Rice Genetics IV - IRRI books - International Rice Research Institute

transcription factor, resulting in activation of reporter gene expression in yeast. DNAfragments encoding either the full-length AVR-Pita or the predicted mature AVR-Pita 176 proteins were cloned into activation domain vectors and tested against a DNAbindingdomain fusion, either to the full-length Pi-ta protein or to a DNA fragmentencoding the Pi-ta LRD (Fig. 1A). Expression of the AVR-Pita 176 fusion protein togetherwith the Pi-ta LRD fusion protein produced a functional transcription factorthat allowed expression of two reporter genes, a histidine biosynthetic gene requiredfor prototrophic growth and the lacZ reporter gene. This suggested that the AVR-Pita 176 protein binds directly to the LRD region from Pi-ta. Interaction, defined byexpression of both reporter genes, was not detected when the AVR protein was fusedto the DNA-binding domain and the LRD protein was fused to the activation domain.Interaction was also not detected with the sensitive pi-ta – LRD A918S , in which serinehad been substituted for alanine at position 918. Furthermore, a virulent avr-pita 176mutant with an amino acid substitution in the putative metalloprotease active site(E177D) failed to interact with either form of the LRD. Thus, the specificity of bindingis consistent with the in vivo specificity in the Pi-ta system, and it seems reasonableto conclude that binding between AVR-Pita 176 and the leucine-rich domain of Pitaplays a role in inducing the plant defense response.Biochemical confirmation of binding between the AVR-Pita 176 and Pi-ta LRDpolypeptides was obtained by a modified “far-western analysis” (Jia et al 2000). Forthis procedure, recombinant forms of AVR-Pita polypeptides, which are insoluble asexpressed in E. coli, were purified, separated using SDS-PAGE gels, transferred to anitrocellulose membrane, and allowed to refold in the presence of ZnCl 2 . Membranescontaining refolded AVR-Pita proteins were incubated with extracts of E. coli proteinscontaining soluble S-tagged Pi-ta or LRD polypeptides and rinsed extensively.Pi-ta polypeptides that were bound to AVR-Pita polypeptides on these membraneswere detected with the S-antibody (Novagen). Again, the LRD domain of Pi-ta boundspecifically to the avirulent form of AVR-Pita 176 . That is, LRD from the resistantallele bound to AVR-Pita 176 , but not to longer forms of the AVR protein, and theLRD A918S from the sensitive allele did not bind under the same conditions. Thus,specific Pi-ta/AVR-Pita binding has been demonstrated in two separate assays.These data, along with the results of the transient expression assays, support thehypothesis that Pi-ta functions as a receptor that binds to the AVR-Pita 176 protein inthe cytoplasm of the invaded plant cell (Fig. 3). In addition, they suggest that theelicitor-binding domain of Pi-ta protein resides within the leucine-rich C-terminalregion and that a single amino acid substitution (A918S) eliminates R gene function.AVR-Pita population analysisSasaki first discovered physiological races of the rice blast pathogen in 1922 (Yamada1985) and, since that time, rice pathogens have been classified into hundreds of physiologicalraces, or pathotypes, that differ in their ability to cause disease on differentrice varieties. According to the gene-for-gene hypothesis, the pathotype of a strain ofthe fungus is determined by its AVR genes, which control sensitivity to various rice REnhancing deployment of genes for blast resistance: . . . 315