Microarray expression studies in the model plant Arabidopsis thaliana infected with the bacterial pathogen Ralstonia solanacearum

Ralstonia solanaearum, a soil borne pathogen infects several important crops causing wilting. In 2000-2001, two eucalyptus isolates, BCCF 401 and BCCF 402 were isolated from plantations in Kwa-Zulu Natal and the Democratic Republic of Congo, respectively. Arabidopsis has been recognised as a host for R. solanacearum and as such has been adopted as a model to understand the plant defence response against this pathogen. The aim of this study was to use microarray expression profiling techniques to elucidate the plant defence response and to identify candidate genes possibly contributing towards resistance against the pathogen. As a means to optimise microarray expression profiling, the differential expression in an Arabidopsis mutant, cir1 (constitutively induced resistance 1) and wild-type plants was investigated using a custom 500-probe microarray. Several genes were found to be induced in cir1 at a significance threshold of –log10(p) equal to 3 (p D559/gmThesis (PhD)--University of Pretoria, 2008.Plant Scienceunrestricte

Microarray experiments: Considerations for experimental design

Microarrays are useful tools to investigate the expression of thousands of genes rapidly. Some researchers remain reluctant to use the technology, however, largely because of its expense. Careful design of a microarray experiment is key to generating cost-effective results. This article explores issues that researchers face when embarking on a microarray experiment for the first time. These include decisions about which microarray platform is available for the organism of interest, the degree of replication (biological and technical) needed and which design (direct or indirect, loop or balanced block) is suitable

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and Related Family: Mechanistic Insights in Plant Disease Resistance

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and related NPR1-like proteins are a functionally similar, yet surprisingly diverse family of transcription co-factors. Initially, NPR1 in Arabidopsis was identified as a positive regulator of systemic acquired resistance (SAR), paralogs NPR3 and NPR4 were later shown to be negative SAR regulators. The mechanisms involved have been the subject of extensive research and debate over the years, during which time a lot has been uncovered. The known roles of this protein family have extended to include influences over a broad range of systems including circadian rhythm, endoplasmic reticulum (ER) resident proteins and the development of lateral organs. Recently, important advances have been made in understanding the regulatory relationship between members of the NPR1-like protein family, providing new insight regarding their interactions, both with each other and other defense-related proteins. Most importantly the influence of salicylic acid (SA) on these interactions has become clearer with NPR1, NPR3, and NPR4 being considered bone fide SA receptors. Additionally, post-translational modification of NPR1 has garnered attention during the past years, adding to the growing regulatory complexity of this protein. Furthermore, growing interest in NPR1 overexpressing crops has provided new insights regarding the role of NPR1 in both biotic and abiotic stresses in several plant species. Given the wealth of information, this review aims to highlight and consolidate the most relevant and influential research in the field to date. In so doing, we attempt to provide insight into the mechanisms and interactions which underly the roles of the NPR1-like proteins in plant disease responses

Transcriptome and hormone profiling reveals Eucalyptus grandis defence responses against Chrysoporthe austroafricana

