Molecular Cloning of PbSTKL1 Gene from Plasmodiophora brassicae Expressed during Clubroot Development

Infection of crucifers by the obligate plant pathogen Plasmodiophora brassicae Woron. results in the formation of clubroot disease in these plants. Plasmodiophora brassicae gene expression during disease development was studied by differential display analysis of total RNA extracted from the roots of Chinese cabbage inoculated with the pathogen. In a series of experiments, 30 differentially expressed bands of cDNA were detected, and the expression of clone no. 17 was confirmed in clubbed roots. Southern blot analysis showed that this clone was a single‐copy gene in the P. brassicae genome. Putative amino acid sequence analysis of the full‐length cDNA of clone no. 17 (4.6 kb, designated PbSTKL1) revealed a serine/threonine kinase‐like domain at the C‐terminal region and a coiled‐coil structure in the middle region of the putative protein. PbSTKL1 expression increased strongly beginning 30 days after inoculation and was coincident with resting spore formation.     

Specific induction of TaAAPT1, an ER- and Golgi-localized ECPT-type aminoalcoholphosphotransferase, results in preferential accumulation of the phosphatidylethanolamine membrane phospholipid during cold acclimation in wheat

Cold acclimation requires substantial alteration in membrane property. In contrast to well-documented fatty acid unsaturation during cold acclimation, changes in phospholipid biosynthesis during cold acclimation are less understood. Here, we isolated and characterized two aminoalcoholphosphotransferase (AAPT) cDNAs, TaAAPT1 and TaAAPT2, from wheat. AAPTs utilize diacylglycerols and CDP-choline/ethanolamine as substrates and catalyze the final step of the CDP-choline/ethanolamine pathway for phosphatidylcholine (PC)/phosphatidylethanolamine (PE) synthesis, respectively. Functionality of TaAAPT1 and TaAAPT2 was demonstrated by heterologous expression in a yeast cpt1Δ ept1Δ double mutant that lacks both AAPT activities. Detailed characterization of AAPT activities from the transformed mutant cells indicated that TaAAPT1 is an ECPT-type enzyme with higher ethanolamine phosphotransferase (EPT) activity than choline phosphotransferase (CPT) activity, while TaAAPT2 is a CEPT-type with the opposite substrate preference. Transient expression of GFP-fused TaAAPT1 and TaAAPT2 proteins in wheat and onion cells indicated they are localized to both the endoplasmic reticulum and Golgi apparatus, suggesting that the final synthesis of PE and PC via the CDP-choline/ethanolamine pathway occurs in these organella. Quantitative PCR analyses revealed that TaAAPT1 expression is strongly induced by cold, while TaAAPT2 was constitutively expressed at lower levels. Measurement of phospholipid content in wheat leaves indicated that PE is more prominently increased in response to cold than PC and accordingly PE/PC ratio increased from 0.385 to 0.530 during 14 days of cold acclimation. Together, these data suggested that an increase in the PE/PC ratio during cold acclimation is regulated at the final step of the biosynthetic pathway.     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

Baseline Sensitivity of Populations of Phytophthora capsici from China to Three Carboxylic Acid Amide (CAA) Fungicides and Sequence Analysis of Cholinephosphotranferases from a CAA‐sensitive Isolate and CAA‐resistant Laboratory Mutants

