Essential role of the small GTPase Rac in disease resistance of rice

Abstract

Production of reactive oxygen intermediates (ROI) and a form of programmed cell death called hypersensitive response (HR) are often associated with disease resistance of plants. We have previously shown that the Rac homolog of rice, OsRac1, is a regulator of ROI production and cell death in rice. Here we show that the constitutively active OsRac1 (i) causes HR-like responses and greatly reduces disease lesions against a virulent race of the rice blast fungus; (ii) causes resistance against a virulent race of bacterial blight; and (iii) causes enhanced production of a phytoalexin and alters expression of defense-related genes. The dominant-negative OsRac1 suppresses elicitor-induced ROI production in transgenic cell cultures, and in plants suppresses the HR induced by the avirulent race of the fungus. Taken together, our findings strongly suggest that OsRac1 has a general role in disease resistance of rice.

Figure 1

Effects of the constitutively active and dominant-negative OsRac1 in transgenic rice plants and cell cultures. (A) Diagrams of vector constructs introduced into rice. pRac1–19V and pRac1–24N are the constitutively active and dominant-negative forms of OsRac1, respectively. pRac1–19V was made by G19V, and pRac1–24N was made by T24N. (B) Cell death, H₂O₂ production, and autofluorescence (AF) in leaf sheath cells of transgenic rice expressing pRac1–19V and pRac1–24N. Plants ca. 2 months old were used for assays. DAB, diaminobenzidine. (C) Effects of a N-acetylchitooligosaccharide elicitor (3 μg/ml) on H₂O₂ production in untransformed and transgenic cell cultures expressing pRac1–19V and pRac1–24N. H₂O₂ levels in media were measured 2 h after addition of the elicitor by a published method (35). NT indicates untransformed control cell cultures. A1 and A5 are transgenic rice cell lines expressing the constitutively active OsRac1, and D41 and D42 are transgenic cell lines expressing the dominant-negative OsRac1. Bars indicate SEM obtained from seven to nine measurements. Student's t test (P < 0.05) was used for statistical analysis of the results.

Figure 2

Responses of transgenic rice plants expressing the constitutively active and the dominant-negative OsRac1 to infection by the rice blast fungus. (A) Responses of the leaf blade of transgenic rice plants expressing the constitutively active OsRac1 to infection by the compatible race 007. NT indicates untransformed control plants, and KA1, KA5, and KA12 are independent transgenic rice plants. A total of eight lines were tested, and results of three lines are shown. Lesions were observed three weeks after infection. (B) Responses of transgenic rice plants expressing the dominant-negative OsRac1 to infection by the incompatible race 031. NT indicates untransformed control plants, and KD1, KD2, and KD4 are independent transgenic rice plants. Lesions were observed 2 weeks after infection. (C) Responses of transgenic rice plants expressing the dominant-negative OsRac1 to infection by the compatible race 007. NT indicates untransformed control plants, and KD1, KD2, and KD4 are independent transgenic rice plants. Lesions were observed 2 weeks after infection. (D) Microscopic observation of the leaf sheath cells in transgenic plants expressing the constitutively active OsRac1 after infection with the compatible race 007. Representative photographs of KA7 (b and e) and KA12 (c and f) are shown. Excised leaf sheaths were infected with the fungus, and photographs were taken 48 h after infection. Arrows indicate appressoria formed from fungal spores. (a–c) Leaf sheath cells under normal light. (d–f) Leaf sheath cells under fluorescence light. (E) Microscopic observation of the leaf sheath cells in transgenic plants expressing the dominant-negative OsRac1 after infection with the incompatible race 031. Infection of the fungus and microscopic observations were performed as in D. Representative photographs of KD4 taken at 48 h (b and e) and 72 h (c and f) after infection are shown. (a–c) Leaf sheath cells under normal light. (d–f) Leaf sheath cells under fluorescence light.

Figure 3

Resistance of transgenic rice expressing constitutively active OsRac1 to a compatible race of rice bacterial blight. (A) Lesion phenotypes of transgenic plants after infection with a compatible race of Xanthomonas oryzae pv. oryzae. Photographs were taken 12 days after inoculation. NT, untransformed control plants; KA1, KA5, and KA12, independent transgenic rice plants expressing the constitutively active OsRac1; and KD1, KD2, and KD4, independent transgenic rice plants expressing the dominant-negative OsRac1. (B) Average lesion length of bacterial blight disease. Leaves were inoculated with the Japanese Xoo race 1. (Bars indicate SEM obtained from four to eight measurements.) Student's t test (P < 0.05) was used for statistical analysis of the results.

Figure 4

Phytoalexin production, alteration of gene expression in transgenic plants, and intracellular localization of OsRac1. (A) Increased production of momilactone A in transgenic rice expressing the constitutively active OsRac1. NT, untransformed control plants; KA1, KA4, KA5, KA7, KA8, KA10, and KA12, independent transgenic rice plants expressing the constitutively active OsRac1; KD1 and KD2, independent transgenic rice plants expressing the dominant-negative OsRac1. (B) Alterations in expression of two defense-related genes in transgenic rice plants expressing the constitutively active OsRac1. D9 is a rice gene homologous with terpenoid cyclase, and POX22.3 is a rice peroxidase induced by a bacterial pathogen (39). Total RNA was isolated from the leaves of transgenic plants and used for Northern blot analysis. Methods for RNA isolation and Northern blot analysis have been described (26). NT, untransformed control plants; and KA1, KA4, KA5, KA7, KA8, KA10, and KA12, independent transgenic rice plants expressing the constitutively active OsRac1. (C) Intracellular localization of OsRac1. (Upper) Various constructs were introduced into rice protoplasts isolated from suspension cells by electroporation. pGFP, GFP alone; pGRac1, a GFP fusion with the wild-type OsRac1; pGRac1–19V, a GFP fusion with the constitutively active OsRac1; pGRac1–212S, a GFP fusion with the constitutively active OsRac1 carrying the C212S mutation; pGRac1–1-24N, a GFP fusion with the dominant-negative OsRac1. C, cytoplasm; and V, vacuole.

Figure 5

Proposed model of disease resistance in rice. OsRac1 is placed downstream of R genes and functions to activate NADPH oxidase to cause an oxidative burst during resistance reactions. OsRac1 is also an intermediate in reactions N-acetylchitooligosaccharide to elicitors such as N-acetylchitooligosaccharides. ROI generated by NADPH oxidase then induce cell death, phytoalexin production, and alteration of gene expression. Production of autofluorescence is OsRac1 dependent. These coordinated changes in cellular metabolisms lead to disease resistance.