Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant

Abstract

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3' end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21-24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

Figure 1

Schematic diagrams representing the chimeric CP:Hc-Pro hairpin RNA construct Ubi-hpCP:Hc-Pro (a) and the target reporter gene construct 35S-GUS:CP:Hc-Pro (b). 35S, Cauliflower mosaic virus 35S promoter; Ubi, maize ubiquitin promoter; HPT, hygromycin phosphate transferase gene; OCS Ter, Agrobacterium octopine synthase gene terminator sequence; Nos Ter, Agrobacterium nopaline synthase gene terminator; CAT INTRON, first intron of castor bean catalase-1 gene; CRE INTRON, the third intron of the Aegilops tauschii go35 NBS-LRR (go35) gene; SpeI, restriction site used for Southern blot analysis; LB, T-DNA left border; RB, right border.

Figure 2

Transient expression assay to evaluate Ubi-hpCP:Hc-Pro construct in rice callus tissue. (a) X-glucuronide staining of rice callus tissue bombarded with (1) 35S-GUS:CP:Hc-Pro, (2) 35S-GUS:CP:Hc-Pro + Ubi-hpCP:Hc-Pro, (3) 35S-GUS:Y-Sat, and (4) 35S-GUS:Y-Sat + Ubi-hpCP:Hc-Pro constructs. Scale bar shows length and width of callus. (b) Histogram showing the average number of blue spots per square millimeter of bombarded rice callus tissue. Error bar represents standard deviation (n = 3).

Figure 3

Southern blot analysis of T₀ transgenic rice plants. DNA extracted from transgenic rice leaves was digested with SpeI restriction enzyme, separated in 0.8% agarose gel, transferred to HyBond-N⁺ Nylon membrane, and hybridized with radioactive labeled CP:Hc-Pro DNA probe. Numbers 1–5 indicate five Ubi-hpCP:Hc-Pro transgenic lines. VC, transgenic rice plants containing the empty vector control transgene. M, DNA marker (1 kb plus Gene Ruler). The arrow indicates the single transgene band in lines 2, 3, and 4.

Figure 4

T₀ transgenic rice plants express 21–24 nt siRNAs. (a) Northern blot hybridization for the detection of siRNA from transgenic rice lines. Twenty-five μg of total RNA extracted from leaves of transgenic plants was hybridized with antisense CP:Hc-Pro RNA probe (the upper panel). The U6 small nuclear RNA was hybridized as a loading control (the lower panel). Lane M represents the 21–24 nt radiolabeled RNA size marker, with a less-exposed picture given on the left to clearly identify the 21 and 24 nt bands. VC is transgenic rice plants containing the empty vector control transgene while, Lanes 1–5 are the same five Ubi-hpCP:Hc-Pro transgenic rice lines shown in Figure 3. (b) Histogram representing the relative intensity of the sRNA band on the Northern blot above.

Figure 5

Ubi-hpCP:Hc-Pro transgene expression and SCMV resistance analysis in T₁ generation. (a) Northern blot hybridization of T₁ progeny of four independent Ubi-hpCP:Hc-Pro lines (#2, #3, #4, and #5) and an empty vector control line (VC) using ³²P-labelled antisense CP:Hc-Pro RNA as probe, which shows Ubi-hpCP:Hc-Pro expression from #2, #3 (except for one plant), #4, and one of #5 plants, but not from the vector control and the other three #5 plants. Lower panel shows RNA loading control. Asterisks indicate plants analysed in (c). (b) Vector control and Ubi-hpCP:Hc-Pro transgene T₁ transgenic rice plants inoculated six weeks before with SCMV. (c) Quantitative real-time RT-PCR analysis of SCMV accumulation in infected VC and hpRNA transgenic T1 plants using CP (left) or Hc-Pro (right) primers.