Identification and expression analysis of C2H2-zinc finger protein genes reveals their role in stress tolerance in Brassica napus.

C2H2-zinc finger (C2H2-ZF) genes are involved in various biological processes in plants including stress response; however, they lack characterization in Brassica napus. We identified 267 C2H2-ZF genes in B. napus and deciphered their physiological properties, subcellular localization, structure, synteny, and phylogeny and investigated the expression of 20 genes in response to different stresses and phytohormone treatments. The 267 genes were distributed on 19 chromosomes; phylogenetic analysis categorized them into five clades. They varied from 0.41 to 9.2 kb in length, had stress-responsive cis-acting elements in promoter regions, and their protein length varied from 9 to 1366 amino acids. About 42% of the genes had one exon, and 88% genes had orthologs in Arabidopsis thaliana. About 97% of the genes were located in nucleus and 3% in cytoplasmic organelles. qRT-PCR analysis showed a different expression pattern of these genes in response to biotic stresses (Plasmodiophora brassicae and Sclerotinia sclerotiorum) and abiotic stresses (cold, drought, and salinity) and hormonal treatments. Differential expression of the same gene was observed under multiple stress conditions, and a few genes showed similar expression in response to more than one phytohormones. Our results suggest that the C2H2-ZF genes can be targeted for the improvement of stress tolerance in canola.

Silicon ameliorates clubroot responses in canola (Brassica napus): A "multi-omics"-based investigation into possible mechanisms.

Clubroot disease, caused by Plasmodiophora brassicae Woronin, results in severe yield losses in Brassica crops, including canola. Silicon (Si) mitigates several stresses and enhances plant resistance to phytopathogens. We investigated the effects of Si on clubroot disease symptoms in canola at two concentrations of Si, Si: soil in 1: 100 w/w (Si1.0) and Si: soil in 1:200 w/w (Si0.5) under greenhouse conditions. In addition, the effects of Si on P. brassicae-induced gene expression, endogenous levels of phytohormones and metabolites were studied using "omics" approaches. Si application reduced clubroot symptoms and improved plant growth parameters. Gene expression analysis revealed increased transcript-level responses in Si1.0 compared to Si0.5 plants at 7-, 14-, and 21-days post-inoculation (dpi). Pathogen-induced transcript-level changes were affected by Si treatment, with genes related to antioxidant activity (e.g., POD, CAT), phytohormone biosynthesis and signalling (e.g., PDF1.2, NPR1, JAZ, IPT, TAA), nitrogen metabolism (e.g., NRT, AAT), and secondary metabolism (e.g., PAL, BCAT4) exhibiting differential expression. Endogenous levels of phytohormones (e.g., auxin, cytokinin), a majority of the amino acids and secondary metabolites (e.g., glucosinolates) were increased at 7 dpi, followed by a decrease at 14- and 21-dpi due to Si-treatment. Stress hormones such as abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) also decreased at the later time points in Si0.5, and Si1.0 treated plants. Si appears to improve clubroot symptoms while enhancing plant growth and associated metabolic processes, including nitrogen metabolism and secondary metabolite biosynthesis.

Genome-wide identification of biotin carboxyl carrier subunits of acetyl-CoA carboxylase in Brassica and their role in stress tolerance in oilseed Brassica napus.

BACKGROUND: Biotin carboxyl carrier protein (BCCP) is a subunit of Acetyl CoA-carboxylase (ACCase) which catalyzes the conversion of acetyl-CoA to malonyl-CoA in a committed step during the de novo biosynthesis of fatty acids. Lipids, lipid metabolites, lipid-metabolizing and -modifying enzymes are known to play a role in biotic and abiotic stress tolerance in plants. In this regard, an understanding of the Brassica napus BCCP genes will aid in the improvement of biotic and abiotic stress tolerance in canola. RESULTS: In this study, we identified 43 BCCP genes in five Brassica species based on published genome data. Among them, Brassica rapa, Brassica oleracea, Brassica nigra, Brassica napus and Brassica juncea had six, seven, seven, 10 and 13 BCCP homologs, respectively. Phylogenetic analysis categorized them into five classes, each with unique conserved domains. The promoter regions of all BCCP genes contained stress-related cis-acting elements as determined by cis-element analysis. We identified four and three duplicated gene pairs (segmental) in B. napus and B. juncea respectively, indicating the role of segmental duplication in the expansion of this gene family. The Ka/Ks ratios of orthologous gene pairs between Arabidopsis thaliana and five Brassica species were mostly less than 1.0, implying that purifying selection, i.e., selective removal of deleterious alleles, played a role during the evolution of Brassica genomes. Analysis of 10 BnaBCCP genes using qRT-PCR showed a different pattern of expression because of exposure of the plants to biotic stresses, such as clubroot and sclerotinia diseases, and abiotic stresses such as drought, low temperature and salinity stresses. CONCLUSIONS: The identification and functional analysis of the Brassica BCCPs demonstrated that some of these genes might play important roles in biotic and abiotic stress responses. Results from this study could lay the foundation for a better understanding of these genes for the improvement of Brassica crops for stress tolerance.

