- Title
- Identification of differentially expressed proteins involved in wheat (Triticum aestivum) resistance against Russian Wheat Aphid (Diuraphis noxia) SA2 using SWATH-MS analysis
- Creator
- Ntlokwana, Sitha Emmanuel
- Date Issued
- 2020
- Date
- 2020
- Type
- Thesis
- Type
- Masters
- Type
- MSc
- Identifier
- http://hdl.handle.net/10353/18872
- Identifier
- vital:42888
- Description
- Wheat (Triticum aestivum) is the second most-consumed cereal crop in the world, second only to maize. In South Africa it is mainly produced in three provinces; the Free State, Western Cape, and the Northen Cape. The average total land area used for wheat cultivation in these three Provinces, is 533 000 hectares, with a resultant annual production of between 1.3 to 2 million tons. The total wheat requirement in South Africa is currently 2.7 million tons according to the production of wheat guidelines published by the Department of Agriculture, Forestry and Fisheries, which is higher than the total annual production. One of the primary reasons that contribute to the lower production levels is insect and pest infestation, in particular, the Russian wheat aphid (Diurophis noxis), which can result in crop loss of up to sixty percent. Russian wheat aphids (RWA) are invasive insects that feed on the phloem of the plant through their specialised thin stylet-like mouthparts and release toxic agents contained in their saliva during feeding, which disrupt cellular functions inside the host plant. This damage leads to symptoms like; chlorosis, necrosis, wilting, stunting and curling of leaves, also known as leaf rolling. This study aimed to identify differentially expressed proteins in resistant and susceptible wheat (Triticum aestivum) cultivars during Russian wheat aphid Biotype South African 2 (RWA-SA2) infestation, in order to identify proteins involved in the wheat resistant mechanism against RWA-SA2. Two wheat cultivars SST398 (resistant to RWA-SA2) and SST356 (susceptible to RWA-SA2) were used, and a total number of 126 plants were planted and divided according to four different harvest times (Control day 0; day 5; day 7 and day 12). The wheat plants were infested with approximately 10 aphids per plant at the three-leaf stage, and placed within an insect cage inside a growth chamber (Conviron, Winnipeg, Canada) set at 24ºC, light intensity of 352 µmol.m-2.sec-1, ambient CO2 levels (410 ppm) and humidity of 60% with a 16h/8h day/night cycle. Wheat leaves were harvested at various time frames, and total protein extraction was performed. The protein samples were reduced with 10 mM dithiothreitol (DTT) and alkylated with 30 mM iodoacetamide (IAA). Sample clean-up and on-bead trypsin digest were performed on megReSyn Hilic columns, over four hours. The resultant peptides were vacuum dried and resuspended in 2% acetonitrile (ACN) before submitting them to LC-MS/MS (SWATH) for analysis. A total number of 611 proteins were differentially expressed, of which 19 were identified to be involved in the resistance response of wheat, and an additional 20 involved in biotic stress responses. This study showed that reactive oxidative species (ROS) such as hydrogen peroxide and hydroxyl radicals (OH-) play a significant role in the early stages of wheat resistance against RWA-SA2 infestation. They are involved in cell wall strengthening, activation of defence genes, involvement of phytohormone signals such as salicylic and jasmonic acid, which also mediates the systemic defences such as, systemic acquired resistance (SAR), leading to the expression of pathogen-related proteins such as (thaumatin-like proteins, oxalate oxidase, defensin, chitinase, and thionins). Although up-regulation of photosynthesis proteins such as (chlorophyll a/b binding protein, photosystem I and II, rubisco and divinyl chlorophyllide a, and 8 vinyl-reductase) were seen on both infested cultivars (susceptible and resistance), higher numbers of these classes of proteins were identified in the resistance cultivar, suggesting that they also play a critical role in resistance. In conclusion, the SWATH analysis used in this study was able to identify numerous proteins involved in the wheat resistance mechanism against RWA-SA2, most of which have not yet been reported to be involved in either biotic stress or RWA-SA2 resistance responses. Future studies are required to biochemically confirm the involvement of these enzymes and proteins the specific metabolic pathways where they are found, in the wheat resistance mechanism against Russian wheat aphid Biotype 2
- Format
- 268 leaves
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | Sitha Dissertation final post external examination Dec 2020.pdf | 5 MB | Adobe Acrobat PDF | View Details Download |