Differential regulation of wheat (Triticum aestivum) callose synthases during Russian wheat aphid (diuraphis noxia)infestation
- Authors: Ngwenya, Nompilo
- Date: 2017
- Subjects: Russian wheat aphid Wheat -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/8100 , 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.
- Full Text:
- Date Issued: 2017
- Authors: Ngwenya, Nompilo
- Date: 2017
- Subjects: Russian wheat aphid Wheat -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/8100 , 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.
- Full Text:
- Date Issued: 2017
Effects of sustained Russian wheat aphid (Diuraphis noxia Mordvilko) feeding on leaf blades of wheat (Triticum aestivum L. cv Adamtas)
- Authors: Matsiliza, Babalwa
- Date: 2003
- Subjects: Russian wheat aphid Wheat -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4213 , http://hdl.handle.net/10962/d1003782
- Description: Penetration of sink as well as source leaves of wheat plants by the Russian wheat aphid, Diuraphis noxia (Mordvilko) was investigated using light, fluorescence and transmission electron techniques, to determine the feeding strategies adopted by the aphid in penetrating and successfully feeding from the phloem, and to assess the structural effects of the probing and feeding behaviour of D. noxia on the feeding sites. Examination of aphid-infested sink, as well as source leaf tissue, showed that D.noxia probed in cells of the vascular bundle more frequently than mesophyll cells. Within the vascular bundle, thin-walled sieve tubes were visited (probed) more than the other cells. In sink leaf material, 68 of 82 (83%) stylets and stylet tracks encountered during the examination of 1000 serial sections (from 5 different plants) terminated in thin-walled sieve tubes and only 14 (17%) in thick-walled sieve tubes. Thin-walled sieve tubes were visited more significantly than thick-walled sieve tubes. However, examination of the aphid.,.infested sink leaf on a per centimetre basis, from the tip of the leaf, revealed that thick-walled sieve tubes in the area closest to the tip (0-2cm from the tip) were as attractive to the aphid as were thin-walled sieve tubes, with no significant difference in the number of times thick- and thin-walled sieve tubes were probed in this area. Some 2-4cm from the tip however, thinwalled sieve tubes were significantly more probed and therefore more attractive than thick-walled sieve tubes. Examination of 2000 serial sections using aphid-infested source leaf tissue, showed that the thin-walled sieve tubes were significantly more probed than thickwalled sieve tubes, along the whole leaf, expressed as a total of all leaves, as well as on a per centimetre basis along the length of the leaf, with 212 (95%) of 222 terminations within the thin-walled sieve tubes and only 10 (5%) in thick-walledsieve tubes. The aphid probed the small vascular bundles (loading bundles) many more times than intermediate or large transport vascular bundles, in sink as well as source leaf. Of a total of 82 stylets and stylet tracks encountered in sink leaf tissue, 31 terminated in small vascular bundles ang the remaining 28 and 16 were located within large and intermediate vascular bundles respectively. In source leaf tissue 121 of 222 stylets and stylet tracks encountered were associated with small vascular bundles and only 58 tracks and 43 tracks with intermediate and large vascular bundles, respectively. The effect of sustained RWA feeding on the transport capacity was examined after the application of 5,6 carboxyfluoresceine diacetate (5,6-CFDA) in control (sink and source leaf tissue) and aphid-infested (source) wheat leaves, using fluorescence microscopy. After 3h acropetal longitudinal transport of 5,6-CF had occurred in sink leaves in longitudinal veins, as well as a lateral transfer via cross veins and subsequent unloading into mesophyll cells close to the tip of the leaf was observed. In control leaf tissue, the fluorescence front was detected up to about 5cm from the point of application and was only associated with the phloem and not unloaded. In contrast, aphid-infested leaf tissue showed very little 5,6-CF transport, being limited to 2cm or less from the point of application. Structural damage to the phloem in general and to the sieve tubes in particular within of control and infested wheat leaves was investigated using transmission electron microscopy (TEM). In addition, leaf strips were mounted in aniline blue to visualise callose deposition using the fluorescence microscopy. At the TEM level. infested leaf tissue showed various abnormalities, which included destruction of cell contents, membrane damage and subsequent loss of cell contents. TEM studies suggest severe osmotic shock resulted from the aphid's probing. Examination of leaf tissue using fluorescence microscopy showed that there was very little characteristic aniline blue-stained callose visible in control leaf tissue, other than the thin diffuse patches along the sieve plates and punctate spots associated with pore plasmodesmatal areas and plasmodesmatal aggregates. In contrast, the aphid-infested leaf tissue was heavily callosed, with callose deposited not only within the phloem tissue but also in neighbouring vascular parE:}nchyma cells as well. The data collectively suggest that D. noxia feeds preferentially within thin-walled sieve tubes, within the small longitudinal vascular bundles in sink , as well source leaf tissue. Based upon the data presented here the thin-walled sieve tubes in the wheat leaf appear to be more attractive to the aphid and that they are probably more functional in terms of transport system and unlo?lding in sink leaves. Aniline blue stained leaf material that had previously hosted large aphid colonies showed evidence of extensive callose deposits 24 to 36h after the aphids were removed, suggesting that the aphids caused severe mechanical damage to the vascular tissue and mesohyll cells as well. Damage (transient or more permanent) and the subsequent deposition of wound callose, disrupted phloem transport and hence the export of photoassimilate from the leaves.
