Comparative proteomics of two citrus varieties in response to infection by the fungus Alternaria alternata

International audienceAlternaria brown spot (ABS) is a disease caused by the necrotrophic fungus Alternaria alternata, which induces necrotic lesions on fruits and young leaves due to the production of the host-specific ACT toxin by the fungus. To better understand the citrus-A. alternata interaction and to identify putative resistance proteins, as well as the receptor of the ACT toxin, citrus plants susceptible ('Minneola' mandarin) and resistant ('Clemenules' tangor) to A. alternata, infected or not (control) with the pathogen were analyzed by proteomics. Protein changes were observed between citrus genotypes after infection, and 150 candidate proteins were obtained. A general scheme of the metabolic processes involved in susceptible and resistant citrus-A. alternata interactions was designed. Susceptible plants presented a high level of proteins involved in stress response at the final stages of the infection, whereas resistant plants presented high level of ROS proteins, metabolic proteins, and proteins involved in the immune system process. Proteins like ferredoxin and cyclophilin are specific to the susceptible variety and may be good candidates as fungal effector-interacting proteins. This is the first citrus-A. alternata proteomics analysis, which has allowed a better understanding of the molecular bases of the citrus response to ABS disease

Distribution of differentially-expressed proteins of sweet orange variety “Westin” in response to <i>Citrus tristeza virus</i> (CTV), according to their expression levels.

<p>The gray segment of the bar corresponds to up-accumulated proteins and in light gray, the down-accumulated ones.</p

Functional categorization and cell location of identified differentially expressed proteins in infected and non-infected sweet orange variety “Westin” subjects.

<p><b>A,</b> Functional categorization of significantly different proteins. <b>B,</b> Cell location of differentially expressed proteins.</p

Analysis of the accumulation of transcripts of the genes encoding enzymes involved in oxidative stress in sweet orange variety “Westin” plants infected and non-infected with CTV.

<p>The columns marked with ** indicate that the values among treatments presented significant differences through Tukey test (p ≤ 0.001. The vertical columns indicate the average values of Cts which were calculated through method 2^-ΔΔCt (LIVAK E SCHMITTGEN, 2001), the bars above the columns indicate the standard error of averages.</p

Peroxidase activity in “Westin” sweet orange infected and non-infected with CTV, with the use of ascorbic acid and guaiacol as electron donors.

<p><b>A, B, and C</b> AsA—exhaustion times in the reaction at 10 mmol.L^-1, 20 mmol.L^-1 and 30 mmol.L^-1, respectively. The arrows indicate the times at which GPX activity was started.</p

Amplification of CTV viral particles through RT-PCR in order to confirm the presence of the virus in infected samples, and to confirm its absence in non-infected samples.

<p><b>M,</b> molecular weight marker; <b>NIC-</b> and <b>IC-,</b> negative controls of the reaction for each of the primers (1 and 2). <b>NI1</b> and <b>I1</b>, non-infected and infected samples (primer 1—CN487/489), respectively. <b>NI2</b> e <b>I2</b>, non-infected and infected samples (primer 2—CN488/491), respectively. The arrows show the amplified bands as per their expected sizes. The reaction was confirmed in a 1% agarose gel.</p

<i>Citrus tristeza virus</i> (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety “Westin”

<div><p>Citrus Tristeza disease, caused by CTV (<i>Citrus tristeza virus</i>), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding <i>CAT</i>, <i>SOD</i>, <i>APX</i> and <i>GPX</i> was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the <i>CAT</i> gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus.</p></div

Differentially expressed proteins among infected and non-infected samples of “Westin” sweet orange, identified through Mass Spectrometry (ms/ms).

<p>* Exclusives Spots from Infected Samples</p><p>The peptides were sequenced through ms/ms</p><p>Score corresponding to the coverage value, as calculated by Mascot.</p><p>Differentially expressed proteins among infected and non-infected samples of “Westin” sweet orange, identified through Mass Spectrometry (ms/ms).</p

Enzyme activity in “Westin” sweet oranges infected and non-infected with CTV.

<p><b>A,</b> Catalase <b>B,</b> Superoxide Dismutase <b>C,</b> Guaiacol Peroxidase. The vertical columns indicate the average absorbance values (n = 4). The bars above the columns represent the standard error of averages. The asterisk (*) indicates that the values presented a significant difference through Tukey test, taking into account p ≤ 0.05.</p