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Effects of silencing green peach aphid (Myzus persicae) genes via RNA interference

Bilgi, Vineeta (2015) Effects of silencing green peach aphid (Myzus persicae) genes via RNA interference. PhD thesis, Murdoch University.

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The green peach aphid (GPA), Myzus persicae (Hemiptera: Aphidae), is a polyphagous insect that feeds on a broad range of more than forty different plant families and also vectors over one hundred plant viruses. Feeding damage, and viral diseases that they transmit, contribute to yield losses in many economically important crops worldwide. The current measures used to control GPAs include genetic resistance and the application of insecticides. Although chemical insecticides provide control and are widely used for managing aphid infestations, they are expensive and some are not environmentally safe. Moreover, insecticides are not always effective because GPAs readily develop resistance. An alternate approach is to exploit the naturally occurring eukaryotic phenomenon of RNA interference (RNAi) to silence vital genes of aphids. RNAi is the sequence-specific degradation of homologous RNA molecules guided by small RNAs and can be triggered by the introduction of double-stranded RNA (dsRNA). This approach is environmentally-friendly, appears to be an effective strategy to silence vital genes of plant pathogens and pests such as nematodes and insects, and could be a competitive alternative to current methods of pest control. However, to develop transgenic plants expressing GPA genes, first suitable gene targets have to be identified and tested for their effects on aphid survival and reproduction through RNAi. Hence, the aims of this research were to identify essential genes of GPAs and to investigate the effects of gene silencing by artificial feeding of dsRNA and delivery of dsRNA through transgenic plants.

Comparative bioinformatics was undertaken for in silico identification of candidate genes of GPA. Six putative functional classes: embryogenesis, osmoregulation, moulting, cuticularisation, feeding, and locomotion were identified based on the life-cycle of GPA. All available expressed sequence tags (ESTs) of GPA were compared to the genomic resources of the free-living nematode, Caenorhabditis elegans to identify genes belonging to the putative functional class and also having lethal RNAi phenotypes. In this way, GPA orthologues of C. elegans genes with an RNAi phenotype belonging to the six putative functional classes were identified and also compared to nucleotide sequences of other insects to identify genes with known or predicted functions. Fifty target genes were identified in silico of which ten were then used for molecular characterisation and RNAi studies. The ten genes chosen for detailed study were named with a prefix Mp representing Myzus persicae and were: MpVha-8 (vacuolar H+ ATPase), MpEat-6 (EATing: abnormal pharyngeal pumping), MpCct-6 (Chaperonin Containing TCP-1), MpLev-11 (LEVamisole resistant; tmy-1), MpSox-2 (alternative splicing transcription factors), MpTnc-2 (Troponin C), MpAqp-4 (aquaporin or aquaglyceroporin related), MpPdi-2 (protein disulphide isomerase), MpPod-2 (polarity and osmotic sensitivity defect), and MpCars-1 (cysteinyl amino-acyl tRNA synthetase).

A study to optimise in vitro RNAi conditions for GPAs was done which involved the assessment of eleven dyes for their suitability in tracing uptake of artificial diet containing sucrose and dsRNA after 24 hr feeding in an artificial feeding chamber. The dyes tested were: fluorescein isothiocyanate, fluorescein diacetate, phloxine B, methylene blue, acid fuschin, fast green, congo red, neutral red, acridine orange, and two food colours, red and yellow. The visibility of dyes inside the aphid body, effects of ingested dye on aphid survival, and effects on the purity of dsRNA in the presence of dye were studied. The optimal concentrations of two vital dyes, neutral red (0.02%) and acridine orange (0.0025%) when mixed with 30% sucrose were easily visualised in the aphid body upon ingestion, did not significantly (p > 0.05) affect aphid survival after 24 hr feeding compared to sucrose-only controls, and also did not affect the quality and purity of dsGFP after 24 hours.

To demonstrate that adding vital dyes to trace uptake of dsRNA allowed effective assessment of target gene silencing, MpVha-8 was used to study the effects of in vitro RNAi. The lowest concentration of neutral red mixed with 30% sucrose and 2 µg/µL of dsMpVha-8 was provided to GPA in an artificial feeding chamber in which aphids were allowed to feed ad libitum. After 24 hr feeding, aphid survival and transcript abundance of the target gene were studied in only those aphids which had fed and showed the presence of dye. A reduction of 21.59% and 29.17% was observed in survival of aphids that fed on dsMpVha-8 with dye and without dye respectively. For both the treatments, there were also long-term effects on aphid survival in comparison to dsGFP-fed aphids when transferred to tobacco; mortality was recorded by day 6 and day 5 in aphids that fed on dsMpVha-8 with dye and without dye respectively. Transcript abundance of the target gene in aphids that fed on dsMpVha-8 mixed in sucrose and dye was compared to that of aphids fed on dsMpVha-8 mixed in sucrose alone and were pooled together. There was a pronounced knockdown of target gene in aphids that had taken up dsRNA with dye. This study demonstrated that the addition of dyes to feed containing dsRNA enabled the assessment of only those aphids that had ingested dsRNA, thus providing a more accurate measure of transcript abundance than when assessing aphids pooled after 24 hr feeding.

