RESEARCH ARTICLE

Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch Alternaria alternata Cai-xia Zhang1,2, Yi Tian1,2, Pei-hua Cong1,2* 1 Key Laboratory of Horticulture Crops Germplasm Resources Utilization, Ministry of Agriculture, P.R. China, 2 Research Institute of Pomology, Chinese Academy of Agricultural Sciences, P.R. China * [email protected]

a11111

Abstract

OPEN ACCESS Citation: Zhang C-x, Tian Y, Cong P-h (2015) Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch Alternaria alternata. PLoS ONE 10(6): e0122233. doi:10.1371/journal.pone.0122233 Academic Editor: Hon-Ming Lam, The Chinese University of Hong Kong, HONG KONG Received: November 22, 2014 Accepted: February 10, 2015 Published: June 18, 2015 Copyright: © 2015 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the National Natural Science Foundation of China (30900968;31201602) and the earmarked fund for the China Agriculture Research System (CARS-28). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Understanding the defence mechanisms used by apple leaves against Alternaria alternate pathogen infection is important for breeding purposes. To investigate the ultrastructural differences between leaf tissues of susceptible and resistant seedlings, in vitro inoculation assays and transmission electron microscopy (TEM) analysis were conducted with two different inoculation assays. The results indicated that the resistant leaves may have certain antifungal activity against A. alternate that is lacking in susceptible leaves. To elucidate the two different host responses to A. alternate infection in apples, the proteomes of susceptible and resistant apple leaves that had or had not been infected with pathogen were characterised using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). MS identified 43 differentially expressed proteins in two different inoculation assays. The known proteins were categorised into 5 classes, among these proteins, some pathogenesis-related (PR) proteins, such as beta-1,3-glucanase, ascorbate peroxidase (APX), glutathione peroxidase (GPX) and mal d1, were identified in susceptible and resistant hosts and were associated with disease resistance of the apple host. In addition, the different levels of mal d1 in susceptible and resistant hosts may contribute to the outstanding anti-disease properties of resistant leaves against A. alternate. Taken together, the resistance mechanisms of the apple host against A. alternate may be a result of the PR proteins and other defence-related proteins. Given the complexity of the biology involved in the interaction between apple leaves and the A. alternate pathogen, further investigation will yield more valuable insights into the molecular mechanisms of suppression of the A. alternate pathogen. Overall, we outline several novel insights into the response of apple leaves to pathogen attacks. These findings increase our knowledge of pathogen resistance mechanisms, and the data will also promote further investigation into the regulation of the expression of these target proteins.

Competing Interests: The authors have declared that no competing interests exist.

