The rice CYP78A gene BSR2 confers resistance to Rhizoctonia solani and affects seed size and growth in Arabidopsis and rice

Abstract

The fungal pathogen Rhizoctonia solani causes devastating diseases in hundreds of plant species. Among these, R. solani causes sheath blight, one of the three major diseases in rice. To date, few genes have been reported that confer resistance to R. solani. Here, rice-FOX Arabidopsis lines identified as having resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and a fungal pathogen, Colletotrichum higginsianum were screened for disease resistance to R. solani. BROAD-SPECTRUM RESISTANCE2 (BSR2), a gene encoding an uncharacterized cytochrome P450 protein belonging to the CYP78A family, conferred resistance to R. solani in Arabidopsis. When overexpressed in rice, BSR2 also conferred resistance to two R. solani anastomosis groups. Both Arabidopsis and rice plants overexpressing BSR2 had slower growth and produced longer seeds than wild-type control plants. In contrast, BSR2-knockdown rice plants were more susceptible to R. solani and displayed faster growth and shorter seeds in comparison with the control. These results indicate that BSR2 is associated with disease resistance, growth rate and seed size in rice and suggest that its function is evolutionarily conserved in both monocot rice and dicot Arabidopsis.

Figure 1

Resistance to Pst DC3000 in BSR2-OX Arabidopsis lines. (a) Phenotypic responses to Pst DC3000 inoculation. Three-week-old plants were inoculated with 2 × 10⁸ cfu/mL of Pst DC3000. The wild-type (WT; Col-0) plants died, but the K02919 (a rice-FOX Arabidopsis T2 line; BSR2-OX) and cpr5-2 (control mutant resistant to Pst DC3000) plants remained green. (b) Growth of Pst DC3000 bacteria in plants. Arabidopsis plants at about the 20-leaf stage were inoculated with Pst DC3000 (10⁶ cfu/mL) by dipping, and the numbers of bacteria in the aerial part of the plants were counted after 4 d. The bacteria in 8 replicates were counted and the differences between WT and two independent BSR2-OX lines (K02919 and RT:BSR2-OX#3) were significant according to a t-test (*P < 0.05; **P < 0.01). Error bars represent the standard deviation.

Figure 2

Resistance to C. higginsianum in BSR2-OX Arabidopsis lines. (a) Phenotypic responses to C. higginsianum inoculation. Three-week-old plants were inoculated with 10⁵ conidia/mL of C. higginsianum. Unlike wild-type (WT; Col-0) plants, K02919 (a rice-FOX Arabidopsis T2 line; BSR2-OX) and Eil-0 (control ecotype resistant to C. higginsianum) plants were still green 13 d after inoculation. (b) Quantification of the relative fungal growth across plant lines. C. higginsianum Actin (ACT) DNA in the Arabidopsis plants were measured using quantitative real-time PCR 3 d after inoculation. The amounts of C. higginsianum genomic ACT DNA relative to Arabidopsis genomic ACT2 DNA in three independent BSR2-OX lines (K02919, RT:BSR2-OX#2 and RT:BSR2-OX#3) were significantly lower than those of WT plants (*P < 0.05 according to a t-test; n = 3–5; error bars represent the standard deviation).

Figure 3

Screening rice-FOX Arabidopsis lines for resistance to R. solani. (a) Evaluation of R. solani resistance using the ratio of lesion length to leaf length. Six-week-old plants were used for drop inoculation with R. solani (MAFF243956; AG1-1A). Seven rice-FOX Arabidopsis lines showing resistance to both Pst DC3000 and C. higginsianum were used: RT:BSR2-OX#1 and #2, RT:AK101795-OX and RT:AK111775-OX are independent retransformed lines for AK072163, AK101795 and AK111775, respectively. K21617, K15424 and K04020 are independent transgenic rice lines with one rice cDNA insert for AK103699, BSR1 (AK070024) and AK066139, respectively. The lesion length and leaf length were measured 7 d after inoculation. Asterisks indicate that values are significantly different from the WT and vector control plants (***P < 0.001, according to a t-test; error bars represent the standard deviation; n = 3). (b) Representative Arabidopsis plants described in (a) immediately after inoculation and 7 d after inoculation. In comparison with the vector control and WT plants, the spread of disease symptoms stopped 7 d after inoculation in the BSR2-OX plants (RT:BSR2-OX#1 and #2). The arrows indicate the inoculation points. R, resistant; S, susceptible.

Figure 4

Resistance to R. solani (AG-1 IA) sheath blight in BSR2-OX and -KD rice lines. Comparisons of (a) inoculated leaves, (b) lesion lengths and (c) relative fungal growth of detached leaf blades from BSR2-OX lines 13 d after drop inoculation with R. solani (MAFF243956; AG1-1A). The second leaf blades from the top leaf at leaf stages 10–13 were used for inoculation. Asterisks indicate that values are significantly different from the WT (*P < 0.05; **P < 0.01, according to a t-test; error bars represent the standard deviation; n = 4). Measurements of relative fungal growth were performed three times with similar results. Comparison of (d) inoculated leaves and (e) lesion lengths of detached leaf blades of BSR2-KD lines 7 d after drop inoculation. The second leaf blades from the top leaf at leaf stages 10–13 were used for inoculation. The lesion lengths in the BSR2-KD plants were longer than those in the WT plants. Tests were performed three times with similar results. Arrows indicate the inoculation points.

Figure 5

Resistance to R. solani (AG-2-2 IIIB) brown sheath blight in BSR2-OX rice lines. Comparison of (a) inoculated leaves and (b) relative fungal growths 11 d after drop inoculation with R. solani (Shimomura 9 isolate; AG-2-2 IIIB). The second leaf blades from the flag leaf at the heading stage were used for inoculation. The relative fungal growth levels in the BSR2-OX plants were lower than those in the WT plants. Tests were performed three times with similar results. The inoculation points are indicated by an arrow.

Figure 6

Morphological traits of BSR2-OX Arabidopsis lines. (a) Representative BSR2-OX phenotypes 77 d after sowing and (b) the number of days to bolting. (c) The comparative sizes, lengths and widths of seeds. Asterisks indicate values that are significantly different from the WT (*P < 0.05, **P < 0.01, ***P < 0.001, according to a t-test; error bars represent the standard deviation; n = 3 (b) and 5 (c)).

Figure 7

Morphological traits of BSR2-OX and -KD rice lines. Leaf stages of (a) BSR2-OX lines 40 d after sowing and (b) BSR2-KD lines 32 d after sowing in comparison with WT at the vegetative stage. (c) Comparison of hulled grain (left) and dehulled grain (right) of BSR2-OX and (d) the lengths of hulled grain and dehulled grain of BSR2-KD rice lines in comparison with WT. Asterisks indicate values that are significantly different from the WT (*P < 0.05, **P < 0.01, ***P < 0.001, according to a t-test; error bars represent the standard deviation; n = 5–12 (a), 4–13 (b), 4 (c) and 5 (d)).