Biochemical and molecular analysis of the early response of Triticum aestivum infected with Puccinia striiformis f.sp. tritici

English: The aim of this study was to establish the oxidative burst and the involvement of protein kinases in the early responses involved in the resistance of a resistant wheat cultivar (Yr1) to Puccinia striiformis f. sp. tritici, thereby establishing the earliest point of recognition and the onset of defense responses to the intruding pathogen by the plant. This time period was then used in an attempt to clone genes involved in the downstream signaling. protein from Oryza sativa and a HGWP repeat containing protein from Oryza sativa respectively. Although no unique motifs were present on the respective polypeptide sequences, the presence of various phosphorylation sites indicate possible regulation through phosphorylation. An XYPPX repeat was present on the polypeptide sequence of 05WVZ03, while an N-glycosylation and an N-myristoylation was present on 05WVZ05 and 05WVZ06 respectively. The isolated cDNA fragments were present as single copy genes in various resistant cultivars, as well as in the susceptible cultivar, Avoset-S, indicating that these genes are not unique to any one resistance cultivar. While naturally expressed in the IR plants, three genes (05WVZ01, 05WVZ05 and 05WVZ06) showed induced expression in the IS plants. The fourth gene (05WVZ03) was apparently expressed as multiple copies within wheat. The expression profiles of none of the clones however indicated a real involvement in signaling. Plants are continuously challenged by a variety of pathogens. To survive these challenges, plants possess an arsenal of defenses, which are activated upon the recognition of the pathogen through certain signaling events. In some cases the difference between resistance and susceptibility lies in the timely activation of signaling. Early signaling events include protein phosphorylation and dephosphorylation and the production of reactive oxygen species (ROS). To establish the earliest time of recognition and the activation of defense responses, the occurrence of the oxidative burst in the plant was first established, whereafter the activation of protein kinases were determined. The oxidative burst was assessed through various enzyme activities e.g. NADPH oxidase, superoxide dismutase (SOD) and peroxidase (POX) activity as well as H2O2 levels. The earliest response of the plants was 18 h.p.i. when total protein kinase activity almost doubled in the infected resistant plants. This was followed by a similar increase in activity 48 h.p.i. Both increases in total protein kinase activity were accompanied by increases in H2O2 levels and glutathione peroxidase activity. However, the second increase at 48 h.p.i. was more significant and is therefore concluded to be the oxidative burst. The recognition of the pathogen, as well as the activation of the defenses therefore occurred between 18 and 48 h.p.i. Once this reaction time was established, differentially expressed cDNA fragments were amplified using DDRT-PCR. Eight different gene fragments were isolated, cloned and sequenced. These isolated cDNA fragments showed different levels of homology to four known polypeptides namely, a Nodulation protein B (fragment) from Rhizobuim sp, a TOBAC Hypothetical protein from Nicotiana tabacum, a Hypothetical