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Penicillium digitatum
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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. 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.

Penicillium digitatum proteomics alpha-sarcin Beetin 27 (BE27)
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    1. Introduction

    Citrus fruits (genus Citrus in family Rutaceae), comprising oranges, grapefruit, mandarins, limes and lemons, are among the most widespread fruit crop worldwide, highly enriched with components beneficial to human health. The great economic value of the citrus fruits market is also based on properties that largely rely on factors affecting both external and internal quality.  While no bacterial postharvest diseases of commercial importance have been reported, citrus fruits are highly susceptible to fungi infection [1]. Among them, the green mould and blue mould, caused by Penicillium digitatum and Penicillium italicum, respectively, represent the two most important diseases in all citrus production during fruits postharvest handling procedures [2][3]. In particular, the postharvest green mould is the main factor affecting citrus fruit decay, leading to huge economic losses worldwide every year and accounting for up to 90% of the total citrus postharvest losses, especially in subtropical climates [3].
    Synthetic fungicides played a key role in crop protection for the control of the citrus postharvest green mould. However, fungicide resistance represents a critical issue worldwide, with still limited approaches for effective disease management [4]. Thus, developing efficient alternative approaches for counteracting postharvest diseases of citrus fruits caused by Penicillium species has become an active field of research. Several promising biological control strategies, including the use of antagonistic microorganisms, the application of naturally-derived bioactive compounds and the induction of natural resistance, have been proposed as potential alternatives to synthetic fungicides for the control of citrus diseases [1].
    These approaches rely on naturally occurring agents such as several secreted proteins from fungi endowed with antiviral, antibacterial, antifungal, and insecticidal activities such as the antifungal protein (AFP) and the ribotoxin α-sarcin from Aspergillus giganteus [5][6][7][8]. Recently, it was reported that α-sarcin, traditionally considered toxic only to animal cells, displays a strong antifungal activity against Penicillium digitatum for its ability to enter into the cytosol and inactivate the ribosomes, thus killing cells and arresting fungus growth [9]. Ribotoxins such as α-sarcin are rRNA endonucleases (EC 4.6.1.23) that catalyse the cleavage of the phosphodiester bond on the 3′ side of the G4325 residue from the rat large rRNA. This nucleotide is located in the Sarcin Ricin Loop (SRL) that is involved in the binding of the elongation factor to the ribosome [9]. In addition, several plant-derived proteins such as the ribosome-inactivating proteins (RIPs) have been extensively studied for their antiviral, antifungal and insecticidal activity mediated by the inhibition of protein synthesis [10][11]. RIPs belong to a class of enzymes (EC 3.2.2.22) that exhibits rRNA N-glycosylase activity. This activity prevents protein synthesis by causing the release of a specific adenine residue in the SRL of the large rRNA. A marked antifungal activity against the green mould Penicillium digitatum has been described for the apoplastic protein beetin 27 (BE27), a RIP isolated from sugar beet (Beta vulgaris L.) leaves. BE27 is able to enter into the cytosol and kill cells, thus arresting the growth of the fungus at a concentration much lower than that present in the apoplast [12].

    2. 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

    Over the past decade, the technological advances in mass spectrometry (MS)-based proteomics, together with the availability of P. digitatum and other fungi genome sequence information, enormously increased the potential to characterize the molecular changes occurring within pathogenic fungi in response to treatments with novel biofungicides. Recently, a label-free quantitative proteomic analysis was performed on Fusarium oxysporum f. sp. cucumerinum mycelia following treatment with canthin-6-one, an alkaloid compound extracted from Ailanthus altissima, with the aim of investigating the molecular mechanisms underlying the antifungal properties of this molecule [13]. A similar approach was applied to decipher the effects of the antifungal peptide ETD151, an analogue of the antifungal insect defensin heliomicin, on the phytopathogenic fungus Botrytis cinerea [14]. In addition, an iTRAQ-based LC-MS/MS analysis was used to evaluate the proteomic profiling of B. cinerea in response to wuyiencin, produced by Streptomyces albulus subsp. wuyiensis, widely used as an antifungal agent in agriculture [15]. The high-throughput capability of mass spectrometry analyses was exploited to evaluate proteomic changes occurring within Penicillium species following treatment with plant-derived pesticides. Recently, proteomic changes occurring following treatment of P. expansum with chitosan, used as a promising alternative for postharvest diseases management, were investigated by two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF MS analysis [16]. A comparative analysis of both mycelial and extracellular proteomes of Penicillium janczewskii was performed to evaluate the impact on the fungus metabolism of the labdanolic acid, a terpenoid from Cistus ladanifer [17]. Moreover, an iTRAQ-based high resolution MS approach was applied for the determination of proteomic alterations in P. expansum spores under decanal stress [18]. Similarly, Penicillium digitatum proteome changes were evaluated in response to the antifungal extract produced by Streptomyces lavendulae strain X33 with the aim of exploring the intrinsic molecular mechanism of the bacterial extract on the fungus [19].