During 2006–2008, 572 isolates of Phytophthora capsici were collected from seven provinces in China, and their sensitivities to three carboxylic acid amides (CAA), dimethomorph, flumorph and pyrimorph were determined. Of these isolates, 90 isolates without a history of exposure to CAA fungicides (CAAs) were used to set up the baseline sensitivity. Baseline EC₅₀ values ranged from 0.122 to 0.203 (mean ± SD, 0.154 ± 0.022) μg ml^?1 for dimethomorph, from 0.301 to 0.487 (mean ± SD, 0.373 ± 0.043) μg ml^?1 for flumorph and from 0.557 to 0.944 (mean ± SD, 0.712 ± 0.082) μg ml^?1 for pyrimorph, respectively. The other 482 isolates were tested with a single discriminatory dose and were completely inhibited at 0.5 μg ml^?1 of dimethomorph. Four CAA‐resistant mutants were generated by repeated exposure to dimethomorph in vitro. As compared to the parental wild‐type isolate, the four CAA‐resistant mutants showed similar fitness in hyphal growth, sporulation in vitro and pathogenicity in vivo. Mutants resistant to CAAs in the in vitro assay caused visible lesions on pepper stems or roots treated with the recommended dose of dimethomorph. Previous studies upon the mode of action of CAAs suggested that these fungicides maybe inhibit phospholipid biosynthesis and that the primary target could be the cholinephosphotranferase (CPT), which is referred to aminoalcoholphosphotransferases (AAPTs). We sequenced and analyzed two CPT (AAPT1 and AAPT2) genes in P. capsici. Based on the cDNA sequence, we found that the AAPT1 and AAPT2 gene span 1538 and 1459 bp and were interrupted by five and three introns, respectively. There was no difference between the parental wild‐type isolate and the four CAA‐resistant mutants in the amino acid sequences of AAPT1 and AAPT2 gene. So, it was assumed that the resistance to dimethomorph was not due to mutations in the amino acid sequence of these two possible target genes.     

Identification of a cDNA clone encoding for a galactose-binding lectin from peanut (Arachis hypogaea) seedling roots

A cDNA clone obtained from developing peanut (Arachis hypogaea) seedling roots, when expressed in Escherichia coli and insect cells (Sf9) gave a 29 kDa subunit protein. The native recombinant protein agglutinates neuraminidase treated human erythrocytes and the agglutination is inhibited by galactose. Nucleotide sequence and predicted amino acid sequence analyses indicate that it is different from peanut seed (PNA and SGL) and nodule (NGLa and NGLb) galactose-binding lectins.     

Characterization of a Moniezia expansa ubiquitin-conjugating enzyme E2 cDNA

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Here we report a new ubiquitin-conjugating enzyme E2 cDNA, which containing a ubiquitin-conjugating enzyme UBCc-domain named UBE2AM. Its cDNA is 899 base pairs in length and contains an open reading frame from nucleotide 171 to 632 encoding 153 amino acids. The result of real time RT-PCR showed that UBEA2 M is expressed in most of M. expansa proglottides and over-expressed in the mature proglottides. Comparison of predicted UBE2AM with UBCc (protein) homologues/orthologous from other species revealed identities between species varying from 97.5 to 99.4% at the amino acid level. Phylogenetic analysis showed the UBE2AM is a member of the eukaryotic UBCc superfamily, which have diverged from a common ancestor and the gene is clustered in the same group with the ubiquitin-conjugating enzyme E2A-like protein from Taenia asiatica.     

Characterization of Squalene synthase Gene from Chlorophytum borivilianum (Sant. and Fernand.)

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.     

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 cDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.     

Expression patterns of a cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis and environmental stress in Ginkgo biloba

The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. A cDNA sequence encoding the CAD gene was isolated from the leaves of Ginkgo biloba L, designated as GbCAD1. The full-length cDNA of GbCAD1 was 1,494?bp containing a 1,074?bp open reading frame encoding a polypeptide of 357 amino acids with a calculated molecular mass of 38.7?kDa and an isoelectric point of 5.74. Comparative and bioinformatic analyses revealed that GbCAD1 showed extensive homology with CADs from other gymnosperm species. Southern blot analysis indicated that GbCAD1 belonged to a multi-gene family. Phylogenetic tree analysis revealed that GbCAD1 shared the same ancestor in evolution with other CADs and had a further relationship with other gymnosperm species. GbCAD1 was an enzyme being pH-dependent and temperature-sensitive, and showing a selected catalyzing. Tissue expression pattern analysis showed that GbCAD1 was constitutively expressed in stems and roots, especially in the parts of the pest and disease infection, with the lower expression being found in two- to four-year-old stem. Further analysis showed the change in lignin content had some linear correlation with the expression level of GbCAD1 mRNA in different tissues. The increased expression of GbCAD1 was detected when the seedling were treated with exogenous abscisic acid, salicylic acid, ethephon, ultraviolet and wounding. These results indicate that the GbCAD1 gene may play a role in the resistance mechanism to biotic and abiotic stresses as well as in tissue-specific developmental lignification.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.