Rice Genetics IV - IRRI books - International Rice Research Institute

tive indica rice varieties supports the hypothesis that a single amino acid substitution,serine for alanine at position 918, eliminates Pi-ta function.AVR-Pita encodes a putative metalloproteaseAVR-Pita was cloned by a map-based approach (Orbach et al 2000) based on its locationadjacent to a telomere (Tel 5) on chromosome 3 of the integrated map of the blastfungus (Nitta et al 1997, Sweigard et al 1993). The AVR-Pita gene encodes a polypeptidewith 223 amino acids and features that are characteristic of fungal-secretedmetalloproteases. Amino acids 173–182 form a characteristic motif of a neutral zincmetalloprotease and mutational analysis of AVR-Pita indicates that maintenance ofthe protease motif is essential for avirulence (G.T. Bryan and B. Valent, unpublishedresults). Homology between the AVR-Pita protein and the metalloprotease NpII fromAspergillus oryzae is confined to the C-terminal 176 amino acids, corresponding tothe mature region of the NPII prepro-protein (Tatsumi et al 1991). Thus, we hypothesizethat AVR-Pita also contains a prepro-region that is processed to an active protease(Fig. 1B). Biochemical demonstration of protease activity, however, remains tobe demonstrated for AVR-Pita.A model for Pi-ta-mediated resistanceTransient expression of Pi-ta and AVR-Pita in plant cells induces resistanceIf the ligand/receptor model for gene-for-gene interactions holds for the Pi-ta/AVR-Pita system, the Pi-ta receptor might bind either the AVR-Pita protein itself or a peptidereleased from a substrate of the putative AVR-Pita protease. Vacuum infiltrationor other apoplastic applications of various forms of recombinant AVR-Pita protein(Fig. 1B) into Pi-ta-containing leaf tissue failed to induce hypersensitive resistance(HR) (G.T. Bryan and B. Valent, unpublished results). These data, along with theputative cytoplasmic location of Pi-ta, suggested that recognition of AVR-Pita mayoccur inside the plant cell.A transient expression assay provides direct evidence that expression of AVR-Pitainside rice cells containing Pi-ta is sufficient to induce HR (Bryan et al 2000, Jia et al2000). For this assay, 1-wk-old intact rice seedlings are biolistically transformed witha GUS reporter gene (E. coli uidA linked to the constitutive 35S promoter from cauliflowermosaic virus). Bombarded cells accumulate β-glucuronidase enzyme (GUS),which is assayed histochemically for accumulation of blue pigment. GUS activity ina transformed cell indicates a “healthy” cell that is not undergoing HR, and reducedGUS activity indicates a cell undergoing HR. Thus, particle bombardment transformationof rice seedlings results in multiple spots of blue pigment in the absence ofHR (Fig. 2).We engineered the full-length AVR-Pita gene and AVR-Pita 176 encoding the predictedmature protease (Fig. 1B) for direct expression in plant cells, and introducedthese constructs along with the GUS reporter gene in the transient assay (Jia et al2000). In these assays, GUS activity was inhibited whenever AVR-Pita 176 and Pi-tawere expressed together in rice cells (Fig. 2). This was true whether the rice varietyEnhancing deployment of genes for blast resistance: . . . 313