David Kenneth Berger

Role of integration host factor in stimulating transcription from the σ54-dependent nifH promoter☆

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.

SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus

BackgroundNAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored.ResultsWe overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area.ConclusionsThis study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.

Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis

Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R2>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.

Comparative analysis of orthologous cellulose synthase promoters from Arabidopsis, Populus and Eucalyptus : evidence of conserved regulatory elements in angiosperms

* The cellulose synthase (CesA) gene family encodes the catalytic subunits of a large protein complex responsible for the deposition of cellulose into plant cell walls. Early in vascular plant evolution, the gene family diverged into distinct members with conserved structures and functions (e.g. primary or secondary cell wall biosynthesis). Although the functions and expression domains of CesA genes have been extensively studied in plants, little is known about transcriptional regulation and promoter evolution in this gene family. * Here, comparative sequence analysis of orthologous CesA promoters from three angiosperm genera, Arabidopsis, Populus and Eucalyptus, was performed to identify putative cis-regulatory sequences. The promoter sequences of groups of Arabidopsis genes that are co-expressed with the primary or secondary cell wall-related CesA genes were also analyzed. * Reporter gene analysis of newly isolated promoter regions of six E. grandis CesA genes in Arabidopsis revealed the conserved functionality of the promoter sequences. Comparative sequence analysis identified 71 conserved sequence motifs, of which 66 were significantly over-represented in either primary or secondary wall-associated promoters. * The presence of conserved cis-regulatory elements in the evolutionary distant CesA promoters of Arabidopsis, Populus and Eucalyptus suggests an ancient transcriptional network regulating cellulose biosynthesis in vascular plants.

MADIBA: A web server toolkit for biological interpretation of Plasmodium and plant gene clusters

BackgroundMicroarray technology makes it possible to identify changes in gene expression of an organism, under various conditions. Data mining is thus essential for deducing significant biological information such as the identification of new biological mechanisms or putative drug targets. While many algorithms and software have been developed for analysing gene expression, the extraction of relevant information from experimental data is still a substantial challenge, requiring significant time and skill.DescriptionMADIBA (MicroArray Data Interface for Biological Annotation) facilitates the assignment of biological meaning to gene expression clusters by automating the post-processing stage. A relational database has been designed to store the data from gene to pathway for Plasmodium, rice and Arabidopsis. Tools within the web interface allow rapid analyses for the identification of the Gene Ontology terms relevant to each cluster; visualising the metabolic pathways where the genes are implicated, their genomic localisations, putative common transcriptional regulatory elements in the upstream sequences, and an analysis specific to the organism being studied.ConclusionMADIBA is an integrated, online tool that will assist researchers in interpreting their results and understand the meaning of the co-expression of a cluster of genes. Functionality of MADIBA was validated by analysing a number of gene clusters from several published experiments – expression profiling of the Plasmodium life cycle, and salt stress treatments of Arabidopsisand rice. In most of the cases, the same conclusions found by the authors were quickly and easily obtained after analysing the gene clusters with MADIBA.

The identification and differential expression of Eucalyptus grandis pathogenesis-related genes in response to salicylic acid and methyl jasmonate

Two important role players in plant defence response are the phytohormones salicylic acid (SA) and jasmonic acid (JA); both of which have been well described in model species such as Arabidopsis thaliana. Several pathogenesis related (PR) genes have previously been used as indicators of the onset of SA and JA signaling in Arabidopsis. This information is lacking in tree genera such as Eucalyptus. The aim of this study was to characterize the transcriptional response of PR genes (EgrPR2, EgrPR3, EgrPR4, EgrPR5, and EgrLOX) identified in Eucalyptus grandis to SA and methyl jasmonate (MeJA) treatment as well as to qualify them as diagnostic for the two signaling pathways. Using the genome sequence of E. grandis, we identified candidate Eucalyptus orthologs EgrPR2, EgrPR3, EgrPR4, EgrPR5, and EgrLOX based on a co-phylogenetic approach. The expression of these genes was investigated after various doses of SA and MeJA (a derivative of JA) treatment as well as at various time points. The transcript levels of EgrPR2 were decreased in response to high concentrations of MeJA whereas the expression of EgrPR3 and EgrLOX declined as the concentrations of SA treatment increased, suggesting an antagonistic relationship between SA and MeJA. Our results support EgrPR2 as potentially diagnostic for SA and EgrPR3, EgrPR4, and EgrLOX as indicators of MeJA signaling. To further validate the diagnostic potential of the PR genes we challenged E. grandis clones with the fungal necrotrophic pathogen Chrysoporthe austroafricana. The tolerant clone showed high induction of EgrPR2 and decreased transcript abundance of EgrPR4. Pre-treatment of the susceptible genotype with 5 mM SA resulted in lesion lengths comparable to the tolerant genotype after artificial inoculation with C. austroafricana. Thus expression profiling of EgrPR2 and EgrPR4 genes could serve as a useful diagnostic approach to determine which of the two signaling pathways are activated against various pathogens in Eucalyptus.