In vitro RNAi studies using the optimised conditions were undertaken to study the effects of silencing nine target genes. These were: MpEat-6, MpCct-6, MpPod-2, MpPdi-2, MpLev-11, MpSox-2, MpTnc-2, MpCars-1, and MpAqp-4. When dsRNA of target genes was delivered to GPA nymphs in a feeding chamber, differences in behaviour, reduction in target transcript levels and survival were found compared to controls. After 24 hr feeding, survival of nymphs fed on all nine dsRNA was between 48-75%, and was significantly lower than treatments of dsGFP and no-dsRNA control. The most obvious behavioural changes were found in aphids fed on dsRNA for genes involved in movement and locomotion. There was paralysis and limited movement for aphids fed on dsMpLev-11, dsMpSox-2 and dsMpTnc-2, and this was accompanied by a reduction in target transcript abundance. When aphids that had fed for 24 hours on dsMpEat-6, dsMpPod-2, dsMpLev-11, dsMpSox-2 and dsMpTnc-2 were transferred to tobacco to study the effects on long-term survival, it was found that aphids survived only until days eight, four, one, one, and three respectively. Conversely, for aphids that fed on dsMpPdi- 2, dsMpCars-1, dsMpCct-6 and dsMpAqp-4, even though there was a gradual decrease in the percentage of aphid survival, complete mortality was not observed over 12 days on tobacco.

In planta RNAi studies were undertaken to study the effects on survival and reproduction of GPA reared on transgenic tobacco plants expressing target genes. For this, tobacco leaf disc transformation using recombinant Agrobacterium tumefaciens harbouring the hairpin expression cassette was done to generate primary transformants (T0). Transgenic T0 plants showing resistance to kanamycin and T1 plants that tested positive for the presence of the neomycin phosphotransferase gene were challenged with nymphs. Amongst the T0 plants tested, dsMpLev-11-expressing tobacco had significantly lower aphid survival as compared to empty vector- and dsGFP-expressing plants. Of all the T0 transgenic plants, dsMpLev-11- expressing tobacco had the most events, a total of nine, which had no aphids surviving by day 16; survival was only until day five or six. The mean number of aphids alive on the T1 events of target dsRNA-expressing tobacco at each time-point (beyond day zero) were found to be significantly lower as compared to the control group of untransformed wild type, GFP and null 6 events. At day 16, the T1 events for each gene that showed the lowest aphid number were: MpSox-2 E8, MpAqp-4 E10, MpLev-11 E12, MpCars-1 E8, MpTnc-2 E8, MpPod-2 E6, MpCct-6 E6, MpEat-6 E8, MpPdi-2 E9, and MpVha-8 E10.

The use of in vitro RNAi studies as a preliminary screen to determine the potential of a candidate gene before progressing into in planta RNAi studies was found to be useful. This was because the effects on survival after feeding on dsRNA through artificial feeding and on transgenic tobacco plants were somewhat similar. For example, aphid survival after feeding on dsMpSox-2 and dsMpLev-11 for 24 hours was 58.3% and 50% respectively and was accompanied by impaired locomotion, reduction in target gene expression, and also affected long-term survival when transferred to tobacco. There was also a reduction in aphid survival on the T1 events MpSox-2 E8 and MpLev-11 E12 as compared to controls. Results of in vitro RNAi for five genes, MpEat-6, MpLev-11, MpSox-2, MpTnc-2 and MpVha-8 complemented the results of in planta RNAi demonstrating the practicality of using an in vitro RNAi screen for selecting genes prior to advancing into host-mediated RNAi.

Except for V-ATPase, which has been tested previously for RNAi effects on aphids including M. persicae, none of the nine target GPA genes studied have ever been tested in GPA and are novel targets. Based on the behavioural changes and effects on aphid survival after in vitro RNAi, and the survival on transgenic T1 events of tobacco, genes were ranked for their efficiency. In an ascending order of their performance these were: MpSox-2, MpLev-11, MpTnc-2, MpPod-2, MpEat-6, MpAqp-4, MpCct-6, MpPdi-2, MpCars-1 and MpVha-8.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Jones, Michael and Fosu-Nyarko, John
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