Identification of lncRNAs in response to infection by Plasmodiophora brassicae in Brassica napus and development of lncRNA-based SSR markers.

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), is yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot-resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with Plasmodiophora brassicae pathotype 3. Pathway enrichment analysis showed that most of the target genes regulated by these DE lncRNAs belonged to plant-pathogen interaction and hormone signaling, as well as primary and secondary metabolic pathways. Comparative analysis of these lncRNAs with 530 previously reported DE lncRNAs, identified using resistance located on BnaA08, detected 12 lncRNAs that showed a similar trend of upregulation in both types of resistant lines; these lncRNAs probably play a fundamental role in clubroot resistance. We identified SSR markers within 196 DE lncRNAs. Genotyping of two DH populations carrying resistance on BnaA03 identified a marker capable of detecting the resistance in 98% of the DH lines. To our knowledge, this is the first report of the identification of SSRs within lncRNAs responsive to P. brassicae infection, demonstrating the potential use of lncRNAs in the breeding of Brassica crops.

Characterization of microRNA-like RNAs associated with sclerotial development in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a model necrotrophic pathogen causing great economic losses worldwide. Sclerotia are dormant structures that play significant biological and ecological roles in the life and disease cycles of S. sclerotiorum and other species of sclerotia-forming fungi. microRNA-like RNAs (milRNAs) as non-coding small RNAs play regulatory roles in fungal development and pathogenicity. Therefore, milRNAs associated with sclerotial development in S. sclerotiorum were investigated in this study. A total of 275 milRNAs with induced expression during sclerotia development were identified, in which 51 were differentially expressed. The target genes of all milRNAs were predicted. The putative functions of the targets regulated by milRNAs were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The expression levels of six selected milRNAs that coordinated with their corresponding targets were validated by qRT-PCR. Among these six milRNAs, Ssc-milR-240 was potentially associated with sclerotial development by epigenetic regulation of its target histone acetyltransferase. This study will facilitate the better understanding of the milRNA regulation associated with sclerotial development in S. sclerotiorum and even other sclerotia-forming fungi. This work will provide novel insights into the molecular regulations of fungal morphogenesis and the candidate targets of milRNAs used for the sustainable management of plant diseases caused by S. sclerotiorum.

Identification and evaluation of suitable reference genes for gene expression analysis in rubber tree leaf.

Gene expression profiles are increasingly applied to investigate molecular mechanism for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber tree, however, little is known in leaf. The main objective of this current study was to identify some stable expression reference genes at various developmental stages of leaf, as well as during abiotic (high and low temperature extremes) and biotic stresses (pathogen stress). Gene expression profilings identified the ubiquitin-proteasome system as excellent potential as reference genes for rubber tree leaf. Among a total of 30 tested genes investigated, 24 new candidate (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new candidate genes had better expression stability comparing with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes for rubber tree leaf under four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data. Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other species.

Identification of lncRNAs Responsive to Infection by Plasmodiophora brassicae in Clubroot-Susceptible and -Resistant Brassica napus Lines Carrying Resistance Introgressed from Rutabaga.

