Rice Genetics IV - IRRI books - International Rice Research Institute

A B CFig. 2. Rice leaves showing localized blue pigment resulting fromGUS enzyme activity after co-bombardment of the GUS reporter genealong with (A) Pi-ta alone, (B) Pi-ta and avirulent AVR-Pita, and (C)Pi-ta and virulent avr-pita – . GUS activity is high in the absence ofhypersensitive resistance (HR) and low in cells undergoing HR.contained the native Pi-ta gene or whether a Pi-ta transgene was co-transformed,along with the GUS reporter and the AVR-Pita 176 genes, into rice varieties lacking Pita.Transient expression of full-length AVR-Pita showed low levels of HR-inducingactivity compared with AVR-Pita 176 , supporting the hypothesis that the AVR-Pita proteinis processed to a mature active form. Mutant avr-pita 176 genes that no longerconfer avirulence to the pathogen in fungal infection assays also fail to induce HR inthe transient assay (Bryan et al 2000, Jia et al 2000). The sensitive pi-ta – allele fromC101A51 fails to induce HR in combination with AVR-Pita. These results suggestthat AVR-Pita and Pi-ta proteins act together inside plant cells to induce defense responses.In addition, these experiments demonstrate that AVR-Pita is the only pathogenprotein that is required for inducing Pi-ta-mediated resistance.The AVR-Pita protein interacts directly with the Pi-ta proteinWe have demonstrated that the Pi-ta LRD region binds to the avirulent AVR-Pita 176protein in the yeast two-hybrid system (Jia et al 2000). In this assay, the DNA bindingdomain and the transcriptional activation domain of a transcription factor are clonedseparately into vectors that produce fusion proteins between each domain and one ofthe proteins being tested. Binding between the two proteins of interest links the DNAbindingdomain to the transcriptional activation domain and reconstitutes an active314 Valent et al