ena-STUDY-The James Hutton Institute-09-01-2018-10:27:31:474-82
Aphid RNAseq host, non-host and artificial diet for Rhopalosiphum padi and Myzus persicae genotype O (clonal lines) at time points 3 hours and 24 hours.
To determine the extent transcriptional plasticity contributes to aphid interactions with host and nonhost plants, we sequenced the transcriptome of R. padi and M. persicae after feeding on an artificial diet for 3 or 24 hours, a host plant for 3 or 24 hours, and a non-host plant for 3 or 24 hours. For R. padi, barley is considered a host and Arabidopsis is considered non-host (Jaouannet, 2015). For M. persicae, Arabidopsis is considered host and barley is considered non-host (Escudero-Martinez, 2017). For both species, the artificial diet consisted of 15 % sucrose, 100 mM L-serine, 100 mM L-methionine and 100 mM L-aspartic acid with a pH of 7.2 (KOH) (Will, 2012).Barley plants (cv Optic) were pre-germinated in Petri dishes with wet filter paper for three days in the dark. Plants were moved to a growth room and grown for 7 days prior to aphid infestation. Arabidopsis plants were sown directly in soil and grown for 5 weeks prior to aphid infestation. Artificial diets were prepared and placed between Parafilm sheets according to (Thorpe, 2016). All plants were grown under 8 hours of light (125 µmol photons/m2.s), at 22 °C and 70% humidity. For transfer of R. padi and M. persicae aphids from stock plants to barley and Arabidopsis, 15 mixed-aged apterous aphids were enclosed in a single clip cage, with one clip cage per plant, and 6 plants per plant-aphid combination per time point (3h and 24h). The clip cage was placed in the middle of the 1st leaf for barley, and covering 1-2 fully expanded leaves for Arabidopsis. For the artificial diet treatment, 100 mixed-aged apterous aphids were used per time point in a single artificial diet container. All aphids were collected 3h and 24h after exposure to plants or diet and flash frozen in liquid nitrogen, and aphids from the 6 individual plants per plant-aphid combination per timepoint were pooled into one single tube. In total, 5 independent biological replicates were performed of the whole experiment. Individual replicates were set up at the same time of day to avoid variability due to the aphid or plant circadian cycle. Host and non-host plants treatments were set up in sequential weeks at approximately 9:00h, with the 3h timepoint collected at 12:00h the same day, and the 24h timepoint at approximately 9:00h the next day. Artificial diet treatments were not set-up in parallel to the plant treatments, but on consecutive days, between 11:00 and 12:00h, with collection of the 3h timepoint occurring between 13:00 to 13:30h the same day, and collection of the 24h timepoint between 11:00 to 12:00h the next day.RNA was extracted from 70-90 aphids with the Qiagen RNeasy Plant Mini Kit® following the manufacturer's protocol. RNA quality was assessed using agarose gel electrophoresis and the Agilent 2100 Bioanalyzer. Approximately, 2.5 µg of total RNA per sample (60 samples total) was submitted to TGAC (The Genome Analysis Centre, Norwich Research Park) for Illumina TrueSeq library preparation and sequencing (100 bp paired end).
To determine the extent transcriptional plasticity contributes to aphid interactions with host and nonhost plants, we sequenced the transcriptome of R. padi and M. persicae after feeding on an artificial diet for 3 or 24 hours, a host plant for 3 or 24 hours, and a non-host plant for 3 or 24 hours. For R. padi, barley is considered a host and Arabidopsis is considered non-host (Jaouannet, 2015). For M. persicae, Arabidopsis is considered host and barley is considered non-host (Escudero-Martinez, 2017). For both species, the artificial diet consisted of 15 % sucrose, 100 mM L-serine, 100 mM L-methionine and 100 mM L-aspartic acid with a pH of 7.2 (KOH) (Will, 2012).Barley plants (cv Optic) were pre-germinated in Petri dishes with wet filter paper for three days in the dark. Plants were moved to a growth room and grown for 7 days prior to aphid infestation. Arabidopsis plants were sown directly in soil and grown for 5 weeks prior to aphid infestation. Artificial diets were prepared and placed between Parafilm sheets according to (Thorpe, 2016). All plants were grown under 8 hours of light (125 µmol photons/m2.s), at 22 °C and 70% humidity. For transfer of R. padi and M. persicae aphids from stock plants to barley and Arabidopsis, 15 mixed-aged apterous aphids were enclosed in a single clip cage, with one clip cage per plant, and 6 plants per plant-aphid combination per time point (3h and 24h). The clip cage was placed in the middle of the 1st leaf for barley, and covering 1-2 fully expanded leaves for Arabidopsis. For the artificial diet treatment, 100 mixed-aged apterous aphids were used per time point in a single artificial diet container. All aphids were collected 3h and 24h after exposure to plants or diet and flash frozen in liquid nitrogen, and aphids from the 6 individual plants per plant-aphid combination per timepoint were pooled into one single tube. In total, 5 independent biological replicates were performed of the whole experiment. Individual replicates were set up at the same time of day to avoid variability due to the aphid or plant circadian cycle. Host and non-host plants treatments were set up in sequential weeks at approximately 9:00h, with the 3h timepoint collected at 12:00h the same day, and the 24h timepoint at approximately 9:00h the next day. Artificial diet treatments were not set-up in parallel to the plant treatments, but on consecutive days, between 11:00 and 12:00h, with collection of the 3h timepoint occurring between 13:00 to 13:30h the same day, and collection of the 24h timepoint between 11:00 to 12:00h the next day.RNA was extracted from 70-90 aphids with the Qiagen RNeasy Plant Mini Kit® following the manufacturer's protocol. RNA quality was assessed using agarose gel electrophoresis and the Agilent 2100 Bioanalyzer. Approximately, 2.5 µg of total RNA per sample (60 samples total) was submitted to TGAC (The Genome Analysis Centre, Norwich Research Park) for Illumina TrueSeq library preparation and sequencing (100 bp paired end).