Clubroot disease, caused by Plasmodiophora brassicae Woronin, is a major threat to the production of Brassica' crops. Resistance to different P. brassicae pathotypes has been reported in the A genome, chromosome A08; however, the molecular mechanism of this resistance, especially the involvement of long noncoding RNAs (lncRNAs), is not understood. We have used a strand-specific lncRNA-Seq approach to catalog lncRNAs from the roots of clubroot-susceptible and -resistant Brassica napus lines. In total, 530 differentially expressed (DE) lncRNAs were identified, including 88% of long intergenic RNAs and 11% natural antisense transcripts. Sixteen lncRNAs were identified as target mimics of the microRNAs (miRNAs) and eight were identified as the precursors of miRNAs. KEGG pathway analysis of the DE lncRNAs showed that the cis-regulated target genes mostly belong to the phenylpropanoid biosynthetic pathway (15%) and plant-pathogen interactions (15%) while the transregulated target genes mostly belong to carbon (18%) and amino acid biosynthesis pathway (19%). In all, 24 DE lncRNAs were identified from chromosome A08, which is known to harbor a quantitative trait locus conferring resistance to different P. brassicae pathotypes; however, eight of these lncRNAs showed expression only in the resistant plants. These results could form the basis for future studies aimed at delineating the roles of lncRNAs in plant-microbe interactions.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein.

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

Regulation of low temperature stress in plants by microRNAs.

Low temperature is one of the most common environmental stresses that seriously affect the growth and development of plants. However, plants have the plasticity in their defence mechanisms enabling them to tolerate and, sometimes, even survive adverse environmental conditions. MicroRNAs (miRNAs) are small non-coding RNAs, approximately 18-24 nucleotides in length, and are being increasingly recognized as regulators of gene expression at the post-transcriptional level and have the ability to influence a broad range of biological processes. There is growing evidence in the literature that reprogramming of gene expression mediated through miRNAs is a major defence mechanism in plants enabling them to respond to stresses. To date, numerous studies have established the importance of miRNA-based regulation of gene expression under low temperature stress. Individual miRNAs can modulate the expression of multiple mRNA targets, and, therefore, the manipulation of a single miRNA has the potential to affect multiple biological processes. Numerous functional studies have attempted to identify the miRNA-target interactions and have elaborated the role of several miRNAs in cold-stress regulation. This review summarizes the current understanding of miRNA-mediated modulation of the expression of key genes as well as genetic and regulatory pathways, involved in low temperature stress responses in plants.

Changes in primary metabolism and associated gene expression during host-pathogen interaction in clubroot resistance of Brassica napus.

The role of primary metabolism during Brassica napus-Plasmodiophora brassicae interaction leading to clubroot resistance has not yet been investigated thoroughly. In this study, we investigated some of the primary metabolites and their derivatives as well as expression of the genes involved in their biosynthesis to decipher this host-pathogen interaction. For this, two sets (clubroot resistant and susceptible) of canola lines were inoculated with P. brassicae pathotype 3A to investigate the endogenous levels of primary metabolites at 7-, 14-, and 21-days after inoculation (DAI). The associated pathways were curated, and expression of the selected genes was analyzed using qRT-PCR. Our results suggested the possible involvement of polyamines (spermidine and spermine) in clubroot susceptibility. Some of the amino acids were highly abundant at 7- or 14-DAI in both resistant and susceptible lines; however, glutamine and the amino acid derivative phenylethylamine showed higher endogenous levels in the resistant lines at later stages of infection. Organic acids such as malic, fumaric, succinic, lactic and citric acids were abundant in the susceptible lines. Conversely, the abundance of salicylic acid (SA) and the expression of benzoate/salicylate carboxyl methyltransferase (BSMT) were higher in the resistant lines at the secondary stage of infection. A reduced disease severity index and gall size were observed when exogenous SA (1.0 mM) was applied to susceptible B. napus; this further supported the role of SA in clubroot resistance. In addition, a higher accumulation of fatty acids and significant upregulation of the pathway genes, glycerol-3-phosphate dehydrogenase (GPD) and amino alcohol phosphotransferase (AAPT) were observed in the resistant lines at 14- and 21-DAI. In contrast, some of the fatty acid derivatives such as phosphatidylcholines represented a lower level in the resistant lines. In conclusion, our findings provided additional insights into the possible involvement of primary metabolites and their derivatives in clubroot resistance.

Proteome- and metabolome-level changes during early stages of clubroot infection in Brassica napus canola.