BACKGROUND : Eucalyptus species and interspecific hybrids exhibit valuable growth and wood properties that make them a highly desirable commodity. However, these trees are challenged by a wide array of biotic stresses during their lifetimes. The Eucalyptus grandis reference genome sequence provides a resource to study pest and pathogen defence mechanisms in long-lived woody plants. E. grandis trees are generally susceptible to Chrysoporthe austroafricana, a causal agent of stem cankers on eucalypts. The aim of this study was to characterize the defence response of E. grandis against C. austroafricana. RESULTS : Hormone profiling of susceptible and moderately resistant clonal E. grandis genotypes indicated a reduction in salicylic acid and gibberellic acid levels at 3 days post inoculation. We hypothesized that these signaling pathways may facilitate resistance. To further investigate other defence mechanisms at this time point, transcriptome profiling was performed. This revealed that cell wall modifications and response to oxidative stress form part of the defence responses common to both genotypes, whilst changes in the hormone signaling pathways may contribute to resistance. Additionally the expression of selected candidate defence response genes was induced earlier in moderately resistant trees than in susceptible trees, supporting the hypothesis that a delayed defence response may occur in the susceptible interaction. CONCLUSION : The ability of a host to fine-tune its defence responses is crucial and the responses identified in this study extends our understanding of plant defence, gained from model systems, to woody perennials.Additional file 1: Table S1. Summary of statistics obtained for transcriptome profiling of TAG5 and ZG14 challenged with C. austroafricana.Additional file 2: Table S2. Summary of significantly differentially expressed genes and their annotations identified from Eucalyptus grandis TAG5 and ZG14.Additional file 3: Figure S1. Molecular function GO terms that are over-represented in TAG5 and ZG14. a – GO terms within the upregulated dataset. b – GO terms within the down-regulated dataset (all terms for this dataset are shown). The y-axis represents the –log2(q-value) and the x-axis represents the GO terms within the datasets. Light and dark grey bars are ZG14 and TAG5 respectively.Additional file 4: Figure S2. Cellular component GO terms that are over-represented in TAG5 and ZG14. a – GO terms within the upregulated dataset. b – GO terms within the down-regulated dataset. The y-axis represents the –log2(q-value) and the x-axis represents the GO terms within the datasets. Light and dark grey bars are ZG14 and TAG5 respectively.Additional file 5: Table S3. List of differentially expressed genes that are common between the susceptible (ZG14) and moderately resistant (TAG5) host.This work was supported by the Genomics Research Institute (GRI) at the University of Pretoria; the National Research Foundation of South Africa (Grant number NBIG 86936); Thuthuka funding (UID:76225); the Forest Molecular Genetics Programme by Mondi and Sappi and the Technology and Human Resources for Industry Programme (UID:80118).http://www.biomedcentral.com/bmcgenomicsam201

eCALIBRATOR : a comparative tool to identify key genes and pathways for eucalyptus defense against biotic stressors

Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under long-term nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5

Insect gallers and their plant hosts : From omics data to systems biology

Gall-inducing insects are capable of exerting a high level of control over their hosts’ cellular machinery to the extent that the plant’s development,metabolism,chemistry,and physiology are all altered in favour of the insect. Many gallers are devastating pests in global agriculture and the limited understanding of their relationship with their hosts prevents the development of robust management strategies. Omics technologies are proving to be important tools in elucidating the mechanisms involved in the interaction as they facilitate analysis of plant hosts and insect effectors for which little or no prior knowledge exists. In this review,we examine the mechanisms behind insect gall development using evidence from omics-level approaches. The secretion of effector proteins and induced phytohormonal imbalances are highlighted as likely mechanisms involved in gall development. However,understanding how these components function within the system is far from complete and a number of questions need to be answered before this information can be used in the development of strategies to engineer or breed plants with enhanced resistance

The Eucalyptus grandis NBS-LRR gene family : physical clustering and expression hotspots

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.Table S1 Full list of Eucalyptus grandis putative NBS-LRR genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and phylogeny clade membership, identification method, raw expression data, log2 fold change values and ANOVA results (p-values). S_F_C, susceptible, fungal treatment, control; S_F_I, susceptible, fungal treatment, inoculated; R_F_C, resistant, fungal treatment, control; R_F_I, resistant, fungal treatment, inoculated; S_I_C, susceptible, insect treatment, control; S_I_I, susceptible, insect treatment, infested; R_I_C, resistant, insect treatment, control; R_I_I, resistant, insect treatment, infested.Table S2 Conserved amino acid sequences for NB-ARC and TIR motifs from MEME analysis with CNL-like and TNL-like gene models in Eucalyptus grandis (Eg) and Arabidopsis thaliana (At; Meyers et al., 2003). The expected amino acid tryptophan (W) is identified in the Kinase 2 subdomain for CNL sequences–underlined.Figure S1 Neighbor joining tree of 480 Eucalyptus grandis NB-ARC domains from complete NBS-LRR genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 495 amino acid sequences (480 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S2 Neighbor joining tree of 616 Eucalyptus grandis NB-ARC domains from all non-TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 631 amino acid sequences (616 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S3 Neighbor joining tree of 396 Eucalyptus grandis NB-ARC domains from all TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 411 amino acid sequences (396 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S4 The definition of a (A) cluster and a (B) supercluster is illustrated using a region (starting at 13 Mb and ending at 18 Mb) on chromosome 4.Figure S5 Physical locations for all complete, partial, and incomplete NBS-LRR gene models that were expressed under challenge of Chrysoporthe austroafricana and Leptocybe invasa on Eucalyptus grandis chromosomes (Mapchart). Variation in means from treatment (ANOVA) were identified based on significance *p < 0.01, **p < 0.001, ***p < 0.0001 (*** are also underlined) and log2 gene expression ratios greater than 1 or smaller than −1 for resistant and susceptible plants. Color distinguishes between different classes (TNL = pink, CNL = green, NL = red, incomplete NL = black, BLAST homolog non-NL = black). Scale bar = Mb. Cluster and supercluster regions are indicated and E. grandis gene IDs are provided.Figure S6 NB-ARC-LRR fused domains (A) and TIR-NB-ARC-LRR fused domains (B). Conserved amino acid sequences are indicated with lines (top). The GKT (Kinase 1) conserved motif is recognized as a P-loop structure important in ATP hydrolysis while the hDD is also well conserved in NB-ARC domains (Kinase 2) as important in co-ordinating Mg2+ as a co-factor (Tameling et al., 2006). These two important sub-domains of NB-ARC are sometimes termed the Walker A and Walker B motifs (Walker et al., 1982) and are identified as A and B, respectively, within the I-Tasser protein structures (bottom) for a representative CNL (Eucgr.L01363) and TNL (Eucgr.C00020) sequence from the Eucalyptus grandis genome.Top up scholarships were generously provided for PT from the University of Sydney and Rural Industries Research and Development Corporation, Australiahttp://www.frontiersin.orgam2016Genetic

