Metabolic aspects of the early response of leaf rust-infected wheat

[摘要] English: The aim of this study was to study various aspects of the defence response of wheat infected by Puccinia triticina. SSH was used to isolate differentially expressed cDNA fragments from P. triticina infected wheat. Once sequenced, two cDNA clones were selected for further analysis. One of the cDNA clones, LRW268, showed similarity to an EST derived from de-etiolated wheat. It showed a 16.5 fold induced expression at 9 hpi in the P. triticina infected wheat. In P. striiformis infected wheat, an induced expression was found in the resistant Avoset-Yr1 plants. Application of chemical elicitors showed an induction of LRW268 expression for plants treated with H2O2, MeJA and menadione. To obtain the full length gene, 5'-RACE was attempted, but it was unsuccessful. However, a contig was assembled using ESTs present in the GenBank database. This yielded a 603 bp contig encoding a 96 amino acid sequence that showed good homology to a RLK interacting protein. The presence of a putative MAPK docking motif and a phosphorylation site indicated that LRW268 could play a role in cell signalling. The presence of LRW268 in different wheat cultivars signified that it could form part of the general defence response of wheat. The second cDNA clone, LRW222, which was similar to an EST from wheat infected with powdery mildew, showed a 4.6 fold induction of expression at 15 hpi in wheat infected with P. triticina, while the susceptible Thatcher cultivar showed an induction at an earlier time interval. P. striiformis infected wheat showed a more constant expression of LRW222. Putative induced expression of LRW222 was observed in H2O2, MeJA and menadione treated wheat. Assembly of a contig using published ESTs yielded a 668 bp contig which encoded an 89 amino acid polypeptide showing homology to various wound-induced protease inhibitors. The presence of a putative MAPK docking motif on LRW222 suggested that it could be a general or pathogen specific protease inhibitor. The effect of light on the wheat defence response was also examined. The photosynthetic capacity of all treatments was measured and fluorescence microscopy performed. Infection caused a decrease in the photosynthetic capacity of the susceptible plants with the resistant plants showing less fluctuation. The infected resistant plants recovered faster and better than the infected resistant plants after the dark incubation. Plants that were additionally dark incubated showed a lower photosynthetic capacity compared to the control treatments. This difference in photosynthetic capacity was not observed on molecular level with photosynthesis related genes showing unaltered expression. The putative expression of certain defence related genes did however show a light dependency. An induced defence was observed in the uninfected plants, confirming a putative volatile signalling event that was detected during an earlier study involving this particular plant/pathogen interaction. The importance of light in wheat resistant towards P. triticina could thus be attributed to the ability of plant to photosynthesise optimally. It must however be emphasized that the expression of all tested genes were not quantitatively determined, since end-point analysis using RT-PCR was used. Future research will include the use of techniques that allows quantitative measurement of gene expression.

[发布机构] University of the Free State