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

Abstract

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

Afrikaans: Die doel van hierdie studie was om die oksidatiewe uitbarsting vastestel en die proteïen kinases wat daarby betrokke is te identifiseer by die vroeë seintransduksie en aanskakeling van verdedigingsreaksies in ‘n weerstandbiedende koring-kultivar (Yr1) na infeksie met Puccinia striiformis f. sp. tritici (streep-roes). Daardeur was die vroegste punt van herkenning van die patogeen deur die plant bewerkstellig. Daardie tyd periode was dan gebruik om gene te identifiseer wat moontlik betrokke kon wees by vroeë seintransduksie. Plante word voortdurend deur patogene bedreig, maar besit die vermoë om hulself te verdedig deur verskeie verdedigingsmeganismes te gebruik. Hierdie meganismes word deur spesifieke seine in die plant na die herkenning van die patogeen geaktiveer. In sekere gevalle lê die verskil tussen weerstandbiedendheid en vatbaarheid vir spesifieke siektes, in die reaksietyd van die plant om die betrokke verdedigingsmeganismes aan te skakel. Proteïen fosforilering en defosforilering, tesame met die produksie van reaktiewe suurstofspesies (die oksidatiewe uitbarsting) vorm deel van vroeë seinoordraging in weerstandbiedende plante na infeksie. Eerstens is bepaal hoe vinnig na infeksie, die patogeen deur die plant herken word. Verder is ook bepaal wanneer die plant se verdedigingsmeganismes aangeskakel is deur vas te stel wanneer die oksidatiewe uitbarsting plaasgevind het, asook deur die betrokkenheid van proteïen kinases in die verdedigingsreaksie te ondersoek. Die oksidatiewe uitbarsting was bepaal deur die aktiwiteite van ‘n verskeidenheid ensieme te bestudeer nl. NADPH oksidase, superoksied dismutase (SOD) en peroksidase (POX). Die H2O2 vlakke is ook gemeet. Die vroegste reaksie in geïnfekteerde weerstandbiedende plante was 18 uur na infeksie waargeneem met ‘n toename in kinase aktiwiteit. ‘n Verdere toename in kinase aktiwiteit was 48 uur na infeksie waargeneem. Beide hierdie toenames was deur toenames in H2O2 vlakke en glutatioon-peroksidase aktiwiteit gevolg. Daar is tot die gevolgtrekking gekom dat die oksidatiewe uitbarsting 48 uur na infeksie plaasgevind het, aangesien die toename in kinase aktiwitiet en H2O2 vlakke hier meer beduidend was. Dus was die vroegste herkenning tesame met die aktivering van die verdedigingsresponse vasgestel as die periode tussen 18 en 48 uur na infeksie. Na die vasstelling van bogenoemde, is cDNA fragmente wat geinduseerd tot uiting kom geamplifiseer d.m.v. DDRT-PCR. Agt cDNA fragmente is geïsoleer, gekloneer en die nukleotied volgordes daarvan bepaal. Hierdie cDNA fragmente het homologie getoon met vier reeds bekende polipeptiede nl. ‘n noduleerings proteïen B fragment van Rhizobuim sp, ‘n TOBAC hipotetiese protein van Nicotiana tabacum, ‘n hipotetiese protein van Oryza sativa en ‘n protein met ‘n HGWP motief van Oryza sativa onderskeidelik. Geen unieke proteïen motiewe was op die onderskeie polipeptiedes gevind nie, alhoewel daar verskeie fosforileringsgebiede teenwoordig was. Die teenwoordigheid van fosforileringsgebiede dui op die moontlike regulering deur fosforilering. ‘n XYPPX herhalende volgorde was teenwoordig op die 05WVZ03 polipeptied, terwyl ‘n Nglukosilerings - en ‘n N-miristolerings – gebied onderskeidelik op 05WVZ05 en 05WVZ06 teenwoordig was. Die geïsoleerde cDNA fragmente is as enkelkopie gene in verskeie weerstandbiedende kultivars asook in die vatbare Avoset-S kultivar teenwoordig. Dit dui daarop dat hierdie gene nie uniek is tot enige van die weerstandbiedende kultivars is nie. Hoewel al die gene tot uiting kom in die weerstandbiedende plante, het drie van hierdie gene nl. 05WVZ01, 05WVZ05 en 05WVZ06 geïnduseerde uiting in die vatbare geïnfekteerde plante vertoon. Die geen, 05WVZ03 word moontlik as veelvuldige kopiëe in koring uitgedruk. Die uitingsprofiele van al die klone toon egter dat nie een ‘n direkte betrokkenheid by vroeë seinoordrag het nie.