Rice copine genes OsBON1 and OsBON3 function as suppressors of broad-spectrum disease resistance

Abstract

Breeding for disease resistance is the most effective strategy to control diseases, particularly with broad-spectrum disease resistance in many crops. However, knowledge on genes and mechanism of broad-spectrum resistance and trade-off between defence and growth in crops is limited. Here, we show that the rice copine genes OsBON1 and OsBON3 are critical suppressors of immunity. Both OsBON1 and OsBON3 changed their protein subcellular localization upon pathogen challenge. Knockdown of OsBON1 and dominant negative mutant of OsBON3 each enhanced resistance to rice bacterial and fungal pathogens with either hemibiotrophic or necrotrophic lifestyles. The defence activation in OsBON1 knockdown mutants was associated with reduced growth, both of which were largely suppressed under high temperature. In contrast, overexpression of OsBON1 or OsBON3 decreased disease resistance and promoted plant growth. However, neither OsBON1 nor OsBON3 could rescue the dwarf phenotype of the Arabidopsis BON1 knockout mutant, suggesting a divergence of the rice and Arabidopsis copine genes. Our study therefore shows that the rice copine genes play a negative role in regulating disease resistance and their expression level and protein location likely have a large impact on the balance between immunity and agronomic traits.

Keywords: bacterial blight; blast; growth; rice immunity; sheath blight; trade-off.

Figure 1

Induction of OsBON1 and OsBON3 by Xoo and characterization of OsBON1‐RNAi and overexpression transgenic plants. (a,b) Induction of OsBON1 (a) and OsBON3 (b) RNA expression by Xoo. Two‐month‐old plants were inoculated by Xoo (strain PXO99A), with mock inoculation (leaf‐clipping with water). Total RNAs were isolated from the inoculated leaves at different time points. Shown are transcript levels of OsBON1 and OsBON3 detected by qRT‐PCR. (c–e) RNA expression levels of OsBON1 (c,e) and OsBON3 (d) in representative lines of OsBON1‐ RNAi (c), OsBON1‐RNAi (d) and OsBON1‐OE (e) compared to the wild‐type TP309 detected by qRT‐PCR. (f) Protein levels of OsBON1 in independent transgenic lines and the wild type detected by Western blot using an anti‐OsBON1 antibody. OsACTIN was used as a control. The rice OsActin1 gene was used as an internal control to normalize expression levels for qRT‐PCR (a–e). Data are shown as means ± SD from three biological replicates (Student's t‐test, *P < 0.05; **P < 0.01; ***P < 0.001) (a–c and e).

Figure 2

OsBON1 negatively regulates disease resistance to Xoo and fungal pathogens M. oryzae and R. solani. Shown are disease resistance phenotypes to Xoo (a–d), M. oryzae (e,f) and R. solani (g,h) in OsBON1‐RNAi and OsBON1‐OE lines. (a,b)Disease symptoms of OsBON1‐ RNAi (a) and OsBON1‐OE lines compared with wild‐type TP309 at 14 dpi with Xoo strain PXO99A. (c) Lesion lengths of OsBON1‐ RNAi and OsBON1‐OE lines compared with wild‐type TP309 at 14 dpi with Xoo strain PXO99A. Data are shown as box plots (n ≥ 50). (d) Xoo strain PXO99A bacterial populations per leaf were measured at 0, 4 and 8 dpi. Values are shown as mean ± SD (n = 3). The bacterial growth curve experiment was repeated twice with similar results. (e) Disease symptoms of the wild‐type TP309, OsBON1‐RNAi lines and OsBON1‐OE lines at 7 dpi with M. oryzae (isolate Hoku1). Scale bars = 2 cm. (f) Average lesion number per leaf of TP309, OsBON1‐RNAi lines and OsBON1‐OE lines at 7 dpi with M. oryzae. Values are means ± SD (n ≥ 30). This experiment was repeated independently at least three times with similar results. (g) Disease scores (from 0 to 5) used in sheath blight resistance at 14 dpi with R. solani AG1‐IA isolate RH‐9. (h) Disease profiles of sheath blight expressed as percentages of six disease scores in TP309, OsBON1‐RNAi and OsBON1‐ OE lines at 14 dpi. Lesion length (c) was measured independently for at least three generations with similar results. Asterisks indicate statistically significant differences in comparison with the wild‐type control (Student's t‐test, *P < 0.05; **P < 0.01; ***P < 0.001). Note that the knockdown and overexpression of OsBON1 significantly increased and decreased Xoo resistance, respectively.

