Analysis of the transcriptional response to Rice Yellow Mottle Virus infection in<Emphasis Type="Italic"> Oryza sativa indica</Emphasis> and<Emphasis Type="Italic"> japonica</Emphasis> cultivars

Several cDNA libraries were constructed using mRNA isolated from roots, panicles, cell suspensions and leaves of non-stressed Oryza sativa indica (IR64) and japonica (Azucena) plants, from wounded leaves, and from leaves of both cultivars inoculated with Rice Yellow Mottle Virus (RYMV). A total of 5549 cleaned expressed sequence tags (ESTs) were generated from these libraries. They were classified into functional categories on the basis of homology, and analyzed for redundancy within each library. The expression profiles represented by each library revealed great differences between indica and japonica backgrounds. EST frequencies during the early stages of RYMV infection indicated that changes in the expression of genes involved in energy metabolism and photosynthesis are differentially accentuated in susceptible and partially resistant cultivars. Mapping of these ESTs revealed that several co-localize with previously described resistance gene analogs and QTLs (quantitative trait loci).     

Genes expressed in zoospores of<Emphasis Type="Italic"> Phytophthora nicotianae</Emphasis>

The genus Phytophthora includes many highly destructive plant pathogens. In many Phytophthora species, pathogen dispersal and initiation of plant infection are achieved by motile, biflagellate zoospores that are chemotactically attracted to suitable infection sites. In order to study gene expression in zoospores, we have constructed a cDNA library using mRNA from zoospores of Phytophthora nicotianae. The library was arrayed and screened using probes derived from mycelium or zoospore mRNA. More than 400 clones representing genes preferentially expressed in zoospores were identified and sequenced from the 5 end of the insert. The expressed sequence tags (ESTs) generated were found to represent 240 genes. The ESTs were compared to sequences in GenBank and in the Phytophthora Genome Consortium database, and classified according to putative function based on homology to known proteins. To further characterize the identified genes, a colony array was created on replicate nylon filters and screened with probes derived from four Phytophthora developmental stages including zoospores, germinating cysts, vegetative mycelium and sporulating hyphae, and from inoculated and uninoculated tobacco seedlings. Data from sequence analysis and colony array screening were compiled into a local database, and searched to identify genes that are preferentially expressed in zoospores for future functional analysis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by C. A. M. J. J. van den Hondel     

Characterisation and expression of monosaccharide transporters in lupins, <Emphasis Type="Italic">Lupinus polyphyllus</Emphasis> and <Emphasis Type="Italic">L. albus</Emphasis>

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

A genomic approach to isoflavone biosynthesis in kudzu (<Emphasis Type="Italic">Pueraria lobata</Emphasis>)

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6′-deoxy- and 6′-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

Two WD-repeat genes from cotton are functional homologues of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis><Emphasis Type="Italic">TRANSPARENT TESTA GLABRA1</Emphasis> (<Emphasis Type="Italic">TTG1</Emphasis>) gene

Cotton fibres are single, highly elongated cells derived from the outer epidermis of ovules, and are developmentally similar to the trichomes of Arabidopsis thaliana. To identify genes involved in the molecular control of cotton fibre initiation, we isolated four putative homologues of the Arabidopsis trichome-associated gene TRANSPARENT TESTA GLABRA1 (TTG1). All four WD-repeat genes are derived from the ancestral D diploid genome of tetraploid cotton and are expressed in many tissues throughout the plant, including ovules and growing fibres. Two of the cotton genes were able to restore trichome formation in ttg1 mutant Arabidopsis plants. Both these genes also complemented the anthocyanin defect in a white-flowered Matthiola incana ttg1 mutant. These results demonstrate parallels in differentiation between trichomes in cotton and Arabidopsis, and indicate that these cotton genes may be functional homologues of AtTTG1.