Promoter mutations of an essential gene for pollen development result in disease resistance in rice

Abstract

Disease resistance and sexual reproductive development are generally considered as separate biological processes, regulated by different sets of genes. Here we show that xa13, a recessive allele conferring disease resistance against bacterial blight, one of the most devastating rice diseases worldwide, plays a key role in both disease resistance and pollen development. The dominant allele, Xa13, is required for both bacterial growth and pollen development. Promoter mutations in Xa13 cause down-regulation of expression during host-pathogen interaction, resulting in the fully recessive xa13 that confers race-specific resistance. The recessive xa13 allele represents a new type of plant disease resistance.

Figure 1.

Identification of xa13. (A) Fine mapping of the xa13 locus on BAC clone 14L03. The figures between markers indicate the numbers of recombination events detected between the xa13 locus and corresponding markers. (B) Association between resistance and the transgene. All of the susceptible transgenic plants have the PCR fragment of Xa13 promoter as in IR24 carrying Xa13, while the resistant plants do not have this fragment. (C) Plasma membrane location of the GFP-XA13 protein revealed by transformation of calli from Zhonghua 11. Bars, 8.0 μm. (Left) GFP-XA13 expression on plasma membrane of callus cells. (Middle) Propidium iodide staining. (Right) Overlaying of left and middle images.

Figure 2.

Expression profiles of xa13 and Xa13 detected by quantitative RT–PCR and the relationship between resistance and levels of xa13- and Xa13-transcripts. Asterisks indicate significant (P < 0.05, t-test) reductions in lesion length between transgenic plants and wild-type plants (ck) measured at 14 d after PXO99 inoculation. Error bars are standard deviations. (A) Xa13-RNAi transformation of Zhonghua 11. Xa13 expression in leaves relative to the untransformed controls was detected at 3 d after inoculation. (B) xa13-RNAi transformation of IRBB13. The xa13 expression in leaves relative to the untransformed controls was detected before pathogen inoculation. (C) Expression levels of Xa13 and xa13 in different tissues, measured relative to xa13 expression in the flag leaves of IRBB13. (D) Expression levels of xa13 and Xa13 relative to the corresponding untreated (0) control in the leaves of near-isogenic lines IRBB13 and IR24, respectively, at 8, 24, and 72 h (8, 24, and 72) after PXO99 inoculation or mock (water) inoculation as the control.

Figure 3.

Sequence comparison of the promoter regions of xa13 and Xa13 from different rice lines. Nucleotide substitution, deletion, or insertion are identified in different rice lines with reference to the sequence of IR24. The triangles with a figure indicate the numbers of nucleotides inserted or deleted and the triangles without a figure represent single-nucleotide insertions/deletions. The green-line-boxed regions represent the fragments relative to the −69 to −86 region of IR24.

Figure 4.

Bacterial growth analysis and the expression pattern of Xa13 in leaves. (A) Growth of Xoo strain PXO99 in leaves of wild-type IRBB13 and T₀ transgenic line D09O3-7 carrying Xa13. Bacterial population was determined from three leaves at each time point by counting colony-forming units (cfu) (Sun et al. 2004). (B) Xa13 promoter-β-glucuronidase (GUS) expression in transgenic rice plant. The blue color indicates that GUS was preferentially expressed in the parenchyma cells surrounding the vascular element (V). Bar, 30 μm.

Figure 5.

Association of Xa13 expression and pollen development. Bars, 30 μm. (Bp) Bicellular pollen grain; (D) dyad; (Dp) degenerated pollen grain; (E) epidermis; (En) endothecium; (Ml) middle layer; (Up) unicellular pollen grain; (T) tapetum; (Tp) tricellular pollen grain; (V) vascular element; (W) anther wall. (A) Pollen development at different stages in wild-type Minghui 63 and Xa13-suppressed T₀ plant D75RMH2, showing that there is no apparent difference between the wild-type and transgenic plants at the unicellular pollen grain stage, whereas various abnormalities are observed at bi- and tricellular pollen grain stages. (B) Expression of Xa13 in anthers of wild-type Minghui 63 and T₀ plants D75RMH2 and D75RMH5 examined by in situ hybridization. The dark blue color in the pollen grains, the cells of anther wall, and the connective vascular element indicates hybridization of Xa13 transcripts.