Differential Expression of Defense/Stress-Related Marker Proteins in Leaves of a Unique Rice Blast Lesion Mimic Mutant (blm) Young-Ho Jung, Randeep Rakwal,*,, § Ganesh Kumar Agrawal, § Junko Shibato, Jung-A Kim,

We analyzed a unique rice (Oryza sativa L.) blast lesion mimic (blm) mutant for differentially expressed proteins in leaves of one- and two-week-old seedlings manifesting the lesion mimic phenotype. Gel-based one- and two-dimensional electrophoresis (1- and 2-DGE) was performed using leaves (blm and wild-type, WT) before (stage 1, S1) and after (stage 2, S2) lesion formation. 1-DGE immunoblotting revealed potent increase in the expression of a key pathogenesis-related (PR) marker biosynthetic enzyme, naringenin 7-O-methyltransferase, involved in rice phytoalexin sakuranetin biosynthesis, and three oxidative-stress-related marker proteins, catalase, ascorbate peroxidase (APX), and superoxide dismutase (SOD) in leaves of the blm mutant. 2-D gel immunoblotting analysis with anti-APX and anti-SOD antibodies revealed newly appearing cross-reacting protein spots in blm. 2-DGE analysis detected 50 Coomassie brilliant blue-stained protein spots differentially expressed in blm. A total of 23 and 44 protein spots was excised for analysis by N-terminal amino acid sequencing and nano-electrospray ionization liquid chromatography mass spectrometry, respectively; 26 nonredundant proteins were identified. The pathogenesis-related class 5 and 10 proteins, including a new OsPR10d protein, were significantly induced in blm. The OsPR5 protein spot was stained with Pro-Q Diamond phosphoprotein gel stain suggesting OsPR5 to be a putative phosphoprotein. Surprisingly, protein spot 20, a lea