Transcriptome analysis of Neotyphodium and Epichloë grass endophytes

Large-scale gene discovery has been performed for the grass fungal endophytes Neotyphodium coenophialum, Neotyphodium lolii, and Epichloë festucae. The resulting sequences have been annotated by comparison with public DNA and protein sequence databases and using intermediate gene ontology annotation tools. Endophyte sequences have also been analysed for the presence of simple sequence repeat and single nucleotide polymorphism molecular genetic markers. Sequences and annotation are maintained within a MySQL database that may be queried using a custom web interface. Two cDNA-based microarrays have been generated from this genome resource. They permit the interrogation of 3806 Neotyphodium genes (NchipTM microarray), and 4195 Neotyphodium and 920 Epichloë genes (EndoChipTM microarray), respectively. These microarrays provide tools for high-throughput transcriptome analysis, including genome-specific gene expression studies, profiling of novel endophyte genes, and investigation of the host grass–symbiont interaction. Comparative transcriptome analysis in Neotyphodium and Epichloë was performed

Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum

Background Capsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms. Methodology/Principal Findings We assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR. Conclusion/Significance DE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.

Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes and pathogenesis-related genes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h postinoculation with S. sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S. sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S. sclerotiorum.

Sexual selection in true fruit flies (Diptera: Tephritidae): transcriptome and experimental evidences for phytochemicals increasing male competitive ability

In male tephritid fruit flies of the genus Bactrocera, feeding on secondary plant compounds (sensu lato male lures = methyl eugenol, raspberry ketone and zingerone) increases male mating success. Ingested male lures alter the male pheromonal blend, normally making it more attractive to females and this is considered the primary mechanism for the enhanced mating success. However, the male lures raspberry ketone and zingerone are known, across a diverse range of other organisms, to be involved in increasing energy metabolism. If this also occurs in Bactrocera, then this may represent an additional benefit to males as courtship is metabolically expensive and lure feeding may increase a fly's short-term energy. We tested this hypothesis by performing comparative RNA-seq analysis between zingerone-fed and unfed males of Bactrocera tryoni. We also carried out behavioural assays with zingerone- and cuelure-fed males to test whether they became more active. RNA-seq analysis revealed, in zingerone-fed flies, up-regulation of 3183 genes with homologues transcripts to those known to regulate intermale aggression, pheromone synthesis, mating and accessory gland proteins, along with significant enrichment of several energy metabolic pathways and gene ontology terms. Behavioural assays show significant increases in locomotor activity, weight reduction and successful mating after mounting; all direct/indirect measures of increased activity. These results suggest that feeding on lures leads to complex physiological changes, which result in more competitive males. These results do not negate the pheromone effect, but do strongly suggest that the phytochemical-induced sexual selection is governed by both female preference and male competitive mechanisms.

A transcriptome approach towards understanding the development of ripening capacity in 'Bartlett' pears (Pyrus communis L.)

Background: The capacity of European pear fruit (Pyrus communis L.) to ripen after harvest develops during the final stages of growth on the tree. The objective of this study was to characterize changes in 'Bartlett' pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity. Results: The softening response of pear fruit held for 14days at 20°C after harvest depended on their maturity. We identified four maturity stages: S1-failed to soften and S2- displayed partial softening (with or without ET-ethylene treatment); S3 - able to soften following ET; and S4 - able to soften without ET. Illumina sequencing and Trinity assembly generated 68,010 unigenes (mean length of 911bp), of which 32.8% were annotated to the RefSeq plant database. Higher numbers of differentially expressed transcripts were recorded in the S3-S4 and S1-S2 transitions (2805 and 2505 unigenes, respectively) than in the S2-S3 transition (2037 unigenes). High expression of genes putatively encoding pectin degradation enzymes in the S1-S2 transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. Moreover, the co-expression of these genes with Exps (Expansins) suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth. K-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster K6 that showed the highest gene expression at S3. AP2/EREBP (APETALA 2/ethylene response element binding protein) and bHLH (basic helix-loop-helix) transcripts were enriched in all three transition S1-S2, S2-S3, and S3-S4. Several members of Aux/IAA (Auxin/indole-3-acetic acid), ARF (Auxin response factors), and WRKY appeared to play an important role in orchestrating the S2-S3 transition. Conclusions: We identified maturity stages associated with the development of ripening capacity in 'Bartlett' pear, and described the transcription profile of fruit at these stages. Our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive (S2) to ethylene-responsive (S3), resulting in fruit softening. The transcriptome will be helpful for future studies about specific developmental pathways regulating the transition to ripening. © 2015 Nham et al.