- Title
- Differential regulation of wheat (Triticum aestivum) callose synthases during Russian wheat aphid (diuraphis noxia)infestation
- Creator
- Ngwenya, Nompilo
- Subject
- Russian wheat aphid Wheat -- Diseases and pests
- Date
- 2017
- Type
- Thesis
- Type
- Masters
- Type
- MSc
- Identifier
- http://hdl.handle.net/10353/8100
- Identifier
- vital:31522
- Description
- Plant production serves as the primary food source for all living beings and is continuously being affected by many biotic and abiotic factors. Wheat, one of the most consumed cereal crops in the world and the second most cultivated crop after maize in South Africa, is continuously being affected by Russian wheat aphid (RWA) since 1978. To counteract the effect of this aphid, the first resistant wheat cultivar was developed in 1984 in South Africa containing the Dn1 resistance gene. However, the mechanism of action of the resistance is still not fully understood to date. The feeding of the RWA on the wheat induces callose deposition especially in susceptible wheat cultivars, linking the β-1.3-polysaccharide (callose) to the resistance mechanism. Callose is synthesized by callose synthases, also known as glucan synthase-like genes (GSL). Not much is known about the mechanism of action of callose synthases or how they are regulated in response to the RWA. Bioinformatics tools, such as those available at NCBI, were used to identify the wheat callose synthases genes. The proteins encoded by the identified wheat callose synthases genes, were then analyzed for conserved domain motifs using the MEME suite and InterproScan, sequence similarities using ClustaL Omega and transmembrane domains using HMMTOP and ProtCamp. These were then compared with those of the already characterized Arabidopsis callose synthase proteins in an attempt to identify the wheat callose synthase(s) that responds to aphid feeding. Further bioinformatics studies were carried out to identify the presence of biotic stress associated cis-acting regulatory elements found in the 1.5 kbp upstream region of the start codon of the Arabidopsis callose synthase genes. Eight partial wheat callose synthase sequences were identified and two of these (TaGSL2 and TaGSL22) showed high similarities to the AtGSL5, which is up-regulated in response to aphid feeding in Arabidopsis. Six of the wheat callose synthase genes were mediated to code for the functional callose synthases proteins: TaGSL3, 8, 12, 19, 22 and 23, and analyzed for conserved protein motifs. Based on the sequence similarities and conserved protein domains, TaGSL2 and TaGSL22 were found to be the most similar to AtGSL5 and most likely to respond to RWA infestation. Cis-acting regulatory element analyses confirmed the possibility of TaGSL22 being responsible for callose deposition in wheat as biotic stress associated cis-acting regulatory elements, Box W1, TC- rich element and W- box were all found in the 1.5 kbp upstream of the TaGSL22 coding region. Callose was quantified in both susceptible and resistant wheat cultivars using the aniline blue fluorescence method. When infested with RWA-SA1, the susceptible wheat cultivar (Tugela) deposited significantly higher amounts of callose, compared to the resistant wheat cultivar (Tugela DN), which deposited little, to no callose with respect to the control samples. Transcription analysis, of the TaGSL2 and TaGSL22 in RWA-SA2 infested Tugela and Tugela DN wheat cultivars, was performed using real time polymerase chain reaction (qPCR). Both TaGSL2 and TaGSL22 genes were up-regulated in Tugela and Tugela DN wheat cultivars in response to RWA-SA2 infestation, with TaGSL22 being more expressed than TaGSL2 in both cultivars, indicating that RWA-SA2 is able to overcome the resistance of Tugela DN and cause callose deposition. However, significantly higher expression of both genes was still observed in the susceptible, Tugela wheat cultivar. This study therefore confirms that callose deposition is associated with RWA-SA1 feeding, only in the susceptible wheat cultivar, Tugela and not the resistant cultivar, Tugela DN. However, during RWA-SA2 feeding, two of the eight identified callose synthases in wheat, are up-regulated in response to RWA-SA2 feeding, in both the resistant and susceptible wheat cultivars with higher levels observed in the susceptible wheat cultivar when compared to the resistant wheat cultivar. TaGSL2 and TaGSL22 are therefore implicated in the callose deposition observed in the susceptible and resistant wheat cultivars, after RWA-SA2 infestation. Further studies are required to confirm the differential regulation of the two wheat callose synthases proteins during RWA infestation and their possible role in the resistance mechanism of the resistant wheat cultivar, Tugela DN.
- Format
- 127 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 | |||
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| View Details | SOURCE1 | MSc (Biochemistry) NGWENYA, N.pdf | 2 MB | Adobe Acrobat PDF | View Details |