Clubroot is a destructive root disease of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. Despite extensive research into the molecular responses of B. napus to P. brassicae, there is limited information on proteome- and metabolome-level changes in response to the pathogen, especially during the initial stages of infection. In this study, we have investigated the proteome- and metabolome- level changes in the roots of clubroot-resistant (CR) and -susceptible (CS) doubled-haploid (DH) B. napus lines, in response to P. brassicae pathotype 3H at 1-, 4-, and 7-days post-inoculation (DPI). Root proteomes were analyzed using nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS). Comparisons of pathogen-inoculated and uninoculated root proteomes revealed 2515 and 1556 differentially abundant proteins at one or more time points (1-, 4-, and 7-DPI) in the CR and CS genotypes, respectively. Several proteins related to primary metabolites (e.g., amino acids, fatty acids, and lipids), secondary metabolites (e.g., glucosinolates), and cell wall reinforcement-related proteins [e.g., laccase, peroxidases, and plant invertase/pectin methylesterase inhibitors (PInv/PMEI)] were identified. Eleven nucleotides and nucleoside-related metabolites, and eight fatty acids and sphingolipid-related metabolites were identified in the metabolomics study. To our knowledge, this is the first report of root proteome-level changes and associated alterations in metabolites during the early stages of P. brassicae infection in B. napus.

Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1.

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrP(c) into a pathogenic isoform PrP(sc). Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrP(c)) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrP(c). POM1 Fab forms a 1:1 complex with huPrP(c) and the measured K(d) of 4.5 × 10(-7) M reveals moderately strong binding between them. Structural comparisons have been made among three prion-antibody complexes: POM1 Fab-huPrP(c), ICSM18 Fab-huPrP(c) and VRQ14 Fab-ovPrP(c). The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion protein in vivo. However, both of the glycosylation sites on huPrP(c) are positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion protein in vivo.

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus).

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

Early-stage responses to Plasmodiophora brassicae at the transcriptome and metabolome levels in clubroot resistant and susceptible oilseed Brassica napus.

Clubroot, a devastating soil-borne root disease, in Brassicaceae is caused by Plasmodiophora brassicae Woronin (P. brassicae W.), an obligate biotrophic protist. Plant growth and development, as well as seed yield of Brassica crops, are severely affected due to this disease. Several reports described the molecular responses of B. napus to P. brassicae; however, information on the early stages of pathogenesis is limited. In this study, we have used transcriptomics and metabolomics to characterize P. brassicae pathogenesis at 1-, 4-, and 7-days post-inoculation (DPI) in clubroot resistant (CR) and susceptible (CS) doubled-haploid (DH) canola lines. When we compared between inoculated and uninoculated groups, a total of 214 and 324 putative genes exhibited differential expression (q-value < 0.05) at one or more time-points in the CR and CS genotypes, respectively. When the inoculated CR and inoculated CS genotypes were compared, 4765 DEGs were differentially expressed (q-value < 0.05) at one or more time-points. Several metabolites related to organic acids (e.g., citrate, pyruvate), amino acids (e.g., proline, aspartate), sugars, and mannitol, were differentially accumulated in roots in response to pathogen infection when the CR and CS genotypes were compared. Several DEGs also corresponded to differentially accumulated metabolites, including pyrroline-5-carboxylate reductase (BnaC04g11450D), citrate synthase (BnaC02g39080D), and pyruvate kinase (BnaC04g23180D) as detected by transcriptome analysis. Our results suggest important roles for these genes in mediating resistance to clubroot disease. To our knowledge, this is the first report of an integrated transcriptome and metabolome analysis aimed at characterizing the molecular basis of resistance to clubroot in canola.

A Proteome-Level Investigation Into Plasmodiophora brassicae Resistance in Brassica napus Canola.

