Deciphering Molecular Determinants Underlying Penicillium digitatum’s Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS

Abstract

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum’s response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum’s response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies.

Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

“…Aliquots of proteins from homogenate (h) and synaptosomal fractions (syn) of 1.5-month-old control and patient hCOs were used for mass spectrometric analysis as previously described [ 17 ]. For each condition, equal amounts of proteins (80 µg in 100 µL of 100 mM triethylammonium bicarbonate) were digested with trypsin and labeled with the following TMT isobaric tags according to the procedure described elsewhere [ 36 ]: 128C and 127C for syn CTR and h CTR samples and 130N and 129N for syn EPM1 and h EPM1 samples, respectively. TMT-labeled samples were then diluted with 2% TFA to the final protein concentration of 0.5 µg/µL for the LC–MS/MS analyses.…”

Antifungal Activity of Ribosome-Inactivating Proteins

“…Sacc. was very sensitive to different type 1 RIPs: sodin 5 [ 109 ], quinoin [ 109 ], BE27 [ 29 , 108 ], dioicin 2 [ 30 ], and PD-S2 [ 30 ]. The one that exerted the greatest effect was BE27 since the growth inhibition was evident at 0.6 µg/mL [ 29 ].…”

“…have been reported as RIPs with antifungal activity [ 89 , 101 ]. On the other hand, the ribotoxin α-sarcin [ 108 ] and the RLPs ageritin [ 114 , 115 ] and eryngitins 3 and 4 [ 116 ] have also shown antifungal activity, indicating that, although never used for that purpose, these proteins could also be tools to defend crops against fungal diseases.…”

Research and development status of antifungal drugs based on proteomics

“…The latest tandem mass tag (TMT) can achieve up to 18-fold labeling, coupled with the use of high-resolution mass spectrometer, it has become a reality to accurately quantify the expression changes of the same protein in a variety of cell states on a large scale [7]. Lucía Citores et al [8] used a high-resolution lcms/ms method based on tandem mass spectrometry (TMT) to differentiate the antifungal drugs produced by organisms.…”