Project description:Reference: De Vos and Jander, 2009 - Plant, Cell, and Environment Aim: Identification of genes responding to aphid saliva. Background: During feeding on phloem sap aphids repeatedly salivate into the sieve element. It is thought that compounds in aphid saliva play a role in sustainable feeding. These compounds may include proteins and small molecules, which can function as virulence factors. Growth conditions Plants: Seeds of wild-type Arabidopsis thaliana (Col-0) were obtained from the were kept in 0.1% Phytagar (Invitrogen, Carlsbad, CA) for 24 h at 4°C prior to planting on Cornell mix with Osmocoat fertilizer. Plants were grown in Conviron growth chambers in 20- x 40-cm nursery flats at a photosynthetic photon flux density of 200 mmol m-2 s-1 and a 16-h photoperiod. The temperature in the chambers was 23°C and the relative humidity was 50%. Plants were grown for 3 weeks and used in experiments before flowering. Aphids: All experiments were conducted with a tobacco-adapted red lineage of M. persicae. Aphids were raised on cabbage (Brassica oleracea) with a 16-h day (150 mmol m-2 s-1 at 24°C) and an 8-h night (19°C) at 50% relative humidity. Experimental set-up/treatment: Fifty aphids were allowed to feed from 50 µL artificial diet, containing sucrose and amino acids (Kim and Jander, 2007) between two layers of Parafilm. After 24 h, artificial diet from 20 aphids and control (0 aphids) diet cups was collected and infiltrated into leaves of intact Arabidopsis plants using a 1-mL syringe without the needle. Plants for control diet and aphid saliva containing diet were grown in the same pot to allow for a paired comparison. Eighteen leaves (3 leaves from 6 plants) treated with control and aphid saliva containing diet were harvested and immediately frozen in liquid nitrogen. This experiment was repeated 3 times to function as independent biological replicates. RNA extraction + processing: RNA was extracted using the Qiagen Plant RNeasy kit. RNA quality and quantity was assessed with an Agilent BioAnalyser 2100. Samples were processed by the Cornell Microarray facility. Whole genome gene expression profiling was done using Affymetrix ATH1 GeneChips. Data analysis: Raw data from the microarrays was normalized at probe-level using gcRMA algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system. Significance of gene expression was determined using the LIMMA (Smyth, 2004) program and raw p values of multiple tests were corrected using False Discovery Rate (FDR).

Project description:In most aphid species, facultative parthenogenetic reproduction allows rapid growth and formation of large single-genotype colonies. Upon predator attack, individual aphids emit an alarm pheromone to warn the colony of this danger. (E)-beta-farnesene (EBF) is the predominant constituent of the alarm pheromone in Myzus persicae (green peach aphid) and many other aphid species. Continuous exposure to alarm pheromone in aphid colonies raised on transgenic Arabidopsis thaliana plants that produce EBF leads to habituation of the aphid population. Whereas naïve aphids are repelled by EBF, habituated aphids show no avoidance response. Individual aphids from the habituated colony can revert back to being EBF-sensitive in three generations, indicating that this behavioral change is not caused by a genetic mutation. Instead, DNA microarray experiments comparing gene expression in naïve and habituated aphids treated with EBF demonstrate an almost complete desensitization in the transcriptional response to EBF. Furthermore, EBF-responsive aphids, but not habituated aphids show significantly lower reproduction in the presence of EBF. Although both naïve and habituated aphids emit EBF upon damage, EBF-responsive aphids display a higher survival rate in the presence of coccinellid predators and thus outperform habituated aphids that do not show an avoidance response. These results provide direct evidence that aphid perception of conspecific alarm pheromone aids in predator avoidance and thereby bestows fitness benefits in survivorship and fecundity. Although habituated M. persicae have equal fecundity on control and EBF-producing plants, such transgenics may have practical applications in agriculture because of increased predation of habituated aphids. Log fold-changes (LogFC) were computed and contigs with P-values ≤ 0.05 were considered to be differentially expressed (see Supplementary file at foot of this record). Agilent 8x15K array previously reported by Ramsey et al., 2007 - BMC Genomics 8: 243. Two-condition experiment using Alexa Fluor 555 and 647 dyes. Biological replicates: 4 control replicates, 4 treated replicates.

Project description:Reference: De Vos and Jander, 2009 - Plant, Cell, and Environment Aim: Identification of genes responding to aphid saliva. Background: During feeding on phloem sap aphids repeatedly salivate into the sieve element. It is thought that compounds in aphid saliva play a role in sustainable feeding. These compounds may include proteins and small molecules, which can function as virulence factors. Growth conditions Plants: Seeds of wild-type Arabidopsis thaliana (Col-0) were obtained from the were kept in 0.1% Phytagar (Invitrogen, Carlsbad, CA) for 24 h at 4°C prior to planting on Cornell mix with Osmocoat fertilizer. Plants were grown in Conviron growth chambers in 20- x 40-cm nursery flats at a photosynthetic photon flux density of 200 mmol m-2 s-1 and a 16-h photoperiod. The temperature in the chambers was 23°C and the relative humidity was 50%. Plants were grown for 3 weeks and used in experiments before flowering. Aphids: All experiments were conducted with a tobacco-adapted red lineage of M. persicae. Aphids were raised on cabbage (Brassica oleracea) with a 16-h day (150 mmol m-2 s-1 at 24°C) and an 8-h night (19°C) at 50% relative humidity. Experimental set-up/treatment: Fifty aphids were allowed to feed from 50 µL artificial diet, containing sucrose and amino acids (Kim and Jander, 2007) between two layers of Parafilm. After 24 h, artificial diet from 20 aphids and control (0 aphids) diet cups was collected and infiltrated into leaves of intact Arabidopsis plants using a 1-mL syringe without the needle. Plants for control diet and aphid saliva containing diet were grown in the same pot to allow for a paired comparison. Eighteen leaves (3 leaves from 6 plants) treated with control and aphid saliva containing diet were harvested and immediately frozen in liquid nitrogen. This experiment was repeated 3 times to function as independent biological replicates. RNA extraction + processing: RNA was extracted using the Qiagen Plant RNeasy kit. RNA quality and quantity was assessed with an Agilent BioAnalyser 2100. Samples were processed by the Cornell Microarray facility. Whole genome gene expression profiling was done using Affymetrix ATH1 GeneChips. Data analysis: Raw data from the microarrays was normalized at probe-level using gcRMA algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system. Significance of gene expression was determined using the LIMMA (Smyth, 2004) program and raw p values of multiple tests were corrected using False Discovery Rate (FDR). 6 samples were used in this experiment

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Myzus persicae Rhopalosiphum padi

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