PLOS ONE | DOI:10.1371/journal.pone.0122233 June 18, 2015

1 / 15

Proteomic Analysis of Anti-Disease Mechanism

Introduction Plants are usually under numerous threats of pathogen infection, and some of them act as hosts to invasive pathogens [1, 2]. Host-pathogen interactions involve complicated defences, generally, host plants express a wide range of resistance-related proteins in response to pathogen attacks, include pathogenesis-related (PR) enzymes [3]. A comprehensive understanding of the proteins induced by pathogens will help reveal the complex molecular mechanisms that mediate plant disease resistance and will aid the development of new strategies to increase disease resistance in some economically important crops. Apple (Malus domestica) is considered a model fruit plant due to its world-wide economic importance; a large number of apple cultivars dominate world fruit production [4, 5]. However, to date, apple cultivation has been limited by many kinds of fungal diseases, and the domestic apple has become an important fruit crop in which to study commercial traits such as disease resistance [6, 7]. Among the many fungal diseases affecting apple trees, Alternaria blotch, which is caused by the pathogen Alternaria alternata, has been a destructive apple disease in China and other East Asian countries [8]. Currently, the disease is spreading worldwide and results in severe negative effects on apple production [9, 10]. A. alternata can cause circular blackish spots on apple leaves in late spring or early summer, resulting in serious defoliation and decreased fruit quality [11, 12]. Currently, management of the Alternaria blotch occurs mainly through traditional chemical control agents instead of resistant cultivars. The secretion of specific sets of proteins has been known to play decisive roles in plant— fungus interactions. More recently, the significance of resistance-related proteins has been reported for many plant-pathogen interactions [13, 14], and some intensive attempts have also been made to engineer resistance to apple disease. Several genes have been isolated that are related to apple disease resistance [3, 15–18]. The functions of some PR enzymes, including chitinases, β-1,3-glucanases and peroxidases, which act directly against pathogens, were also confirmed by some assays [15–17]. Whereas previous studies on the interaction between plants and pathogenic microorganisms were focused largely on some model plants and pathogens, relatively few have been performed using apple and its pathogens. Over the last few years, the use of proteomic analysis has drastically expanded for the identification of stress-related proteins based on two-dimensional electrophoresis (2-DE) and mapping the dynamics of differential expression involved in the host-plant response to biotic stresses, such as pathogen-crop interactions [19, 20]. In apple, the molecular mechanisms of disease resistance against A. alternata have not been illustrated clearly. Although most apple resistance genes have been identified [21], little is known about their biological roles. To date, very few proteomic analyses of apple hostpathogen interactions have been reported. Considering that Alternaria blotch is the most common disease influencing apple production, studies of the host self-defence mechanisms that closely relate to anti-disease properties should be performed. A recent study showed that susceptible and resistant apples exhibit different patterns of gene expression in response to A. alternata infection [7]. To understand the host defence mechanisms induced by the pathogen, we investigated the specific stress-related proteins that mediate interactions between A. alternata and its host and aimed to understand the molecular mechanisms of plant-pathogen interactions. The proteins identified as associated with the antifungal mechanisms of the host plant against A. alternata may be novel links to the antifungal mechanisms of resistant apples. This study may provide clues to the molecular mechanisms of apple resistance to A. alternata, accelerating the process of apple molecular breeding and providing a theoretical basis and technical reference for future genetic improvement studies of high-quality, disease-resistant apple varieties.

PLOS ONE | DOI:10.1371/journal.pone.0122233 June 18, 2015

2 / 15

Proteomic Analysis of Anti-Disease Mechanism

Materials and Methods Plant materials and fungal pathogen The plant material used in this study came from an 8-year-old apple seedling population consisting of 110 seedlings derived from a cross of ‘Huacui’ and ‘Golden Delicious’, which was grown in an experimental orchard at the Institute of Pomology at the Chinese Academy of Agricultural Sciences (CAAS; Xingcheng, China). The evaluation of a disease rating by field inoculation was previously conducted on these 110 seedlings in 2008 and 2009 [7]. Based on these evaluation results, two of the 110 seedlings with obvious resistance differences, including one highly susceptible seedling and one highly resistant seedling, were chosen as hosts for this study. The aggressive strain of A. alternata used in this study was provided by the Fruit Plant Protection Research Center at the Institute of Pomology at CAAS. To culture this fungal strain for in vitro assays, A. alternata mycelium was incubated on petri plates containing potato dextrose agar (Sigma Chemical Co., St. Louis, MO) at 25°C to harvest spores.

In vitro inoculation assays of the pathogen on host plants The inoculation assays were performed as described by [8] with some modifications. The samples were harvested 48 h after inoculation; both infected and control samples were harvested at the same time after inoculation. Three replicates for each treatment were performed, and each replicate contained 30 leaves. The entire experiment was repeated three times to ensure reliable results. Samples were harvested, immediately frozen in liquid nitrogen, and then ground to a fine powder prior for protein extraction.

