English: The interaction between wheat (Triticum aestivum) and the Russian wheat aphid (Diuraphis noxia) was investigated on a molecular level. The resistant cultivar, Tugela DN1, was infested with the Russian wheat aphid and tissue harvested at 3 hour time intervals for 24 hours. Differential display RT-PCR was used to clone genes that were inducibly expressed using primers specific to the conserved subdomain VIb of the kinase domain of protein kinases. Differentially expressed genes were found as early as three hours after infestation. These fragments were cloned and sequenced. A total of 11 differentially expressed cDNA fragments were analyzed. Four showed homology to genes encoding proteins involved in normal cellular metabolism, such as the Rubisco large subunit, indole-3-gylcerol phosphate lyase that produces indole that could act as a volatile messenger molecule, triose-phosphate isomerase that is involved in glycolysis and 2- isopropylmalate synthase that is involved in leucine biosynthesis. Of the other cDNAs, three clones showed homology to several hypothetical proteins with no defined functions, while the other three showed no homology at all with any other known genes. One of the unknown clones (D20) was selected for further analysis. The gene was inducibly expressed only in the infested resistant plants, but not in the infested susceptible plants. The expression of the gene was induced within 3 h after infestation reaching a maximum expression level 6 h.p.i. The gene is present as a single copy within the genomes of susceptible and 5 different resistance lines. An attempt to clone the full length gene was only partially successful since after the sequences were combined homology was found with several hypothetical genes from rice and maize. However, no identity and function could be given.
Afrikaans: Tydens hierdie studie is die interaksie tussen koring (Triticum aestivum) en die Russiese koringluis (Diuraphis noxia) op molekulêre vlak ondersoek. Tugela DN1, wat weerstandbiedend is teen die Russiese koringluis, is met die luise geïnfesteer. Blaarweefsel is elke 3 uur versamel oor ‘n tydperk van 24 uur. Gene wat gedurende die tydperk differensieël tot uiting gekom het, is met behulp van DD RT-PCR geamplifiseer. Die inleiers wat gebruik was, kodeer spesifiek vir die hoogs gekonserveerde subdomein VIb van plant proteïenkinases. Verskeie fragmente is gevind waarvan die transkripsie binne drie uur na infestering geïnduseer is. Differensieël uitgedrukte cDNA-fragmente is gekloneer en die nukleotiedvolgordes daarvan bepaal. Elf gekloneerde cDNA-fragmente is verder ge-analiseer. Vier van die fragmente het homologie getoon met gene waarvan die gekodeerde proteïene ‘n rol speel tydens metabolisme. Ingesluit hierby was gene wat kodeer vir die groot subeenheid van Rubisco wat betrokke is by fotosintese, indool-3- gliserolfosfaatliase wat indool sintetiseer wat kan dien as ‘n vlugtige seinmolekule, triose-fosfaatisomerase wat betrokke is by glikoliese en 2-isopropielmalaatsintase wat vermoedelik betrokke is by leusienbiosintese. Van die oorblywende ses klone het drie homologie getoon met verskeie hipotetiese proteïene waarvoor daar tans nog geen funksie bestaan nie. Die oorblywende drie het egter geen homologie met enige bekende gene getoon nie. Een van die onbekende klone (D20) was gekies vir verdere studie. Die uiting van die kloon word slegs in die weerstandbiedende geïnfesteerde plante geïnduseer en glad nie in die geïnfesteerde vatbare plante nie. Induksie van uiting was binne drie ure na infesteering. Die maksimum uitsingsvlak is ses ure na infesteering bereik. Die geen kom voor as ‘n enkelkopie in die genome van vatbare, sowel as vyf verskillende weerstandbiedende, koringlyne. ‘n Poging om die DNA-volgorde van die volledige geen te bepaal, was egter slegs gedeeltelik suksesvol. Nadat alle beskikbare DNA volgordes saamgevoeg was, kon homologie alleenlik met verskeie hipotetiese proteïene uit rys en mielies verkry word. Tans kan daar egter nog geen funksie of positiewe identifikasie aan die gekloneerde D20 cDNA-fragment gegee word nie.