Visser, B.Huang, Ju-ChiVan der Westhuizen, A. J.2015-09-012015-09-01200820082008http://hdl.handle.net/11660/1114English: 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.Afrikaans: Die doel van die studie was om verskillende aspekte van die verdedigingsrespons van koring na infeksie deur Puccinia triticina, te ondersoek. cDNA fragmente wat na infeksie differensieël tot uiting gekom het, is met behulp van SSH geïsoleer. Nadat die klone se basisvolgorde bepaal is, is twee cDNA klone vir verdere analise gekies. Een cDNA kloon, LRW268, het ooreenkoms met ʼn EST wat uit gedeëtioleerde koring gekloneer was, getoon. Kloon LRW268 se uitdrukking was 9 uur na infeksie 16.5-voudig hoër. Geïnduseerde uiting was ook gevind in Avoset-Yr1 koring wat met P. striiformis geïnfekteer was. Verder het die bespuiting van koring met H2O2, MeJA en menadioon ook tot hoër uitdrukking gelei. Hoewel onsuksesvol, is daar met 5’-RACE probeer om die volledige geen te kloneer. Uiteindelik is ’n contig wat die volledige geen verteenwoordig, deur middel van ESTs soos gevind in die GenBank databasis, saamgestel. Die voorgestelde geen was 603 bp lank en het vir ’n polipeptied van 96 aminosure kodeer. Die polipeptied het goeie ooreenkoms met ’n proteïen wat aan RLKs bind, getoon. Aangesien die voorgestelde polipeptied moontlike MAPK-bindingsmotiewe en fosforileringssetels besit, dui dit daarop dat dit by seinoordraging betrokke kan wees. Aangesien die geen ook verder in verskillende kultivars teenwoordig is, is dit ʼn aanduiding dat dit moontlik ’n rol te speel het tydens die algemene verdedigingsrespons van koring. ʼn Tweede cDNA kloon, LRW222, wat ooreenkoms toon met ʼn EST uit poeieragtige meeldou-geïnfekteerde koring, het ʼn 4.6-voudige geïnduseerde uitdrukking na 15 uur in geïnfekteerde Thatcher+Lr34 koring getoon. Die geïnduseerde uitdrukking was vroeër in die vatbare Thatcher kultivar. Koring geïnfekteer met P. striiformis het ʼn meer konstante uiting getoon. Moontlike geïnduseerde uitdrukking is gevind in koring wat behandel was met H2O2, MeJA en menadioon. ’n Contig saamgestel uit beskikbare ESTs was 668 bp lank en het kodeer vir ’n polipeptied van 89 aminosure. Die polipeptied het groot ooreenkomste getoon met verskeie wond-geïnduseerde protease inhibitore. Die verder teenwoordigheid van ’n moontlike MAPK bindingsmotief het weereens daarop gedui dat die gekodeerde polipeptied of ’n algemene of patogeen-spesifieke protease inhibitor kan wees. Laastens is die invloed van lig op koring se verdedigingsrespons ook ondersoek. Die fotosintetiese kapasiteit van alle behandelings was gemeet en fluoresensiemikroskopie is gebruik om infeksiestrukture te tel. Infeksie het gelei tot ʼn afname in fotosintetiese kapasiteit van vatbare koring terwyl die weerstandbiedende plante minder fluktuasies getoon het. Laasgenoemde plante het ook vinniger en beter na die donker inkubasie herstel in vergelyking met die geïnfekteerde vatbare plante. Die plante wat addisioneel aan donker blootgestel was, se fotosintetiese kapasiteit was laer as die van die kontrole plante. Die uitdrukking van verskeie verdedigingsverwante gene het ook ’n lig-afhanklikheid getoon. ’n Aangeskakelde verdedigingsrespons in die ongeïnfekteerde plante was moontlik as gevolg van seinoordraging deur vlugtige stowwe wat al voorheen in dieselfde plant/patogeen interaksie waargeneem is. ʼn Effektiewe verdedigingsrespons van weerstandbiedende koring teen P. triticina infeksie is dus afhanklik van die plant se vermoë om optimaal te fotosintetiseer. Dit is egter belangrik om te noem dat die uitdrukking van alle gene in die studie nie kwantitatief gemeet is nie, aangesien RT-PCR gebruik is wat net ’n eindpuntproduk lewer. Toekomstige studies sal egter van ander tegnieke gebruik maak om te verseker dat alle uitdrukkingstudies kwantitatief is.enWheat -- Disease and pest resistanceLeaf rust of wheatCellular signal transductionPuccinia striiformisTriticum aestivumPuccinia triticinaThesis (Ph.D. (Genetics))--University of the Free State, 2008ThesisUniversity of the Free State