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

[摘要] English: The aim of this study was to establish the oxidative burst and the involvement ofprotein kinases in the early responses involved in the resistance of a resistant wheatcultivar (Yr1) to Puccinia striiformis f. sp. tritici, thereby establishing the earliest pointof recognition and the onset of defense responses to the intruding pathogen by theplant. This time period was then used in an attempt to clone genes involved in thedownstream signaling. protein from Oryza sativa and a HGWP repeat containing protein from Oryza sativarespectively.Although no unique motifs were present on the respective polypeptide sequences, thepresence of various phosphorylation sites indicate possible regulation throughphosphorylation. An XYPPX repeat was present on the polypeptide sequence of05WVZ03, while an N-glycosylation and an N-myristoylation was present on05WVZ05 and 05WVZ06 respectively.The isolated cDNA fragments were present as single copy genes in various resistantcultivars, as well as in the susceptible cultivar, Avoset-S, indicating that these genesare not unique to any one resistance cultivar. While naturally expressed in the IRplants, three genes (05WVZ01, 05WVZ05 and 05WVZ06) showed inducedexpression in the IS plants. The fourth gene (05WVZ03) was apparently expressed asmultiple copies within wheat. The expression profiles of none of the clones howeverindicated a real involvement in signaling.Plants are continuously challenged by a variety of pathogens. To survive thesechallenges, plants possess an arsenal of defenses, which are activated upon therecognition of the pathogen through certain signaling events. In some cases thedifference between resistance and susceptibility lies in the timely activation ofsignaling. Early signaling events include protein phosphorylation anddephosphorylation 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 theactivation of protein kinases were determined.The oxidative burst was assessed through various enzyme activities e.g. NADPHoxidase, superoxide dismutase (SOD) and peroxidase (POX) activity as well as H2O2levels. The earliest response of the plants was 18 h.p.i. when total protein kinaseactivity almost doubled in the infected resistant plants. This was followed by a similarincrease in activity 48 h.p.i. Both increases in total protein kinase activity wereaccompanied by increases in H2O2 levels and glutathione peroxidase activity.However, the second increase at 48 h.p.i. was more significant and is thereforeconcluded to be the oxidative burst. The recognition of the pathogen, as well as theactivation of the defenses therefore occurred between 18 and 48 h.p.i.Once this reaction time was established, differentially expressed cDNA fragmentswere amplified using DDRT-PCR. Eight different gene fragments were isolated,cloned and sequenced. These isolated cDNA fragments showed different levels ofhomology to four known polypeptides namely, a Nodulation protein B (fragment) fromRhizobuim sp, a TOBAC Hypothetical protein from Nicotiana tabacum, a Hypothetical

[发布机构] University of the Free State