. These candidate genes were expressed during seedling development, most prominently a putative secondary metabolite transporter. Taken together, these findings support the testable hypothesis that breeding for plant resistance to insects involves targeting genes for regulation and transport of defensive metabolites, in addition to core biosynthesis genes.     

Pothos vietnamensis sp. nov. (Araceae–Pothoideae–Potheae) from Vietnam

Pothos vietnamensis V. D. Nguyen & P. C. Boyce is described and illustrated from northern Vietnam as a new species of the Pothos supergroup, and it is compared with the two most similar species: P. kerrii and P. pilulifer to which P. vietnamensis is comparable by having a very small fertile portion (< 5 mm diameter) on the spadix. Ecology, habitat, population size and conservation status are also discussed.     

Granaticins and their biosynthetic gene cluster from <Emphasis Type="Italic">Streptomyces vietnamensis</Emphasis>: evidence of horizontal gene transfer

Streptomyces vietnamensis, a recently designated species isolated from tropical forest soil, was found to be a new granaticin producer. The granaticin biosynthetic gene cluster (gra) and flanking genes from S. vietnamensis were cloned and sequenced by a sequential cloning strategy. All biosynthetic genes were found as expected. The high overall homology of the gra cluster from S. vietnamensis to that of Streptomyces violaceoruber Tü22 indicated a recent common ancestor of the two clusters. However, a flanking gene orf35 was missing from the gra cluster of S. vietnamensis, and high frequency of insertions and deletions of short fragment (shorter than 63 bp) were observed throughout the sequenced region compared to that of S. violaceoruber Tü22. These revealed a rapid evolution of the gra cluster and suggested that small insertions and deletions might be one of the basic evolution mechanisms for streptomycete genomes. The phylogenetic incongruence between 16S rDNA and the gra cluster and the scattered distribution of the granaticin producers within Streptomyces implicated horizontal gene transfer (HGT) being involved in the gra cluster dispersion. The remnants of orf35 found in S. vietnamensis present a scenario on how the antibiotic gene clusters evolved after HGT. The contemporary gra cluster residing in S. vietnamensis could be interpreted as a combination of HGT and highly variable vertical transmission.     

构建酿酒酵母细胞工厂高效合成摩尔酸

[目的] 摩尔酸作为齐墩果烷型三萜化合物具有抗HIV、抗炎等多种生物学活性,其前体物质是计曼尼醇,本研究基于合成生物学策略构建酿酒酵母细胞工厂高效合成摩尔酸。[方法] 运用CRISPR/Cas9技术,首先分别整合不同来源的氧化鲨烯环化酶（OSCs）,筛选高产计曼尼醇底盘细胞;进一步异源表达长春花来源的细胞色素P450氧化酶（CYP716AL1）和麻风树来源的细胞色素P450还原酶（JcCPR）,构建摩尔酸生物合成途径;并通过CYP716AL1和不同来源的CPR适配研究以及过表达甲羟戊酸（MVA）代谢途径中关键酶的方式提高摩尔酸的产量。[结果] 整合苹果来源的氧化鲨烯环化酶MdOSC获得的重组菌株计曼尼醇产量最高,达68.3 mg/L;以此为底盘细胞进一步整合CYP716AL1和JcCPR实现了摩尔酸的生物合成,产量为15.0 mg/L;共表达CYP716AL1和拟南芥来源的CPR获得的重组菌株摩尔酸产量最高,达到24.3 mg/L;最后过表达MVA代谢途径中的关键酶法呢基焦磷酸合酶（ERG20）和鲨烯环氧酶（ERG1）,获得的重组菌株摩尔酸产量高达34.1 mg/L。[结论] 本研究实现了摩尔酸的高效生物合成,为构建高产齐墩果烷型三萜酿酒酵母细胞工厂提供了理论和技术依据。     

Characterization of CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM Homologs in Rice (Oryza sativa L.)

To enhance our understanding of brassinosteroid (BR) biosynthesis in rice, we attempted to identify putative rice homologs of Arabidopsis CYP90A1/ CPD and related mutants. Two candidate genes, designated CYP90A3/OsCPD1 and CYP90A4/OsCPD2, are located on chromosomes 11 (2.0 cM) and 12 (1.9 cM), respectively. Based on sequence similarity with the Arabidopsis CYP90A1/CPD gene, we predict that the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 gene products function as C-23α hydroxylases in the BR biosynthesis pathway. Both are broadly expressed in wild-type rice, and their expression is regulated by a feedback mechanism. A retrotransposon insertion mutant of CYP90A3/OsCPD1, oscpd1-1, did not produce any BR-deficient phenotype or feedback upregulation of genes for BR biosynthesis enzymes. These results indicate that if, as predicted, the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 genes do function in the BR biosynthesis pathway, they may each have enough capacity to catalyze BR biosynthesis on their own. As a consequence, the oscpd1-1 mutant may not be deficient in endogenous BRs. Interestingly, BR biosynthesis enzymes except C-6 oxidase are encoded by plural genes in rice but by single genes in Arabidopsis (again, except C-6 oxidase). On the basis of these findings, we discuss the differences in BR biosynthesis between rice and Arabidopsis.     

Characterization of a Rice Gene Family Encoding Type-A Diterpene Cyclases

We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.