Clubroot of Brassicaceae, an economically important soil borne disease, is caused by Plasmodiophora brassicae Woronin, an obligate, biotrophic protist. This disease poses a serious threat to canola and related crops in Canada and around the globe causing significant losses. The pathogen is continuously evolving and new pathotypes are emerging, which necessitates the development of novel resistant canola cultivars to manage the disease. Proteins play a crucial role in many biological functions and the identification of differentially abundant proteins (DAP) using proteomics is a suitable approach to understand plant-pathogen interactions to assist in the development of gene specific markers for developing clubroot resistant (CR) cultivars. In this study, P. brassicae pathotype 3 (P3H) was used to challenge CR and clubroot susceptible (CS) canola lines. Root samples were collected at three distinct stages of pathogenesis, 7-, 14-, and 21-days post inoculation (DPI), protein samples were isolated, digested with trypsin and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. A total of 937 proteins demonstrated a significant (q-value < 0.05) change in abundance in at least in one of the time points when compared between control and inoculated CR-parent, CR-progeny, CS-parent, CS-progeny and 784 proteins were significantly (q < 0.05) changed in abundance in at least in one of the time points when compared between the inoculated- CR and CS root proteomes of parent and progeny across the three time points tested. Functional annotation of differentially abundant proteins (DAPs) revealed several proteins related to calcium dependent signaling pathways. In addition, proteins related to reactive oxygen species (ROS) biochemistry, dehydrins, lignin, thaumatin, and phytohormones were identified. Among the DAPs, 73 putative proteins orthologous to CR proteins and quantitative trait loci (QTL) associated with eight CR loci in different chromosomes including chromosomes A3 and A8 were identified. Proteins including BnaA02T0335400WE, BnaA03T0374600WE, BnaA03T0262200WE, and BnaA03T0464700WE are orthologous to identified CR loci with possible roles in mediating clubroot responses. In conclusion, these results have contributed to an improved understanding of the mechanisms involved in mediating response to P. brassicae in canola at the protein level.

Proteomic analysis of egg white proteins during storage.

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white-shell eggs were used in the present study to determine the proteome-level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22 ± 2 °C) when the maximum number of proteome-level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.

Functional characterization of four APETALA2-family genes (RAP2.6, RAP2.6L, DREB19 and DREB26) in Arabidopsis.

APETALA2 (AP2) transcription factors (TFs) play very important roles in plant growth and development and in defense response. Here, we report functional characterization of four AP2 TF family genes [(RAP2.6 (At1g43160), RAP2.6L (At5g13330), DREB 26 (At1g21910) and DREB19 (At2g38340)] that were identified among NaCl inducible transcripts in abscisic acid responsive 17 (ABR17) transgenic Arabidopsis in our previous microarray analyses. DREB19 and DREB26 function as transactivators and localize in the nucleus. All four genes were abundant in early vegetative and flowering stages, although the magnitude of the expression varied. We observed tissue specific expression patterns for RAP2.6, RAP2.6L, DREB19 and DREB26 in flowers and other organs. RAP2.6 and RAP2.6L were responsive to stress hormones like jasmonic acid, salicylic acid, abscisic acid and ethylene in addition to salt and drought. DREB19 and DREB26 were less responsive to stress hormones, but the former was highly responsive to salt, heat and drought. Overexpression of RAP2.6 in Arabidopsis resulted in a dwarf phenotype with extensive secondary branching and small siliques, and DREB26 overexpression resulted in deformed plants. However, overexpression of RAP2.6L and DREB19 enhanced performance under salt and drought stresses without affecting phenotype. In summary, we have demonstrated that RAP2.6, RAP2.6L, DREB26 and DREB19 are transactivators, they exhibit tissue specific expression, and they participate in plant developmental processes as well as biotic and/or abiotic stress signaling. It is possible that the results from this study on these transcription factors, in particular RAP2.6L and DREB19, can be utilized in developing salt and drought tolerant plants in the future.

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody.

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, ß = 95.6°.

Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: a host-specific toxin from the fungal pathogen Pyrenophora tritici-repentis.

Tan spot, caused by Pyrenophora tritici-repentis, is an important foliar disease of wheat. The fungus produces the host-specific, chlorosis-inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48-72 h. Analysis by 2-DE revealed a total of 102 protein spots with significantly altered intensities 12-36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer-treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.

The proteome of liquid Sclerotial exudates from Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) is a necrotrophic plant pathogen that is capable of infecting more than 400 plant species worldwide. The sclerotium plays important roles in the disease and fungal life cycles. The exudation of liquid droplets is a common feature during sclerotial development, but little is known regarding the nature of these exudates. A proteome-level study was performed in order to gain a better understanding of the types of proteins present in the exudates. Fifty-six proteins were identified and classified into several functional categories, including amino acid metabolism, carbohydrate metabolism, lipid and secondary metabolism, as well as energy, signal transduction, and those with unknown functions. The roles of the identified proteins are discussed within the context of sclerotial development and fungal virulence. Our results may facilitate additional studies aimed at characterizing the function of these proteins in the formation of sclerotia and the life cycle of S. sclerotiorum.