Project description:Fatty acid-amino acid conjugates (FACs) in the oral secretion (OS) of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) in N. attenuata. The second fully expanded leaf of rosette stage plants were wounded by rolling a fabric pattern wheel three times on each side of the midvein and the wounds were immediately supplied with either 20 uL of 0.02% (v/v) Tween-20/water (solvent control) or 10 uL of synthetic N-linolenoyl-glutamic acid (18:3-Glu; 0.03 nmol/µL in 0.02% (v/v) Tween-20/water). Tissue was collected after 30 min of the treatments and frozen in liquid nitrogen. Total RNA was extracted by the phenol/chloroform-LiCl method and poly(A)+-RNA was purified from total RNA. Subsequent steps for the construction of the SuperSAGE libraries and 454 sequencing were performed as previously described using double NlaIII digestions (Molina et al., 2008, BMC Genomics, 9:553). Two SuperSAGE libraries were generated after wounding and FAC elicitation of Nicotiana attenuata leaves. Leaves were harvested after 30 min of the treatments, total RNA extracted, polyA+ mRNA isolated and used for cDNA synthesis and SuperSAGE analysis as described my Matsumura et al (2003).
Project description:Fatty acid-amino acid conjugates (FACs) in the oral secretion (OS) of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) in N. attenuata. The second fully expanded leaf of rosette stage plants were wounded by rolling a fabric pattern wheel three times on each side of the midvein and the wounds were immediately supplied with either 20 uL of 0.02% (v/v) Tween-20/water (solvent control) or 10 uL of synthetic N-linolenoyl-glutamic acid (18:3-Glu; 0.03 nmol/µL in 0.02% (v/v) Tween-20/water). Tissue was collected after 30 min of the treatments and frozen in liquid nitrogen. Total RNA was extracted by the phenol/chloroform-LiCl method and poly(A)+-RNA was purified from total RNA. Subsequent steps for the construction of the SuperSAGE libraries and 454 sequencing were performed as previously described using double NlaIII digestions (Molina et al., 2008, BMC Genomics, 9:553).
Project description:Identification of transcripts that change expression in roots of Nicotiana attenuata plants with reduced expression of HSPRO and in association with Piriformospora indica. Gene expression in roots of Nicotiana attenuata wild type and ir-hspro seedlings was measured at 14 days after inoculation with Piriformospora indica. Three independent experiments were performed with wild type plants and three independent experiments were performed with ir-hspro plants.
