Rice Genetics IV - IRRI books - International Rice Research Institute

an effective inducer of host resistance against blast infection (Midoh and Iwata 1996).Expression of PBZ1 and PR1 was highly activated in leaves of the three cdr mutantswhen the lesion was developed. In contrast, very low expression of these two geneswas detected in leaves of the wild type. In mutant leaves showing no visible lesions,PBZ1 expression was much lower. Thus, PBZ1 expression was closely correlatedwith development of the lesion. These results indicate that defense-related genes arehighly induced in leaves of the cdr mutants exhibiting lesion formation.The level of momilactone A in the leaves of the cdr mutants that showed lesionswas increased approximately 100-fold in cdr1 and Cdr3 and more than 400-fold incdr2. In leaves that showed only a few lesions, much lower levels of momilactone Awere detected. These results clearly indicate that the level of this major phytoalexin ofrice in these mutants is much higher than that found in the wild type.Enhanced production of ROS in response to calyculin A,an inhibitor of protein phosphatase, in the cdr mutantsIt was previously shown that calyculin A, an inhibitor of protein phosphatase 1, inducesROS in cell suspension cultures of rice (Kuchitsu et al 1995). Thus, we examinedits effect on H 2 O 2 production in cell suspension cultures of the mutants and thewild type. The wild-type cells generated H 2 O 2 after treatment with calyculin A. Theobserved H 2 O 2 production by calyculin A was inhibited by DPI, a potent inhibitor ofthe NADPH oxidase, suggesting that the plasma membrane NADPH oxidase wasinvolved in the H 2 O 2 production in the cell cultures of rice. When suspension cellcultures of cdr1 and cdr2 were treated with calyculin A, increased production of H 2 O 2was observed and this enhanced production of H 2 O 2 was inhibited by DPI. No H 2 O 2production was observed if they were not treated with the protein phosphatase inhibitor.In contrast to cdr1 and cdr2, no enhancement of H 2 O 2 production was observed insuspension cell cultures of Cdr3.The results obtained from the studies using the cell cultures suggest that the twomutants, cdr1 and cdr2, have alterations in the signaling cascade leading to activationof NADPH oxidase. Since these two mutations are both recessive, they may be mutationsof factors that normally suppress NADPH oxidase in the absence of signalsfrom pathogens. In contrast, Cdr3 may have alterations downstream of NADPH oxidase.Identification of proteins whose phosphorylation levelswere increased in cdr mutantsTo study proteins whose phosphorylation levels were increased by the cdr mutations,we compared profiles of 32 P-labeled protein spots separated by two-dimensional gelelectrophoresis. We identified four protein spots that were phosphorylated by calyculinA treatment and the degree of phosphorylation was increased in the cdr mutants. Onespot was shown to be more phosphorylated in the cdr1 than in the wild type. For theother three protein spots, phosphorylation increased equally in both cdr1 and cdr2compared with the wild type. We attempted to sequence these polypeptides after proteasetreatment and obtained information on the amino acid sequences. Based on theMolecular signaling in disease resistance of rice 331