Browsing by Author "Weiller, Florent"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
- ItemCell wall profiling of tobacco leaves and grapes in the context of Botrytis cinerea infection(Stellenbosch : Stellenbosch University, 2021-03) Weiller, Florent; Moore, John P.; Vivier, Melane A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The plant cell wall has been shown to be at the centre of plant-biotic stress interactions. In the case of a pathogen encounter (e.g. fungi), the cell wall acts as the second barrier after the cuticle to stop pathogen penetration and tissue colonisation. The plant cell wall matrix composition and architecture influences the nature of resistance and susceptible responses to fungal pathogens. Botrytis cinerea, the fungus responsible for grey mold disease, causes worldwide crop losses. Understanding factors that differentiate susceptible from resistant plants is essential to develop new plant protection strategies against B. cinerea. In this study, genetically engineered plants with known resistance phenotypes, compared to the untransformed controls (with susceptible phenotypes) were used to understand the leaf cell wall changes either afforded by the transgenes introduced and/or the Botrytis infection process. The approach to investigate cell wall changes during Botrytis infection was also expanded to grapevine berry fruit. The method of choice was Comprehensive Microarray Polymer Profiling (CoMPP) technology, a high-throughput method for tracking cell wall matrix polysaccharide and protein composition and it was combined with monosaccharide profiling and various imaging tools. In the first part of this study, the leaves of nine tobacco (Nicotiana tabacum) lines: four overexpressing the known defence gene, grapevine polygalacturonase-inhibiting protein 1 (VviPGIP1), four overexpressing another known defence gene, the tobacco cinnamyl alcohol dehydrogenase 14 (NtCAD14) and the wild type SR1 were screened for cell wall compositional differences due to genetic overexpression in the absence of infection. Total lignin and monolignols were assayed using Py-GC-MS, but results showed limited variations between the different plant lines. CoMPP and GC-MS analysis for cell wall composition and monosaccharide content showed variation in homogalacturonan (HG) methylesterification patterning between the various transgenic plant lines. Arabinogalactan proteins (AGPs) and extensins increased in the majority of the VviPGIP1 lines. These results suggest that PGIP overexpression probably influences pectinmethylesterase (PME) enzyme activity and affects glycoprotein organisation. Following these results, more in-depth analysis continued during infection of PGIP plants. Infection experiments were conducted using B. cinerea on leaves of VviPGIP1 tobacco lines, compared to the wild type as control. Lesion size differences were observed from 96 hours post inoculation (hpi) with reduced lesions in the transgenic lines compared to the wild type, confirming the known resistance responses of the transgenic plants to the fungus. Cell wall alterations were followed in the 72 hpi period, showing HG degradation with RG-I signal increase, whereas the cellulose-xyloglucan network was mostly unaffected. Extensins and AGPs accumulated at and around the lesions, while distal non-infected leaves showed matrix changes from 72 hpi, with reorganisation of the cellulose/xyloglucan network and in the homogalacturonan methylesterification patterning, indicative of a systemic response. A typical Botrytis infection with cell wall depectination exposed less hemicellulose polysaccharides. In parallel, the plant seemed to build a defensive response to the infection with accumulation of defence related proteins at the lesion and in distal leaves. Unlike the wild type, the transgenic plants response was efficient in restricting Botrytis progression from 72 hpi onwards. In the last part of this study berries, at three developmental stages (veraison, post-veraison and ripe), from four Vitis vinifera cultivars (Cabernet Sauvignon, Sauvignon Blanc, Dauphine and Barlinka grape berries), were infected with B. cinerea. Ripe grapes from all four cultivars developed symptoms and showed cell wall degradation with cultivar differences were noted, while very few successful B. cinerea infections on veraison and post-veraison grapes were observed. This is in line with the generalised view that grapevine berries display ontogenic resistance in the greener developmental stages, although there are known exceptions. Cabernet Sauvignon was the least susceptible cultivar to B. cinerea and displayed a delayed lesion development in our experimental conditions. The infected grapes from all the cultivars were characterised by altered HG methylesterification patterning, extensin reorganisation, as well as glucan accumulation as the infection progressed. This work has brought new insights to existing efforts to fully characterise the role of the grapevine PGIP1 gene in plant cell wall modifications, in the presence and absence of B. cinerea infection. It provides further proof that small changes in the cell wall matrix contribute to the possible priming of plants to resist and overcome infection. Moreover, the similarities and differences observed between tobacco leaves and grape berry cell walls during Botrytis infection will help to conduct targeted experiments and to complement the existing models on plant cell wall – B. cinerea interactions.
- ItemOverexpression of VviPGIP1 and NtCAD14 in tobacco screened using glycan microarrays reveals cell wall reorganisation in the absence of fungal infection(MDPI, 2020-07-15) Weiller, Florent; Gerber, Lorenz; Trygg, Johan; Fangel, Jonatan U.; Willats, William G. T.; Driouich, Azeddine; Vivier, Melane A.; Moore, John P.The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC–MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.