MA TORRESl, H ONOUCHI2, S HAMADA2, C MACHIDA2, Y MACHIDA2, KE HAMMOND-KOSACKl and JDG JONESl lThe Sainsbury Laboratory, John lnnes Centre, Colney Lane, Norwich, NR4 7UH, UK; 2Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan. Background and objectives
The objectives of our research are the identification of components of the NADPH oxidase in plants and to elucidate the role of this enzyme in ROS production and in plant defence response. The production of reactive oxygen species (ROS) is one of the earliest events detected in the plant defence response activated following pathogen infection [1]. Various lines of evidence suggest that a plasma membrane NADPH oxidase, analogous to that which generates the respiratory burst in mammalian phagocytes [2], is the most likely source of this ROS detected in plants upon elicitation. A rice gene homologue to gp91phox, one of the membrane components of the mammalian NADPH oxidase, has recently been identified in our laboratory [3]. Based on the homology between the rice gene OsrbohA (for Oryza sativa respiratory burst oxidase homologue A) and the human protein gp91phox, we designed a PCRbased strategy to find homologues of this component of the NADPH oxidase in Arabidopsis. Results and conclusions
By using degenerate primers, we identified PCR products corresponding to 6 different genes in Arabidopsis, that we called Atrboh A-F . Interestingly, products corresponding to homologues D, E and F were amplified when cDNA was used as a template, while products corresponding to homologues A, B and C were only obtained from the genomic DNA template. The genomic clones containing the homologues A-E, as well as cDNAs corresponding to homologues D and F, have been identified and sequenced. The alignment of the deduced protein sequences reveals that all 6 genes are highly conserved, especially in the regions identified in the carboxyl end of gp91phox as cofactor binding sites. However each Arabidopsis gene is longer than gp91phox; while the human protein has 570 aa, the Arabidopsis ones range from 843 to 948 aa. Interestingly, in the amino termini of each Arabidopsis homologue, not present in gp91phox, there is a calcium binding motif which suggests a direct regulatory effect of calcium in the activity of the NADPH oxidase in plants. To elucidate the role of the NADPH oxidase in ROS production and in the activation of other defence responses, we are focussing our efforts in generating mutants of these Atrboh genes. In one strategy, we are using a PVX based constuct (Amplicon) [4] carrying the 3' end of AtrbohD to gene silence this homologue and possibly the other homologues because the region fused to the amplicon contains motifs conserved between all the Rboh genes. In a second strategy, we have generated a dominant interfering mutant allele by overexpressing a full length AtrbohD CDNA carrying a pro(415)>his mutation. In humans this mutation is known to inactivate the NADPH oxidase but does not affect complex assembly [2]. In a third strategy, we are trying to identify plants containing transposon insertions inside the Atrboh genes by screening pools of DNA derived from plants containing dspm insertions. If via either of these strategies plant resistance in a normally incompatible interaction is severely compromised, this will be good proof of both the involvement of a NADPH oxidase complex in the generation of the active oxygen species in plants during pathogen attack and its absolute requirement for resistance. References
1. Hammond-Kosack KE, Jones JDG, 1996. Plant Cell 8,1773-1791. 2. Roos D, Deboer M, Kuribayashi F, Meischi C, Weening RS, Segal AW, Ahlin A, Nemet K, Hossle JP, Bernatowska-Matuszkiewicz E, Middleton-Price H, 1996. Blood 87, 1663-1681. 3. Groom QJ, Torres MA, Fordhamskeiton AP, Hammond-Kosack KE, Robinson NJ, Jones JDG, 1996. Plant Journal 10, 512-522. 4. Angell SM, Baulcombe DC, 1997. The EMBO Journal 16, 3675-3684.