Inhibition of OsSWEET11 function in mesophyll cells improves resistance of rice to sheath blight disease

Abstract

Pathogen-host interaction is a complicated process; pathogens mainly infect host plants to acquire nutrients, especially sugars. Rhizoctonia solani, the causative agent of sheath blight disease, is a major pathogen of rice. However, it is not known how this pathogen obtains sugar from rice plants. In this study, we found that the rice sugar transporter OsSWEET11 is involved in the pathogenesis of sheath blight disease. Quantitative real-time polymerase chain reaction (qRT-PCR) and β-d-glucuronidase expression analyses showed that R. solani infection significantly enhanced OsSWEET11 expression in leaves amongst the clade III SWEET members. The analyses of transgenic plants revealed that Ossweet11 mutants were less susceptible, whereas plants overexpressing OsSWEET11 were more susceptible, to sheath blight compared with wild-type controls, but the yield of OsSWEET11 mutants and overexpressors was reduced. SWEETs become active on oligomerization. Split-ubiquitin yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays showed that mutated OsSWEET11 interacted with normal OsSWEET11. In addition, expression of conserved residue mutated AtSWEET1 inhibited normal AtSWEET1 activity. To analyse whether inhibition of OsSWEET11 function in mesophyll cells is related to defence against this disease, mutated OsSWEET11 was expressed under the control of the Rubisco promoter, which is specific for green tissues. The resistance of transgenic plants to sheath blight disease, but not other disease, was improved, whereas yield production was not obviously affected. Overall, these results suggest that R. solani might acquire sugar from rice leaves by the activation of OsSWEET11 expression. The plants can be protected from infection by manipulation of the expression of OsSWEET11 without affecting the crop yield.

Keywords: OsSWEET11; mesophyll cell; resistance; rice; sheath blight disease.

Figure 1

Expression patterns of OsSWEET11 induced by Rhizoctonia solani AG1‐1A infection. (A) Expression levels of OsSWEET11 were analysed after 0, 24, 48 and 72 h of R. solani infection. Solid potato dextrose agar (PDA) medium was used as a control (Mock), and R. solani AG1‐1A cultured on the surface of PDA was used for the infection of rice leaves and sheaths. Ten leaves from each time point were sampled for RNA extraction. The experiments were repeated three times, and the data represent the means ± standard error (SE) (n = 3). Significant differences at P < 0.05 are indicated by different letters. (B) Schematic representation of the plasmid construct used for β‐d‐glucuronidase (GUS) expression in the transgenic plants, in which the OsSWEET11 promoter drives GUS expression. (C) Analysis of GUS expression in leaf and sheath after 48 h of mock and AG1‐1A infection.

Figure 2

Response of OsSWEET11 mutants and rice overexpression lines to Rhizoctonia solani AG1‐1A. (A) Leaves from wild‐type (WT) plants, OsSWEET11 mutants (Ossweet11‐1 and Ossweet11‐2) and OsSWEET11 rice overexpression lines (OsSWEET11 OX2 and OsSWEET11 OX4) were inoculated with R. solani AG1‐1A and photographed after infection. Six leaves from each line were analysed, and the experiments were repeated three times. (B) The lesion scales were analysed for R. solani AG1‐1A‐infected leaves by determining the lesion area on the leaf surface. Data represent the means ± standard error (SE) (n > 10). Significant differences at P < 0.05 are indicated by different letters.

Figure 3

Alignment of amino acid sequences of the selected SWEETs and functional analysis of the conserved residues in glucose transport. (A) Sequence alignment of OsSWEET2b, AtSWEET1a, OsSWEET11, OpSWEET, OgSWEET, TaSWEET, ZmSWEET and SbSWEET. The conserved residues are shown in red boxes. The triangle indicates the residues tested for glucose transport function. (B) Growth assays of mutant AtSWEET1 proteins expressed in EBY4000 yeast strain were performed on YNB (Yeast Nitrogen Base without Amino Acids) medium containing 2% glucose or maltose. AtSWEET1 mutants carrying P23A, G76D, P162A, P191T and Q202D led to the loss of glucose transport activity. Empty (pDRf1) vector and AtSWEET1 were used as negative and positive controls, respectively. The positions of the conserved residues in OsSWEET11 are shown in the left panel. The yeast cells were grown at 28 °C for 3 days.