The Eucalyptus grandis NBS-LRR gene family : physical clustering and expression hotspots

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.Table S1 Full list of Eucalyptus grandis putative NBS-LRR genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and phylogeny clade membership, identification method, raw expression data, log2 fold change values and ANOVA results (p-values). S_F_C, susceptible, fungal treatment, control; S_F_I, susceptible, fungal treatment, inoculated; R_F_C, resistant, fungal treatment, control; R_F_I, resistant, fungal treatment, inoculated; S_I_C, susceptible, insect treatment, control; S_I_I, susceptible, insect treatment, infested; R_I_C, resistant, insect treatment, control; R_I_I, resistant, insect treatment, infested.Table S2 Conserved amino acid sequences for NB-ARC and TIR motifs from MEME analysis with CNL-like and TNL-like gene models in Eucalyptus grandis (Eg) and Arabidopsis thaliana (At; Meyers et al., 2003). The expected amino acid tryptophan (W) is identified in the Kinase 2 subdomain for CNL sequences–underlined.Figure S1 Neighbor joining tree of 480 Eucalyptus grandis NB-ARC domains from complete NBS-LRR genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 495 amino acid sequences (480 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S2 Neighbor joining tree of 616 Eucalyptus grandis NB-ARC domains from all non-TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 631 amino acid sequences (616 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S3 Neighbor joining tree of 396 Eucalyptus grandis NB-ARC domains from all TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 411 amino acid sequences (396 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S4 The definition of a (A) cluster and a (B) supercluster is illustrated using a region (starting at 13 Mb and ending at 18 Mb) on chromosome 4.Figure S5 Physical locations for all complete, partial, and incomplete NBS-LRR gene models that were expressed under challenge of Chrysoporthe austroafricana and Leptocybe invasa on Eucalyptus grandis chromosomes (Mapchart). Variation in means from treatment (ANOVA) were identified based on significance *p < 0.01, **p < 0.001, ***p < 0.0001 (*** are also underlined) and log2 gene expression ratios greater than 1 or smaller than −1 for resistant and susceptible plants. Color distinguishes between different classes (TNL = pink, CNL = green, NL = red, incomplete NL = black, BLAST homolog non-NL = black). Scale bar = Mb. Cluster and supercluster regions are indicated and E. grandis gene IDs are provided.Figure S6 NB-ARC-LRR fused domains (A) and TIR-NB-ARC-LRR fused domains (B). Conserved amino acid sequences are indicated with lines (top). The GKT (Kinase 1) conserved motif is recognized as a P-loop structure important in ATP hydrolysis while the hDD is also well conserved in NB-ARC domains (Kinase 2) as important in co-ordinating Mg2+ as a co-factor (Tameling et al., 2006). These two important sub-domains of NB-ARC are sometimes termed the Walker A and Walker B motifs (Walker et al., 1982) and are identified as A and B, respectively, within the I-Tasser protein structures (bottom) for a representative CNL (Eucgr.L01363) and TNL (Eucgr.C00020) sequence from the Eucalyptus grandis genome.Top up scholarships were generously provided for PT from the University of Sydney and Rural Industries Research and Development Corporation, Australiahttp://www.frontiersin.orgam2016Genetic