The rare sugar d-allose acts as a triggering molecule of rice defence via ROS generation

Only d-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to d-allose. d-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-d-allose, a structural derivative of d-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding d-allose kinase to increase d-allose 6-phosphate synthesis were more sensitive to d-allose, but E. coli AlsI encoding d-allose 6-phosphate isomerase expression to decrease d-allose 6-phosphate reduced sensitivity. A d-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, d-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of d-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of d-allose to d-allose 6-phosphate, and treatment with d-allose might prove to be useful for reducing disease development in rice.


Fig. S4. Metabolic pathway of D-allose in Escherichia coli.
Metabolic pathway of D-allose in E. coli described by Kim et al. (1997) was shown.   expression in leaves at 0 to 24 h after treatment with 5 mM D-allose (D-All) or D-glucose (D-Glc), relative to control (no sugar) (Con) (± SE, n = 4).
(C) Subcellular localization of GFP-tagged OsG6PDH1 and OsG6PDH2 in plant cells.
Epidermal layers of tobacco leaves were bombarded with particles coated with constructs to express GFP alone (upper), OsG6PDH1-GFP (middle), and OsG6PDH2-GFP (lower).    a Kinetic parameters were determined using G6PDH-coupled assay for A6P with a maximum concentration of 5 mM (Wakao and Benning, 2005). ND: not detected. b G6PDH activity with G6P with 10 mM DTT.

Transient localization assay
G6PDH1-or G6PDH2-GFP fusion proteins were expressed using a particle gun-mediated DNA delivery to tobacco epidermal cells and imaged using epifluorescence system DP70-SET-A, differential interference contrast optics and an Olympus BX51 microscope (Olympus, Tokyo, Japan). After bombardment, tissues were incubated for 24 h at 24˚C in the dark; ca. 100 cells/construct were examined for GFP localization in at least three independent experiments. All methods are as described by Yamasaki and Akimitsu (2007).

Genotypic determination of homozygote for Tos17 insertion
Total DNA from leaves of WT and Tos17 mutant lines were isolated using NecleoSpin plant II (MACHEREY-NAGEL, Hoerdt, France) and manufacturer's instructions. PCR was run using KOD DNA polymerase (TOYOBO, Osaka, Japan) in a thermal cycler with initial PCR activation at 94˚C for 2 min followed by 30 cycles of 3-step cycling (denaturation at 98˚C for 10 sec annealing at 60˚C for 30 sec, and extension at 68˚C for 1 min) with gene-specific primers used for PCR (Table S1).

Recombinant G6PDH assays
Activity of recombinant rice G6PDHs was measured spectrophotometrically (340 nm) at 25°C by detecting NADP reduction via G6PDH reaction, which is coupled with G6P production, as described by Wakao and Benning (2005)

Construction of overexpression vectors
F GTCGACATGTCAGGAGGATCTTCACCAA R GTCGACAAGGGTGGGTGGTA F, forward direction; R, reverse direction. Extra nucleotides attached to introduce restriction sites are underlined. Kinetic parameters were determined using G6PDH-coupled assay for G6P (Wakao and Benning, 2005).