Figure 4

Assessment of the interaction between wild‐type (WT) and mutated OsSWEET11 and inhibition of glucose transport by co‐expression of WT and mutant AtSWEET1 proteins in yeast EBY4000. (A) Interaction between OsSWEET11 and mOsSWEET11 was analysed using a split‐ubiquitin yeast two‐hybrid system. mSWEET11 was cloned into the Nub vector, whereas OsSWEET11 was cloned into the Cub (C‐terminal ubiquitin domain driven by methionine‐repressible MET25 promoter and fused to the artificial PLV transcription factor) or Nub vector. NubWT (Nub) and NubG (N‐terminal ubiquitin domain carrying a glycine mutation) were used as positive and negative controls, respectively. (B) Observation of the yellow fluorescent protein (YFP) fluorescence. Reconstitution of the YFP fluorescence from OsSWEET11‐nYFP + OsSWEET11‐cCFP or OsSWEET11‐nYFP + mOsSWEET11‐cCFP (left, fluorescence channel; right, bright field). Co‐expression of OsSWEET11‐nYFP + cCFP or OsSWEET11‐nYFP + AtSWEET1‐cCFP was used as negative control. Bars, 20 μm. (C) Co‐immunoprecipitation (Co‐IP) was performed to analyse the interaction between OsSWEET11 and mOsSWEET11 in tobacco leaves. OsSWEET11‐Myc or OsSWEET11‐Myc + mOsSWEET11‐GFP was transformed into tobacco leaves using Agrobacterium‐mediated transformation. The total protein and Myc antibody‐immunoprecipitated proteins were analysed using Western blot analysis with either Myc or green fluorescent protein (GFP) antibodies. CBB (Coomassie Brilliant Blue) staining was used as a loading control. (D) The p112AINE empty vector or p112AINE‐SWEET1 construct was co‐transformed with pDRf1 empty vector or pDR‐mAtSWEET1 construct into the yeast strain, EBY4000. Growth assays of yeast cells co‐expressing WT and mutant AtSWEET1 protein, mAtSWEET1, were performed on YNB (Yeast Nitrogen Base without Amino Acids) medium containing 2% glucose or maltose. The yeast cells were grown at 28 °C for 3 days.

Figure 5

Expression levels of OsSWEET11 and disease symptoms in the transgenic plants expressing mOsSWEET11 under the control of the Rubisco promoter. (A) Schematic diagram showing the construct used for the generation of transgenic plants. The Rubisco promoter was used to drive mOsSWEET11, a mutated form of OsSWEET11. (B) The expression levels of OsSWEET11 were analysed in the roots, leaves, sheaths and flowers of wild‐type and three independent transgenic plants (pRub‐mOsSWEET11 #1, #2 and #6) by quantitative real‐time polymerase chain reaction (qRT‐PCR). The experiments were repeated three times. Significant differences at P < 0.05 are indicated by different letters. (C) The leaves of wild‐type and three independent transgenic plants (pRub‐mOsSWEET11 #1, #2 and #6) were infected with Rhizoctonia solani AG1‐1A and photographed after 3 days of infection. Six leaves from each line were analysed, and the experiments were repeated three times. (D) The lesion scales were analysed for the R. solani AG1‐1A‐infected leaves shown in (C) by determination of the lesion area on the leaf surface. Data represent means ± standard error (SE) (n > 10). Significant differences at P < 0.05 are indicated by different letters.

Figure 6

Morphology and grain weight of wild‐type plants, OsSWEET11 mutants, overexpressors and pRub‐mOsSWEET11 transgenic plants. (A) Four‐month‐old wild‐type plants, OsSWEET11 overexpressors and pRub‐mOsSWEET11 transgenic plants were photographed. Scale bar, 10 cm. (B) The 1000‐grain weight was analysed using the seeds from the different plants. (C) Seeds harvested from wild‐type and pRub‐mOsSWEET11 transgenic plants were photographed. Scale bar, 1 cm. (D) The seed length and width in wild‐type and pRub‐mOsSWEET11 transgenic plants were analysed. More than 100 seeds were analysed. (E) The tiller number in wild‐type and pRub‐mOsSWEET11 transgenic plants was analysed. More than 20 plants were analysed. (F) The grain number in wild‐type and pRub‐mOsSWEET11 transgenic plants was analysed. More than 20 plants were analysed. Significant differences at P < 0.05 are indicated by different letters.

Figure 7

Schematic representation of the hypothesized function of OsSWEET11 during Rhizoctonia solani infection. Rhizoctonia solani infection activates the expression of OsSWEET11 via an unknown mechanism to efflux sucrose to the apoplasm, and cell wall invertase catabolizes sucrose to glucose and fructose. Further, glucose can be taken up into R. solani cells. The expression of mutated OsSWEET11 via the Rubisco promoter may generate a non‐functional OsSWEET11 complex. The wild‐type (WT) OsSWEET11 induced by R. solani may form heterotrimers consisting of WT OsSWEET11 and mutated OsSWEET11, which may be expressed under the Rubisco promoter, and the non‐functional OsSWEET11 complex may inhibit sugar efflux.