Figure 3

OsBON1 affects expression of rice defence‐responsive genes. RNA expression of PR1a (a) and PR4 (b) was detected in 2‐month‐old OsBON1‐RNAi and OsBON1‐OE transgenic and wild‐type TP309 before and after inoculation with Xoo PXO99A. The OsActin1 gene was used as an internal control, and expression levels were normalized to 0 h of TP309. Data are means ± SD (n = 3). Asterisks indicate significant difference in comparison with the wild‐type plants (Student's t‐test, *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4

OsBON1 promotes rice growth and development. (a,b) Morphology (a) and tiller number (b) of 2‐month‐old TP309 and OsBON1‐RNAi plants. (c,d) Morphology (c) and tiller number (d) of 2‐month‐old TP309 and OsBON1‐OE plants. (e,f) Morphology (e) and plant height (f) of TP309, OsBON1‐RNAi and OsBON1‐OE mature plants. Data on tiller number and plant height are shown as box plots (n ≥ 30). Asterisks indicate statistically significant differences in comparison with the wild‐type control (Student's t‐test, ***P < 0.001) (b, d and f). Scale bars = 20 cm (a, c and e).

Figure 5

High temperature inhibits the stunted growth phenotype and PR1a overexpression of OsBON1‐RNAi plants. (a,b) Morphology of 2‐week‐old TP309 and OsBON1‐RNAi plants grown at 26 °C (a) and 32 °C (b). Note that the 32 °C growth inhibited the dwarfing of OsBON 1‐RNAi plants. Scale bars = 8 cm. (c,d) Plant heights of TP309 and OsBON1‐RNAi lines grown at 26 °C (c) and 32 °C (d). Data are means ± SD (n ≥ 20). No significant difference was detected at 32° C. (e,f) Expression levels of OsPR1a revealed by qRT‐PCR in the wild‐type TP309 and OsBON1‐RNAi lines at 26 °C (e) and 32 °C (f). The OsActin1 gene was used as an internal control. Note that the constitutive activation of OsPR1a in the OsBON1‐RNAi lines was compromised under high temperature (32 °C). No significant difference was detected at 32 °C. Asterisks indicate statistically significant difference in comparison with the wild‐type control (Student's t‐test, *P < 0.05; **P < 0.01) (c and e).

Figure 6

OsBON3 negatively regulates rice disease resistance. (a) Protein levels of OsBON3 in representative OsBON3‐OE and OsBON3‐eGFP transgenic lines and wild types detected by Western blot using an anti‐BON3 antibody. (b) Disease symptoms of OsBON3‐OE lines in comparison with the wild‐type NIP at 14 dpi with Xoo PXO99A. (c) Lesion lengths of OsBON3‐OE and NIP at 14 dpi with Xoo PXO99A. Data are shown in box plots (n ≥ 50). (d) Bacterial growth measured at 0, 4 and 8 dpi with Xoo PXO99A. Data are means ± SD (n  =  3). The bacterial growth curve experiment was repeated twice with similar results. (e) Lesion mimic phenotype of 12‐week‐old OsBON3‐eGFP plants. Scale bars = 1 cm. (f) DAB staining of H₂O₂ accumulation in twelve‐week‐old OsBON3‐eGFP leaves. Scale bars = 1 cm. (g,h) Disease resistance of OsBON3‐eGFP lines compared with the wild type at 14 dpi with Xoo PXO99A. Shown here are disease symptoms (g) and lesion length (h). Lesion length (h) data are shown as box plots (n ≥ 50). Lesion length measurement was repeated independently for 3 generations with similar results. Asterisks indicate statistically significant difference in comparison with the wild‐type control (Student's t‐test, *P < 0.05; **P < 0.01; ***P < 0.001) (c,d and h).

Figure 7

Pathogen infection alters subcellular localization of OsBON1 and OsBON3. (a) Fluorescence signals of OsBON1‐YFP and OsBON3‐YFP expressed in rice protoplasts and N. benthamiana leaves. Both proteins are mainly localized to the plasma membrane in contrast to the control protein YFP. Scale bars, 10 μm (rice protoplasts) or 50 μm (tobacco leaves). (b) Fluorescence signals of OsBON1‐YFP and OsBON3‐YFP in root cells of stable transgenic lines of Ubi::OsBON1‐eGFP and Ubi::OsBON3‐eGFP. Plasmolysis was induced using 30% sucrose. Scale bars = 50 μm. (c) Localization of OsBON1‐eGFP protein after infection by Xoo. Roots of OsBON1‐eGFP seedlings were infected with Xoo or mock‐inoculated with water. Shown are confocal images taken at 48 hpi. Scale bars = 50 μm. (d) Localization change of OsBON1‐eGFP during M. oryzae infection. Shown are laser confocal images taken at 0 h (control) and 36 h after leaf sheath inoculation with M. oryzae. The transgenic plants with the plasma membrane‐located OsAnn‐eGFP were used as a control. Arrows indicate biotrophic interfacial complex (BIC). Scale bars = 20 μm.