- Full Text:
- Date Issued: 2003
- Authors: Matsiliza, Babalwa
- Date: 2003
- Subjects: Russian wheat aphid Wheat -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4213 , http://hdl.handle.net/10962/d1003782
- Description: Penetration of sink as well as source leaves of wheat plants by the Russian wheat aphid, Diuraphis noxia (Mordvilko) was investigated using light, fluorescence and transmission electron techniques, to determine the feeding strategies adopted by the aphid in penetrating and successfully feeding from the phloem, and to assess the structural effects of the probing and feeding behaviour of D. noxia on the feeding sites. Examination of aphid-infested sink, as well as source leaf tissue, showed that D.noxia probed in cells of the vascular bundle more frequently than mesophyll cells. Within the vascular bundle, thin-walled sieve tubes were visited (probed) more than the other cells. In sink leaf material, 68 of 82 (83%) stylets and stylet tracks encountered during the examination of 1000 serial sections (from 5 different plants) terminated in thin-walled sieve tubes and only 14 (17%) in thick-walled sieve tubes. Thin-walled sieve tubes were visited more significantly than thick-walled sieve tubes. However, examination of the aphid.,.infested sink leaf on a per centimetre basis, from the tip of the leaf, revealed that thick-walled sieve tubes in the area closest to the tip (0-2cm from the tip) were as attractive to the aphid as were thin-walled sieve tubes, with no significant difference in the number of times thick- and thin-walled sieve tubes were probed in this area. Some 2-4cm from the tip however, thinwalled sieve tubes were significantly more probed and therefore more attractive than thick-walled sieve tubes. Examination of 2000 serial sections using aphid-infested source leaf tissue, showed that the thin-walled sieve tubes were significantly more probed than thickwalled sieve tubes, along the whole leaf, expressed as a total of all leaves, as well as on a per centimetre basis along the length of the leaf, with 212 (95%) of 222 terminations within the thin-walled sieve tubes and only 10 (5%) in thick-walledsieve tubes. The aphid probed the small vascular bundles (loading bundles) many more times than intermediate or large transport vascular bundles, in sink as well as source leaf. Of a total of 82 stylets and stylet tracks encountered in sink leaf tissue, 31 terminated in small vascular bundles ang the remaining 28 and 16 were located within large and intermediate vascular bundles respectively. In source leaf tissue 121 of 222 stylets and stylet tracks encountered were associated with small vascular bundles and only 58 tracks and 43 tracks with intermediate and large vascular bundles, respectively. The effect of sustained RWA feeding on the transport capacity was examined after the application of 5,6 carboxyfluoresceine diacetate (5,6-CFDA) in control (sink and source leaf tissue) and aphid-infested (source) wheat leaves, using fluorescence microscopy. After 3h acropetal longitudinal transport of 5,6-CF had occurred in sink leaves in longitudinal veins, as well as a lateral transfer via cross veins and subsequent unloading into mesophyll cells close to the tip of the leaf was observed. In control leaf tissue, the fluorescence front was detected up to about 5cm from the point of application and was only associated with the phloem and not unloaded. In contrast, aphid-infested leaf tissue showed very little 5,6-CF transport, being limited to 2cm or less from the point of application. Structural damage to the phloem in general and to the sieve tubes in particular within of control and infested wheat leaves was investigated using transmission electron microscopy (TEM). In addition, leaf strips were mounted in aniline blue to visualise callose deposition using the fluorescence microscopy. At the TEM level. infested leaf tissue showed various abnormalities, which included destruction of cell contents, membrane damage and subsequent loss of cell contents. TEM studies suggest severe osmotic shock resulted from the aphid's probing. Examination of leaf tissue using fluorescence microscopy showed that there was very little characteristic aniline blue-stained callose visible in control leaf tissue, other than the thin diffuse patches along the sieve plates and punctate spots associated with pore plasmodesmatal areas and plasmodesmatal aggregates. In contrast, the aphid-infested leaf tissue was heavily callosed, with callose deposited not only within the phloem tissue but also in neighbouring vascular parE:}nchyma cells as well. The data collectively suggest that D. noxia feeds preferentially within thin-walled sieve tubes, within the small longitudinal vascular bundles in sink , as well source leaf tissue. Based upon the data presented here the thin-walled sieve tubes in the wheat leaf appear to be more attractive to the aphid and that they are probably more functional in terms of transport system and unlo?lding in sink leaves. Aniline blue stained leaf material that had previously hosted large aphid colonies showed evidence of extensive callose deposits 24 to 36h after the aphids were removed, suggesting that the aphids caused severe mechanical damage to the vascular tissue and mesohyll cells as well. Damage (transient or more permanent) and the subsequent deposition of wound callose, disrupted phloem transport and hence the export of photoassimilate from the leaves.
- Full Text:
- Date Issued: 2003
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