Grapevine powdery mildew is caused by Erysiphe necator, an Ascomycete fungus and an obligate biotroph restricted to growth on its grapevine host. Biotrophic pathogens form a stable association with host cells without directly causing cell death, and take up nutrients from, in the case of powdery mildew ( PM ), host epidermal cells ( Rumbolz et al., 2000 ). As the fungus grows, its increasing biomass becomes a strong nutrient sink capable of altering assimilate flow and storage in the host. To identify host genes that may mediate nutrient delivery to powdery mildew infected tissues and therefore may contribute to disease susceptibility, a candidate gene approach using degenerate and RT - PCR, and a nontargeted approach using microarray analysis was instigated. Once identified, " susceptibility genes " could be targeted for manipulation to provide alternative resistance strategies based on reduced susceptibility in the future. In addition to genes encoding pathogenesis and stress related proteins, microarray analysis revealed that transcript levels of a putative metal transporter and a cell wall structural protein were elevated in infected berry skin, while aquaporin water channels and genes associated with photosynthesis were generally repressed. Degenerate PCR was used to isolated new cell wall invertase, monosaccharide and amino acid transporter genes and initial RT - PCR revealed that expression of genes involved in sugar mobilisation were the most significantly modulated by powdery mildew infection. Previously unreported hexose transporters ( HTs ), ( VvHT3, VvHT4 and VvHT5 ) and a cwINV ( VvcwINV ) had been isolated from cDNA prepared from powdery mildew infected grapevine leaves. Full length clones of grapevine HTs and cwINV were obtained by RACE PCR. Heterologous expression of the three new HTs in yeast confirmed that VvHT4 and VvHT5 mediated glucose uptake, while VvHT3 did not function in the yeast system. However, transient expression of a translational fusion of the VvHT3 protein with green florescence protein in onion epidermal cells indicated that it is targeted to the plasma membrane of plant cells. Quantitative RT - PCR analysis of these new genes, together with previously reported grapevine HTs and cytoplasmic and vacuolar invertases, indicated that expression of VvcwINV and VvHT5, were significantly up - regulated by PM infection, while a vacuolar invertase was strongly down - regulated by PM infection. Invertase activity assays were in agreement with these findings, showing elevated sucrolytic activity in insoluble fractions and reduced sucrolytic activity in soluble fractions. These results suggest that apoplasmic phloem unloading of sucrose in the infected leaf is elevated and that VvHT5 is induced to recover the additional hexoses from the apoplasm. Basic localisation studies indicated that VvHT5 and VvcwINV are not induced specifically in powdery mildew infected leaf regions, but are induced in a more diffuse distribution within infected leaves. To determine if induction of VvHT5 and VvcwINV is specific to PM infection or if other stimuli may also mediate these responses, leaves were inoculated with downy mildew or stressed by wounding. Transcript levels of VvHT5 and VvcwINV were elevated by wounding and downy mildew infection, suggesting that the induction of these genes may be part of a general stress response. To explore the signalling pathways that may underlie these responses, leaves were treated with the plant growth regulators ethylene, jasmonate and abscisic acid. Exogenous application of ethylene and methyl jasmonate only marginally affected the expression of the genes studied, however foliar application of abscisic acid ( ABA ) induced gene expression changes similar to those observed in response to powdery mildew infection and wounding. Promoter sequences of VvHT3, VvHT4, VvHT5 and VvcwINV were isolated and analysed for the presence of regulatory elements. Compared with the promoters of VvHTs that were not induced by pathogen infection or wounding, the VvHT5 and VvcwINV promoters contained numerous motifs associated with induction by ABA including ABRE, Myc and Myb binding elements. The path of sugar loading into the mesocarp of grape berries during ripening is still poorly understood and few molecular components associated with this process have been described. Quantitative RT - PCR was used to monitor the expression of five HTs and VvcwINV during Cabernet sauvignon and Shiraz berry development and ripening. Of the three new HTs reported here, the expression of VvHT3 is most consistent with a potential role in sugar loading, while VvHT5 is induced late in this process. VvcwINV transcript levels were high pre - ripening and also during the later stages of ripening, therefore based on this expression pattern, a role for this enzyme during ripening is not clearly evident. These results are discussed in terms of an apoplasmic step in phloem unloading in ripening grape berries. This study has provided new insights into the molecular and biochemical processes associated with the formation of carbohydrate sink metabolism in response to stress stimuli, and sugar delivery to grape berries during ripening. ABA - dependant pathways may mediate the stress - associated induction of VvcwINV and VvHT5, presumably to recruit additional carbohydrates to the affected organ to energise repair and defence responses. At this stage it is unknown if this response is beneficial to pathogen nutrition, however potentially, modification of genes associated with carbohydrate sink metabolism could provide an alternative way to engineer resistance to this pathogen. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.
| Identifer | oai:union.ndltd.org:ADTP/263765 |
| Date | January 2006 |
| Creators | Hayes, Matthew Allan |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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