Proteomic Analysis of Fusarium oxysporum-Induced Mechanism in Grafted Watermelon Seedlings

Abstract

Grafting can improve the resistance of watermelon to soil-borne diseases. However, the molecular mechanism of defense response is not completely understood. Herein, we used a proteomic approach to investigate the molecular basis involved in grafted watermelon leaf defense against Fusarium oxysporum f.sp. niveum (FON) infection. The bottle gourd rootstock-grafted (RG) watermelon seedlings were highly resistant to FON compared with self-grafted (SG) watermelon plants, with a disease incidence of 3.4 and 89%, respectively. Meanwhile, grafting significantly induced the activity of pathogenesis-related proteases under FON challenge. Proteins extracted from leaves of RG and SG under FON inoculation were analyzed using two-dimensional gel electrophoresis. Thirty-nine differentially accumulated proteins (DAPs) were identified and classified into 10 functional groups. Accordingly, protein biosynthetic and stress- and defense-related proteins play crucial roles in the enhancement of disease resistance of RG watermelon seedlings, compared with that of SG watermelon seedlings. Proteins involved in signal transduction positively regulated the defense process. Carbohydrate and energy metabolism and photosystem contributed to energy production in RG watermelon seedlings under FON infection. The disease resistance of RG watermelon seedlings may also be related to the improved scavenging capacity of reactive oxygen species (ROS). The expression profile of 10 randomly selected proteins was measured using quantitative real-time PCR, among which, 7 was consistent with the results of the proteomic analysis. The functional implications of these proteins in regulating grafted watermelon response against F. oxysporum are discussed.

Keywords: Citrullus lanatus; Fusarium oxysporum f.sp. niveum; bottle gourd; proteomics; rootstock grafting.

FIGURE 1

Physiological responses of the grafted watermelon seedlings infected with FON. Changes of β-1,3-glucanase (A) and chitinase (B) activities in the leaves of RG and SG plants. Vertical bars labeled with different letters are significantly different at p < 0.05. Error bars are based on three biological replicates.

FIGURE 2

Representative 2-DE gels of proteins extracted from leaves of grafted watermelon seedlings. Proteins extracted from leaves of (A) SG-C, (B) RG-C, (C) SG-FON at 240 hpi, and (D) RG-FON at 240 hpi were focused on IPG strips (11 cm, pH 4–7 NL) and separated by SDS-PAGE (12.5%). Arrows mean the differentially accumulated proteins among RG and SG responding to FON infection.

FIGURE 3

Venn diagram of differentially accumulated proteins (DAPs) in RG and SG infected with FON. DAPs were analyzed based on SG-FON vs. SG-C and RG-FON vs. RG-C libraries, respectively.

FIGURE 4

Functional category (A) and subcellular location (B) of DAPs in leaves of grafted watermelon in response to FON infection.

FIGURE 5

Protein–protein interaction (PPI) network as elucidated through the STRING 11.0 online software with a confidence score of 0.4 using Arabidopsis thaliana. The network nodes represent proteins, and the edges indicate the predicted functional associations. The clusters mean the highly interacting proteins involved in photosystem, energy metabolism, translation, and C metabolism.

FIGURE 6

Relative expression level of genes corresponding to 10 randomly selected DAPs in leaves of grafted watermelon in response to FON infection. The value of the relative expression level was normalized to 18SrRNA. Error bars were based on three technical replicates. USP (spot L2), universal stress protein; TrxH (spot L6), thioredoxin h; GS (spot L9), glutamine synthetase; TLP (spot L15), thaumatin-like protein; HOP (spot L21), Hsp70–Hsp90 organizing protein; JIP (spot L22), jasmonate-induced protein; APXT (spot L23), L-ascorbate peroxidase T; ARG (spot L28), arginase; FNR (spot L31), ferredoxin–NADP reductase; IRL (spot L35), isoflavone reductase-like protein.