Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch <i>Alternaria alternata</i>

<div><p>Understanding the defence mechanisms used by apple leaves against <i>Alternaria alternate</i> 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 <i>A</i>. <i>alternate</i> that is lacking in susceptible leaves. To elucidate the two different host responses to <i>A</i>. <i>alternate</i> 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 <i>A</i>. <i>alternate</i>. Taken together, the resistance mechanisms of the apple host against <i>A</i>. <i>alternate</i> 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 <i>A</i>. <i>alternate</i> pathogen, further investigation will yield more valuable insights into the molecular mechanisms of suppression of the <i>A</i>. <i>alternate</i> 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.</p></div

Venn diagram showing the distribution of protein spots expressed as indicated in Fig 2.

<p>Venn diagram showing the distribution of protein spots expressed as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122233#pone.0122233.g002" target="_blank">Fig 2</a>.</p

Differentially expressed proteins identified from resistant (R) and susceptible (S) leaves.

<p>Differentially expressed proteins identified from resistant (R) and susceptible (S) leaves.</p

2-DE analysis of proteins induced by the <i>A</i>. <i>alternata</i> pathogen in resistant (R) and susceptible (S) leaves.

<p>Total protein (800 μg) was separated on 2D gels (pH 4–7) and stained with CBB R-350. Approximate molecular masses and pIs are indicated in the margins. Circles indicate the 43 proteins identified by MALDI-TOF-TOF/MS that changed in abundance more than 1.5-fold between controls and treated samples. The affected proteins are numbered, and the numbers correspond to the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122233#pone.0122233.g003" target="_blank">Fig 3</a>. This figure represents three biological replicates.</p

Functional categorisation of the identified proteins that are differentially regulated in resistant (R) and susceptible (S) leaves infected with the <i>A</i>. <i>alternata</i> pathogen.

<p>A total of 43 identified proteins were assigned to the functional categories. The Roman numerals of the categories correspond to the functional categories described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122233#pone.0122233.g003" target="_blank">Fig 3</a>. The percentage represents the proportion of proteins in each category.</p

Gene expression stability (M) of candidate genes calculated by geNorm.

<p>(A) Abiotic stresses treated root tissues from cultivar ‘Gala’; (B) <i>P</i>. <i>ultimum</i> infected #58 roots; (C) <i>P</i>. <i>ultimum</i> infected #75 roots; (D) <i>R</i>. <i>solani</i> infected #115 roots. The least stable genes are on the left, while the most stable genes are on the right.</p

Description of candidate reference genes and primer sequences for qRT-PCR.

<p>Description of candidate reference genes and primer sequences for qRT-PCR.</p

Evaluation of selected reference genes using RNA-Seq data.

<p>Evaluation of selected reference genes using RNA-Seq data.</p

Stability ranking of 15 candidate reference genes of four treatments.

<p>Stability ranking of 15 candidate reference genes of four treatments.</p

Cycle threshold (Ct) values of the fifteen tested genes across all samples.

<p>(A) Abiotic stresses treated root tissues of cultivar ‘Gala’; (B) <i>P</i>. <i>ultimum</i> infected #58 roots; (C) <i>P</i>. <i>ultimum</i> infected #75 roots; (D) <i>R</i>. <i>solani</i> infected #115 roots. For each box, the upper and lower edges indicate the 25th and 75th percentiles, while whisker caps represent the maximum and minimum values. The line across the box depicts the median.</p