Mapping QTL conferring resistance in maize to gray leaf spot disease caused by Cercospora zeina

BackgroundGray leaf spot (GLS) is a globally important foliar disease of maize. Cercospora zeina, one of the two fungal species that cause the disease, is prevalent in southern Africa, China, Brazil and the eastern corn belt of the USA. Identification of QTL for GLS resistance in subtropical germplasm is important to support breeding programmes in developing countries where C. zeina limits production of this staple food crop.ResultsA maize RIL population (F7:S6) from a cross between CML444 and SC Malawi was field-tested under GLS disease pressure at five field sites over three seasons in KwaZulu-Natal, South Africa. Thirty QTL identified from eleven field trials (environments) were consolidated to seven QTL for GLS resistance based on their expression in at least two environments and location in the same core maize bins. Four GLS resistance alleles were derived from the more resistant parent CML444 (bin 1.10, 4.08, 9.04/9.05, 10.06/10.07), whereas the remainder were from SC Malawi (bin 6.06/6.07, 7.02/7.03, 9.06). QTLs in bin 4.08 and bin 6.06/6.07 were also detected as joint QTLs, each explained more than 11% of the phenotypic variation, and were identified in four and seven environments, respectively. Common markers were used to allocate GLS QTL from eleven previous studies to bins on the IBM2005 map, and GLS QTL “hotspots” were noted. Bin 4.08 and 7.02/7.03 GLS QTL from this study overlapped with hotspots, whereas the bin 6.06/6.07 and bin 9.06 QTLs appeared to be unique. QTL for flowering time (bin 1.07, 4.09) in this population did not correspond to QTL for GLS resistance.ConclusionsQTL mapping of a RIL population from the subtropical maize parents CML444 and SC Malawi identified seven QTL for resistance to gray leaf spot disease caused by C. zeina. These QTL together with QTL from eleven studies were allocated to bins on the IBM2005 map to provide a basis for comparison. Hotspots of GLS QTL were identified on chromosomes one, two, four, five and seven, with QTL in the current study overlapping with two of these. Two QTL from this study did not overlap with previously reported QTL.

Gene-for-Gene Tolerance to Bacterial Wilt in Arabidopsis

Bacterial wilt caused by Ralstonia solanacearum is a disease of widespread economic importance that affects numerous plant species, including Arabidopsis thaliana. We describe a pathosystem between A. thaliana and biovar 3 phylotype I strain BCCF402 of R. solanacearum isolated from Eucalyptus trees. A. thaliana accession Be-0 was susceptible and accession Kil-0 was tolerant. Kil-0 exhibited no wilting symptoms and no significant reduction in fitness (biomass, seed yield, and germination efficiency) after inoculation with R. solanacearum BCCF402, despite high bacterial numbers in planta. This was in contrast to the well-characterized resistance response in the accession Nd-1, which limits bacterial multiplication at early stages of infection and does not wilt. R. solanacearum BCCF402 was highly virulent because the susceptible accession Be-0 was completely wilted after inoculation. Genetic analyses, allelism studies with Nd-1, and RRS1 cleaved amplified polymorphic sequence marker analysis showed that the tolerance phenotype in Kil-0 was dependent upon the resistance gene RRS1. Knockout and complementation studies of the R. solanacearum BCCF402 effector PopP2 confirmed that the tolerance response in Kil-0 was dependent upon the RRS1-PopP2 interaction. Our data indicate that the gene-for-gene interaction between RRS1 and PopP2 can contribute to tolerance, as well as resistance, which makes it a useful model system for evolutionary studies of the arms race between plants and bacterial pathogens. In addition, the results alert biotechnologists to the risk that deployment of RRS1 in transgenic crops may result in persistence of the pathogen in the field.

Quantitative phenotyping of grey leaf spot disease in maize using real-time PCR

Grey leaf spot is an important maize foliar disease caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina. Although methods exist to detect these Cercospora species in maize, current techniques do not allow quantification of the fungi in planta. We developed a real-time SYBR® Green PCR assay for quantification of grey leaf spot disease in maize based on the amplification of a fragment of a cytochrome P450 reductase (cpr1) gene. In planta fungal DNA content was normalised to a maize glutathione S-transferase III gene (gst3) to yield values of ng Cercospora DNA/mg maize DNA. The assay was specific to the two Cercospora spp., and we observed no amplification of the cpr1 fragment in non-target maize leaf pathogens or saprophytes. The assay was employed to quantify C. zeina in glasshouse inoculated maize plants and grey leaf spot infected field plants of resistant and susceptible maize lines. In both instances, C. zeina DNA content correlated with symptomatic leaf lesion area, and the susceptible maize line contained significantly more C. zeina DNA than the resistant line. Sequence differences between the C. zeina and C. zeae-maydis cpr1 amplicons enabled us to perform melt curve analyses to identify the Cercospora species causing grey leaf spot at a particular location. This assay has application in the early detection and quantification of Cercospora spp., both of which are important tools in grey leaf spot disease management and maize breeding programmes.

Investigating the molecular underpinnings underlying morphology and changes in carbon partitioning during tension wood formation in Eucalyptus

Tension wood has distinct physical and chemical properties, including altered fibre properties, cell wall composition and ultrastructure. It serves as a good system for investigating the genetic regulation of secondary cell wall biosynthesis and wood formation. The reference genome sequence for Eucalyptus grandis allows investigation of the global transcriptional reprogramming that accompanies tension wood formation in this global wood fibre crop. We report the first comprehensive analysis of physicochemical wood property changes in tension wood of Eucalyptus measured in a hybrid (E. grandis × Eucalyptus urophylla) clone, as well as genome-wide gene expression changes in xylem tissues 3 wk post-induction using RNA sequencing. We found that Eucalyptus tension wood in field-grown trees is characterized by an increase in cellulose, a reduction in lignin, xylose and mannose, and a marked increase in galactose. Gene expression profiling in tension wood-forming tissue showed corresponding down-regulation of monolignol biosynthetic genes, and differential expression of several carbohydrate active enzymes. We conclude that alterations of cell wall traits induced by tension wood formation in Eucalyptus are a consequence of a combination of down-regulation of lignin biosynthesis and hemicellulose remodelling, rather than the often proposed up-regulation of the cellulose biosynthetic pathway.