A gene expression microarray for Nicotiana benthamiana based on de novo transcriptome sequence assembly
ABSTRACT: Gene expression microarray was designed, based on two recently published versions of the N. benthamiana transcriptome (v.3 and v.5). Based on homology searches and hybridization tests we identified the sense strand in 106,684 transcriptome contigs and used this information to design a Nb-105k microarray on Agilent eArray platform. By the hybridization of N. benthamiana roots vs leaves RNA samples we showed high specificity and sensitivity of the new microarray in detection of differentially expressed transcripts. Agilent microarrays in 2x105k format (two microarrays per slide) were used for hybridization of labeled RNA from leaves vs roots of N. benthamiana plants. Four biological replicates were used for two-color design in a dye-swap manner.
Project description:Size fractionated small RNA from total RNA extracts of Cicer arietinum leaves and from Nicotiana benthamiana infected by Cymbidium ringspot virus were mixed in a ratio of 1000 to 1 in amount, respectively. The RNA was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit Please see www.illumina.com for details of the sequencing technology. Short RNA fractionation and characterization
Project description:• A comparison of the transcriptomes of russeted vs. waxy apple exocarps previously highlighted a tight relationship between a gene encoding a MYB-type transcription factor, MdMYB93, and some key suberin biosynthetic genes. The present work assesses the role of this transcription factor in the suberization process. • A phylogenetic analysis of MdMYB93 and Arabidopsis thaliana MYBs was performed and the function of MdMYB93 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight the MdMYB93-regulated genes. UPLC-TripleTOF and GC-MS were used to investigate alterations in phenylpropanoid, soluble free lipid, and lipid polyester contents. • A massive accumulation of suberin and its biosynthetic precursors in MdMYB93 agro-infiltrated leaves was accompanied by a remobilization of phenylpropanoids and an increased amount of lignin precursors. Gene expression profiling displayed a concomitant alteration of lipid and phenylpropanoid metabolism, cell wall development, and extracellular transport, with a large number of induced transcripts predicted to be involved in suberin deposition. • The present work supports a major role of MdMYB93 in the regulation of suberin deposition in russeted apple skins, from the synthesis of monomeric precursors, their transport, polymerization, and final deposition as suberin in primary cell wall. In order to draw a consistent picture of a gene expression profile of the MYB93 induced was performed comparing of 4 biological replicates of Nicotiana benthamiana leaves infiltrated with Pearleygate103 35S::MdMYB93 and 4 biological replicates of control plants infiltrated with P19 only.
Project description:To investigate the mechanism under the cross protection between HLSV and TMV, microarray analysis was conducted to examine the transcriptional levels of global host genes during cross protection, using Tobacco Gene Expression Microarray, 4x44k slides. The transcriptional level of some host genes corresponded to accumulation level of TMV. Some host genes were up-regulated only by HLSV. The gene expression in Nicotiana benthamiana during cross protection was measured at 12 days post HLSV or mock buffer inoculated as well as TMV infected N. benthamiana which pre-inoculated HLSV or mock bufer at 3 dpi (which is equal to 15 dpi by HLSV).
Project description:Small RNA expression from Nicotiana benthamiana leaves was profiled with the primary goal of ascertaining microRNA isoform diversity for known, conserved families. A secondary goal was to provide a baseline small RNA expression atlas for this species and tissue. Two biological replicates of leaves from growth-room grown plants. The two libraries were each run twice on different runs, so there are a total of four datasets. For most analyses, it will be appropriate to combine the run1 and run2 versions for the libraries.
Project description:We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation-based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs were the same in different plant species and in the absence of RDR6. We used the TerminatorTM 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were not complementary to highly abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double-stranded RNA and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double-stranded RNA or by RNA-dependent RNA polymerase. Size-fractionated (19-24 nt) small RNA from early systemic leaves of CymRSV-infected Nicotiana benthamiana total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification, the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.
Project description:Drought is one of the major factor that limits crop production and reduces yield. To understand the early response of plants under nearly natural conditions, pepper plants were grown in a greenhouse and drought stressed by withholding water for one week. Plants adapted to the decreasing water content of the substrate by adjustment of their osmotic potential in roots by accumulation of raffinose, glucose, galactinol and proline. In contrast in leaves levels of fructose, sucrose and also galactinol increased. Due to the water deficit cadaverine, putrescine, spermidine and spermine accumulated in leaves whereas the concentration of polyamines was reduced in roots. These polyamines are suggested to rather act as stress protectants than for osmotic adjustment. To understand the molecular basis of the response to this early drought stress better, four suppression subtractive hybridisation libraries from leaves and roots were constructed. Microarray technique was used to identify differentially expressed genes. A total of 109 unique ESTs were detected. The diversity of the putative functions of all identified genes confirms the complexity of the plant response to drought stress. Keywords: Transcription profiling Two-condition experiment in roots and leaves, control leaves (CL) vs. drought-stressed leaves (DL) and control roots (CR) vs. drought-stressed roots (DR). Biological replicates: 4 control (1-4), drought-stressed (1-4), independently grown and harvested. One swap replicate per array.
Project description:Phosphorus, in its orthophosphate form (Pi), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole genome molecular mechanisms contributing to plant acclimation to Pi deficiency remain largely unknown. White lupin (Lupinus albus L.) has evolved unique adaptations for growth in Pi deficient soils including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to Pi supply. We de novo assembled 277,224,180 Illumina reads from 12 cDNA libraries to build the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences 50,734 were transcriptionally active (RPKM = 3) representing approximately 7.8% of the Lupinus albus genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to Pi deficiency with a = 2-fold change and a p-value = 0.05. Twelve sequences were consistently differentially expressed due to Pi deficiency stress in three species, making them ideal candidates to monitor the Pi status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in Pi deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to Pi deficiency. Examination of 2 different tissue types (roots and leaves) under phosphorus (P) -sufficient or P-deficient condition with 3 biological replications per condition in white lupin (Lupinus albus).
Project description:We monitored by RNAseq the transcriptomic response of roots and leaves of Triticum aestivum cv chinese Spring during a long term interaction with Funneliformis mossae (2 months) with or without a pathogen infection by infiltration of Xanthomonas translucens CFBP 2054. The control condition of roots and leaves wheat without mycorhizal fungi is in E-MTAB-5891 (material produced simultaneously and treated at the same time).