Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice

Abstract

Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

Figure 1

Diagrammatic representation of gene constructs and molecular evaluation of transgenic rice lines. (a) (I) Schematic representation of T-DNA construct harboring OsCHI11 gene under the green tissue-specific promoter P _D54O-544 used for rice transformation. (II) Diagrammatic representation of T-DNA construct AtNPR1 gene placed under the control of maize green tissue-specific PEPC promoter. (III) Diagrammatic representation of the T-DNA construct used to transform mature embryogenic calli of jaldi-13 rice variety. OsCHI11 and AtNPR1 genes under the control of the rice green tissue-specific promoter (P _D54O–544) and maize green tissue-specific promoter (PEPC), respectively. (b) (I) PCR analysis of T₂ transgenic rice plants with partial gene-specific (OsCHI11) primers which amplified 490 bp product. (II) PCR analysis of T₂ transgenic rice plants with partial gene-specific (AtNPR1) primers which amplified 1.7Kbp product. (III) PCR based screening of T₂ transgenic and non-transformed WT performed with partial gene specific primer (OsCHI11) which amplified 490 bp product. (IV) PCR analysis performed with partial gene specific primer (AtNPR1) showing amplification of 1.7Kbp product. PC-Positive control and NC- Negative control. (c) (I) Southern blot analysis of T₂ transgenic plants (C): genomic DNA digested with SalI restriction enzyme and probed with 1.1 kbp HPT gene fragment. (II) Southern blot analysis T₂ transgenic plants (N): genomic DNA digested by EcoRI restriction enzyme and probed with 1.1 kbp HPT gene fragment. (III) Southern hybridization analysis of T₂ transgenic plants (C-N): genomic DNA was digested with SalI restriction enzyme and probed with 1.1 kbp HPT (hygromycin phosphotransferase) gene fragment. WT represents wild type.

Figure 2

Expression analysis and activity assay of transgenic and wild type plants. (a) Semi quantitative RT-PCR analysis of selected T₂ transgenic OsCHI11 overexpressing rice lines using gene specific primers taking β-tubulin as reference control.(b) Relative quantity of rice OsCHI11 mRNA in leaves of T₂ (C8-9-1, C5-9-8, C1-2-4) overexpressing and WT rice lines as determined by RT-PCR (Real time PCR). (c) Semi quantitative RT-PCR analysis of selected T₂ transgenic AtNPR1 rice lines using gene-specific primers taking β-tubulin as internal control. (d) Relative quantity of Arabidopsis NPR1 mRNA in leaves of T₂ (N4-3-2, N7-9-4, C1-8-7) overexpressing and WT rice lines as determined by RT-PCR (Real time PCR). (e) Semi quantitative RT-PCR analysis of selected T₂ transgenic OsCHI11-AtNPR1 rice lines using gene-specific (AtNPR1) primer with β-tubulin as internal control. (f) Semi quantitative RT-PCR analysis of selected T₂ transgenic OsCHI11-AtNPR1 rice lines using gene-specific (OsCHI11) primer where β-tubulin acts as internal control. (g) Relative abundance of AtNPR1 mRNA in T₂ transgenic (CN5-2-1, CN4-2-2, CN2-5-3) pyramided lines and untransformed WT was determined by real-time PCR using SYBR green.(h) Relative quantity of OsCHI11 mRNA in T₂ transgenic (CN5-2-1, CN4-2-2, CN2-5-3) rice lines and non-transformed WT was determined real -time PCR using SYBR green. Values represent the mean ± SE of three independent experiments. (i) In-gel chitinase activity assay of T₂ transgenic and non-transformed wild type (WT) plants. (j) In-gel chitinase activity assay of T₂ pyramided transgenic and non-transformed wild type (WT) plants. (k) Activity assay (‘In-solution’) of chitinase in T₂ transgenic and WT plants by dinitrosalycylic acid (DNSA) method. Activity was measured by spectrophotometer with an optical density of 530 nm (OD₅₃₀). (l) ‘In-solution’ activity assay of chitinase in T₂ transgenic and WT plants by dinitrosalycylic acid (DNSA) method. Chitinase activity was measured by spectrophotometer with an optical density of 530 nm (OD₅₃₀). Values are presented as mean ± SE (n = 3).

