Proteomic Analysis of the Resistance Mechanisms in Sugarcane during Sporisorium scitamineum Infection

Abstract

Smut disease is caused by Sporisorium scitamineum, an important sugarcane fungal pathogen causing an extensive loss in yield and sugar quality. The available literature suggests that there are two types of smut resistance mechanisms: external resistance by physical or chemical barriers and intrinsic internal resistance mechanisms operating at host⁻pathogen interaction at cellular and molecular levels. The nature of smut resistance mechanisms, however, remains largely unknown. The present study investigated the changes in proteome occurring in two sugarcane varieties with contrasting susceptibility to smut-F134 and NCo310-at whip development stage after S. scitamineum infection. Total proteins from pathogen inoculated and uninoculated (control) leaves were separated by two-dimensional gel electrophoresis (2D-PAGE). Protein identification was performed using BLASTp and tBLASTn against NCBI nonredundant protein databases and EST databases, respectively. A total of thirty proteins spots representing differentially expressed proteins (DEPs), 16 from F134 and 14 from NCo310, were identified and analyzed by MALDI-TOF/TOF MS. In F134, 4 DEPs were upregulated and nine were downregulated, while, nine were upregulated and three were downregulated in NCo310. The DEPs were associated with DNA binding, metabolic processes, defense, stress response, photorespiration, protein refolding, chloroplast, nucleus and plasma membrane. Finally, the expression of CAT, SOD, and PAL with recognized roles in S. scitamineum infection in both sugarcane verities were analyzed by real-time quantitative PCR (RT-qPCR) technique. Identification of genes critical for smut resistance in sugarcane will increase our knowledge of S. scitamineum-sugarcane interaction and help to develop molecular and conventional breeding strategies for variety improvement.

Keywords: ISR; RT-qPCR; Sporisorium scitamineum; proteomics; smut; sugarcane.

Figure 1

2-DE SDS-PAGE gel pictures of sugarcane varieties F134 and NCo310 with their controls. (A) control (F134), (B) treatment (F134), (C) control (NCo310), and (D) treatment (NCo310). Yellow for the protein spots of interest, red for upregulated proteins, and blue for downregulated proteins.

Figure 2

Peptide mass fingerprinting of differentially expressed proteins associated with sugarcane varieties F134 and NCo310 after Sporisorium scitamineum inoculation.

Figure 3

qRT-PCR analysis of differentially expressed genes in leaf tissue of sugarcane varieties F134 and NCo310 during sugarcane–S. scitamineum interaction. (A) Catalase (SuCAT), (B) superoxide dismutase (SuSOD), and (C) phenylalanine ammonia lyase (SuPAL). Data were normalized to the GAPDH expression level. All data points are the mean ± SE (n = 3).

Figure 4

Analysis of enzyme activities in leaf and root tissues of sugarcane varieties F134 and NCo310 infected with S. scitamineum stress. (A,B) Superoxide dismutase, (C,D) phenylalanine ammonia lyase, and (E,F) Catalase. All data points (with the subtraction of their controls) are the mean ± SE (n = 3).

Figure 5

Analysis of phytohormone levels in leaf and root tissues of sugarcane varieties F134 and NCo310 infected with S. scitamineum stress. (A,B) Cytokinin and (C,D) ethylene. All data points (with the subtraction of their controls) are the mean ± SE (n = 3).