Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches’ broom disease in cacao

Abstract Background Moniliophthora perniciosa is a phytopathogenic fungus responsible for witches’ broom disease of cacao trees (Theobroma cacao L.). Understanding the molecular events during germination of the pathogen may enable the development of strategies for disease control in these economically important plants. In this study, we determined a comparative proteomic profile of M. perniciosa basidiospores during germination by two-dimensional SDS-PAGE and mass spectrometry. Results A total of 316 proteins were identified. Molecular changes during the development of the germinative tube were identified by a hierarchical clustering analysis based on the differential accumulation of proteins. Proteins associated with fungal filamentation, such as septin and kinesin, were detected only 4 h after germination (hag). A transcription factor related to biosynthesis of the secondary metabolite fumagillin, which can form hybrids with polyketides, was induced 2 hag, and polyketide synthase was observed 4 hag. The accumulation of ATP synthase, binding immunoglobulin protein (BiP), and catalase was validated by western blotting. Conclusions In this study, we showed variations in protein expression during the early germination stages of fungus M. perniciosa. Proteins associated with fungal filamentation, and consequently with virulence, were detected in basidiospores 4 hag., for example, septin and kinesin. We discuss these results and propose a model of the germination of fungus M. perniciosa. This research can help elucidate the mechanisms underlying basic processes of host invasion and to develop strategies for control of the disease

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

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

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

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

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

Protein Profile.

<p>Differential profile (according to the statistical analysis performed by the software) of the expression of identified proteins between non infected (A) and infected (B) treatments of “Westin” sweet orange.</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