Figure 3

Real-time PCR analysis of some differentially expressed PR, SA and JA pathway genes in single transgene containing as well as pyramided rice lines. Total RNA isolated from leaves of both transgenic T₂ and non-transformed WT plant, inoculated with sheath blight fungus R. solani, and harvested at 24 and 48 hpi (hours post infection). Experiment performed by SYBR green-based quantitative real-time PCR, using β-tubulin as internal control. Expression of (a) PR10A, (b) RC24, (c) OsPR1b, (d) OsPR5, (e) OsAOS2, (f) OsPAL, (g) OsMAPK6 and (h) OsNH1 gene in transgenic and wild type plants. Each bar represents the mean ± SE of three independent experiments.

Figure 4

Oxidative damage and activities of different antioxidant enzymes in WT and T₂ transgenic rice plants after sheath blight infection. (a) (I) In situ detection of O²⁻ by NBT staining in WT and T₂ transgenic lines. (II) In situ detection of H₂O₂ by DAB staining in WT and T₂ transgenic rice lines. (b) APX activity; (c) CAT activity; (d) POD activity; (e) SOD activity; (f) Determination of lipid hydroperoxide content in both transgenic and WT plants; (g) Determination of MDA accumulation in leaves of WT and T₂ transgenic rice plant lines. Leaves were collected from transgenic and wild type (WT) plants after 2 days post infection (dpi). Data represent means ± SE calculated from three replicates. Three independent experiments performed.

Figure 5

Evaluation of T₂ transgenic lines against sheath blight disease along with non-transformed control through In vitro and In vivo plant bioassay. (a) Mycellial agar disc bioassay showing reduced infection cushion formation in T₂ transgenic lines (CN5-2-1, N4-3-2, C8-9-1) than in the wild-type (WT). Experiments replicated three times. (b) Representative images of reduced lesion formation in transgenic leaves (CN5-2-1, N4-3-2, and C8-9-1) relative to WT in mycellial agar disc bioassay. (c) Bar diagram showing percentage of affected area after 72 hrs post infection in transgenic leaf samples than wild type (WT) in RS -toxin bioassay. (d) Images showing less affected area in transgenic leaves (CN5-2-1, C8-9-1, and N4-3-2) compared to WT control in the toxin bioassay. (e) Image showing reduced sheath blight symptoms development in rice tillers in T₂ transgenic plants, than that in WT control after 21 dpi (Days post infection). (f) Percentage of infected area on tillers on T₂ transgenic (CN5-2-1, C8-9-1, and N4-3-2) and WT plants after 21 dpi. (g) Images showing sheath blight symptoms on leaf blade of transgenic (CN5-2-1, C8-9-1, and N4-3-2) and non-transformed control. (h) Lesion size on leaf blade of WT and transgenic lines after 21 dpi. (i) Lesion size on leaf sheath of both WT and transgenic lines (CN5-2-1, C8-9-1, and N4-3-2) after 21 dpi. Values are presented as mean of 10 replicas ± SE. (j) Representative images showing reduction in symptoms development on rice sheath of transgenic lines as compared to WT after 21 dpi.

Figure 6

Whole plant bioassay of T₂ transgenic and wild type (WT) plants with sheath blight fungus, R. solani. (a) Percent Disease Index (PDI) value in T₂ transgenic plants with respect to wild type control at 7, 14 and 21 dpi (days post inoculation). The values represent as the mean ± SE (n = 15). (b) Representative images showing typical sheath blight symptoms development on rice tillers of T₂ transgenic (CN5-2-1, C8-9-1, and N4-3-2) and non transformed wild type control. Pictures were taken at 7, 14 and 21 dpi. Red arrows indicate sheath blight symptoms.

Figure 7

Enhanced resistance of transgenic lines to highly virulent isolates of R. solani and seed germination analysis. (a) Bar diagram showing percentage of survival tillers in T₂ transgenic lines (CN5-2-1, C8-9-1, and N4-3-2) compared to wild type (WT) after 21 dpi. Bar represents mean ± SE of three independent experiments. (b) Images showing more number of surviving tillers in T₂ transgenic lines (CN5-2-1, C8-9-1, and C4-3-2) compared to WT after 21 dpi. (c) Percentage of filled grains per panicle on transgenic and non-transformed control after 21 dpi. Bar represents mean ± SE of five independent experiments. (d) Images showing more grain count per panicle in transgenic plants after sheath blight infection with respect to WT at 21 dpi. (e) Images showing seed germination and phenotypic evaluation of T₃ transgenic and wild type (WT) control seeds at juvenile stage.