Transcriptome profiling of wheat differentially expressed genes exposed to different chemotypes of Fusarium graminearum.

KEY MESSAGE: The study is an overview of the behavior of the wheat transcriptome to the Fusarium graminearum fungus using two different chemotypes. The transcriptome profiles of seven putative differentially expressed defense-related genes were identified by SSH and further examined using qPCR. Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by several species of the fungus fusarium, is important in all wheat growing regions worldwide. The most dominant species in Canada is Fusarium graminearum (Fg). F. graminearum isolates producing mycotoxins such as 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON). The objective of this study was to investigate the effect of the different chemotypes of Fg on the transcriptome pattern of expressed wheat genes. A cDNA library was constructed from infected "Sumai 3" spikes harvested at different times after inoculation with a macroconidia suspension. Employing suppression subtractive hybridization (SSH), the subtracted cDNA library was differentially screened by dot-blot hybridization. Thirty-one clones were identified; one was isolated and characterized, and transcriptome profiling of seven up-regulated putative defense-related genes was performed using quantitative real-time reverse-transcriptase PCR. These genes may be involved in the wheat-pathogen interactions revealing transcript accumulation differences between the non-diseased, 3ADON-, and 15ADON-infected plants. Additionally, significant differences in gene expression were observed between 3ADON- and 15ADON-infected plants which highlight the significance of a particular chemotype in FHB disease.

Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

Inhibition of RuBisCO cloned from Thermosynechococcus vulcanus and expressed in Escherichia coli with compounds predicted by Molecular Operation Environment (MOE).

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of a thermophilic cyanobacterium, Thermosynechococcus vulcanus, was cloned and expressed in Escherichia coli. The purified enzyme had higher thermostability than RuBisCOs isolated from mesophilic cyanobacteria. Prediction of the tertiary structure was performed using the software Molecular Operating Environment (MOE). The predicted structure did not give any clue about the basis of thermostability. Then, the molecular docking of substrates and inhibitors in the catalytic site were carried out to test analogs for consistency of ribulose 1,5-bisphosphate, a RuBisCO substrate. The analogs were searched in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and 99 compounds were selected for the docking. The mol files from LIGAND Database in KEGG were changed to a three dimensional (3D) structure for use in docking simulation. The docking simulation was performed on ASEDock of MOE, and the SiteFinder command suggested about 20 candidates for the docking site of the compounds. Based on the homology of these candidate sites with the xylulose 1,5-bisphosphate (XBP)-binding site of RuBisCO isolated from Synechococcus PCC 6301, one site was selected for the docking simulation. The 40 compounds with the highest docking energies included synthetic organic substances that had never been demonstrated to be inhibitors of RuBisCO. The total docking energies were -102 kcal/mol, -104 kcal/mol, -94.0 kcal/mol, and -57.7 kcal/mol for ribulose 1,5-bisphosphate (RuBP), etidronate, risedronate, and citrate respectively. Kinetic analysis of RuBisCO revealed a K(m) value of 315 microM for RuBP, and K(i) values of 1.70, 0.93, and 2.04 mM for etidronate, risedronate, and citrate respectively. From these values, the binding energies were estimated to be -4.85, -3.84, -4.20, and -3.73 kcal/mol for RuBP, etidronate, risedronate, and citrate respectively. The differences between the values estimated from experimental data and by simulation may mainly depend on the dissimilarity of the environment for the protein and ligands between the experiments and the simulation. The results obtained here suggested a few new inhibitors, which might be useful as tools for studying the relationship between the structure and the function of RuBisCO.

A bacterial transgene for catalase protects translation of d1 protein during exposure of salt-stressed tobacco leaves to strong light.

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum 'Xanthi') and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [(35)S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplast's translational machinery to salt stress by scavenging hydrogen peroxide.