Transmission electron microscopy (TEM) analysis The selected control and inoculated leaves were sampled and collected 48 h after inoculation. The samples were cut into 2 to 3 mm2 pieces, fixed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0) at 4°C for 24 h, washed 3 times in 0.1 M sodium phosphate buffer, post-fixed in 1% OsO4 in 0.1 M sodium phosphate buffer for 4 h at 4°C, and then washed in 0.1 M sodium phosphate buffer. Following dehydration in a graded acetone series, the samples were embedded in Epon-Araldite-DDSA and sectioned at 2 μm thickness. Sections on grids were stained with lead citrate for 2.5 min before observation by TEM (Hitachi H-7500, Hitachi Co. Ltd., Tokyo, Japan).

Protein extraction and 2-DE The protein extraction was performed as described by [22] and [23] with some optimisation. In short, 2 g of frozen lyophilised tissue powder was resuspended in 4 mL of ice-cold extraction buffer (30% sucrose w/v, 20 mM Tris-HCl (pH 8.0), 10 mM EGTA, 1 mM DTT, 1% Triton X100 v/v, 2% β-mercaptoethanol v/v, and 1 mM PMSF). After the sample was vortexed for 10 min at room temperature, an equal volume of precooled Tris—HCl (pH 7.5)-saturated phenol was added, and then the mixture was further vortexed for 20 min. After centrifugation (15,000 g at 4°C for 20 min), the upper phase was collected and transferred to a new centrifuge tube. Proteins were precipitated from the phenol phase with three volumes of 100 mM ammonium acetate in methanol overnight at -20°C. The protein pellets were subsequently rinsed three times with cold acetone containing 13 mM DTT. After centrifugation, the rinsed pellets were air-dried and resuspended in lysis buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (v/v) IPG buffer, and 1% (w/v) DTT]. The protein solution was either used immediately for 2-D

PLOS ONE | DOI:10.1371/journal.pone.0122233 June 18, 2015

3 / 15

Proteomic Analysis of Anti-Disease Mechanism

electrophoresis or maintained at -80°C prior to use. The protein concentration was determined using a 2-D Quant Kit (GE Healthcare). 2-DE was performed according to [22] with some modifications. A sample containing 800 μg of total protein was loaded onto an immobilised pH gradient (IPG) strip (18 cm, pH 4–7 linear, GE Healthcare) and rehydrated for 12 h at room temperature. Then, the strips were subjected to isoelectric focussing (IEF) in an Ettan IPGphor system according to the following procedure: 300 V for 1 h, 600 V for 1 h, 1,000 V for 1 h, 5,000 V for 1 h, and 10,000 V for 6 h. After IEF, the strips were transferred to perform the SDS-PAGE or were stored at -20°C. Prior to the second dimension analysis, the strips were equilibrated for 15 min in 10 mL of equilibration solution (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.002% bromophenol blue) containing 1% DTT w/v, followed by incubation in 4% iodoacetamide w/v in the same solution for 15 min. The separation of proteins in the second dimension was performed using SDS polyacrylamide gels (12.5%) on the Ettan DALT System (GE Healthcare): 0.5 w / gel for 30 min and 10 w/ gel for 5 h. After electrophoresis, the gels were stained with Coomassie Brilliant Blue (CBB) R-350.

Image and data analysis The 2-D gels were scanned at a resolution of 600 dpi, and the image analysis was conducted using Image Master 2D Platinum Version 7.0 software (GE Healthcare). The Mr of each protein spot in the gel was determined by referencing protein markers. For each treatment, three images were obtained, representing 3 independent biological replicates, and these replicates were grouped as a class to calculate the average volume of all protein spots. The standard values of protein spots on the three replicate 2D gels from each treatment were exported to SPSS Version 13.0 (Lead Technologies, Chicago, Illinois, USA) for statistical analysis. Only those with significant and consistent changes were counted as differentially accumulated proteins (>1.5-fold, p

Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch Alternaria alternata.

Understanding the defence mechanisms used by apple leaves against Alternaria alternate pathogen infection is important for breeding purposes. To inves...
4MB Sizes 0 Downloads 8 Views