    In this work, the molecular determinants of Penicillium digitatum response to α-sarcin and BE27, two inhibitors of protein synthesis, were investigated. We further evaluated protein changes following treatment with the commonly used fungicide thiabendazole (TBZ), a microtubule-destabilizing drug that inhibits mitosis [20] with the aim to compare P. digitatum response to biological and chemical fungicides. An advanced quantitative proteomic approach based on Tandem Mass Tag (TMT) isobaric labelling and nano-liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC MS/MS) was used to identify and quantify the expression levels of proteins from treated and untreated P. digitatum mycelia (Figure 1).

    Figure 1. Schematic workflow applied for proteomic analysis of P. digitatum treated with fungicide compounds (i.e., α-sarcin, BE27 and TBZ).

    Collectively, we identified candidate proteins potentially associated with α-sarcin, BE27 and TBZ treatment outcomes in P. digitatum. These were mainly involved in cell wall degradation and fungal morphogenesis, stress response, antioxidant and detoxification mechanisms and metabolic pathways. Although a similar trend in protein changes was driven by the three antifungal treatments, a distinct regulation in response to α-sarcin and BE27 treatments was also observed for a subset of proteins. 

    We focused primarily on differentially expressed proteins potentially impairing P. digitatum growth and virulence such as cell wall-degrading enzymes (CWDEs) and proteases. In phytopathogenic fungi, an arsenal of catalytic proteins supports nutrient acquisition, conidial formation, substrate colonization and host invasion [21]. We found several CWDEs and proteases that were differentially regulated upon protein toxins and TBZ antifungal treatments such as pectinesterases, pectate lyases, polygalacturonases, glucanases and peptidases. The importance of CWDEs in the virulence and pathogenesis of P. digitatum has been reported during the infection of oranges [22] and on postharvest citrus [23].

    On the pathogen side, we observed an upregulation of CWDEs belonging to the classes of pectinesterases and pectate lyases when treated with both biofungicides and TBZ, while some differences among treatments were related to the expression levels of polygalacturonases and glucanases as well as some peptidases. These results suggest that α-sarcin and BE27 toxins may trigger a specific response in modulating cell wall integrity. In this framework, the proteins involved in cell wall morphogenesis and remodelling were also differentially regulated, such as Hydrophobin and GPI-anchored membrane protein. Hydrophobins are surface-active proteins produced by filamentous fungi. They have a role in fungal growth as structural components and fungi’s interaction with their environment. They are essential for aerial growth and the attachment of fungi to solid supports. Spores of filamentous fungi are also covered by Hydrophobins that renders the conidial surface hydrophobic and wet-resistant [24]. In our experimental model, the upregulation of Hydrophobin may, thus, potentially also affect the observed effects on mycelium growth and morphogenesis. Other proteins that are functionally involved in maintaining cell wall integrity, such as GPI-anchored membrane proteins [25] and C-4 methyl sterol oxidase Erg25 [26][27] were differentially regulated by both protein toxins or by BE27 alone, respectively. These sources of evidence support the idea that toxins could affect the maintenance of the P. digitatum plasma membrane’s stability and fluidity.
    Some proteins that are differentially regulated by antifungal treatments were related to energetic metabolism and amino acid metabolism, pointing to a global metabolic reprogramming during P. digitatum’s response to antifungal treatments. A subset of these proteins is involved in the biosynthesis of vitamins and cofactors such as thiamine (Vitamin B1) and its precursor thiazole. These molecules are precursors of the active thiamine diphosphate (TPP), an essential coenzyme of several metabolic enzymes including Pyruvate decarboxylase, which was also upregulated following both protein toxins and TBZ treatments. These results implied that a response in the expression of key enzymes involved in thiamine biosynthesis is elicited, which is likely to counteract the metabolic stress triggered by the three anti-fungal agents. Interestingly, consistent with our results, thiamine metabolism was also reported to be affected by the antifungal extract of the S. lavendulae strain X33 on the mycelial growth of P. digitatum [19].
    Treatments with fungicides also trigger a response in some P. digitatum mediators of the redox system and stress-response homeostasis, such as the upregulation of Catalase in response to TBZ. Catalase is a well-known antioxidant enzyme produced by fungi as an infection strategy [28]. In P. digitatum, it was suggested that Catalase eliminates hydrogen peroxide produced by the plant as a defence mechanism [29]. Besides the role in plant infection, in agreement with our findings, catalase was also upregulated in P. digitatum treated with the X33 antifungal extract [19]. Proteins related to antioxidant defence response were also differentially regulated in responses to chitosan in P. expansum [30]. In our experimental system, further changes in the expression levels of Glyoxalase and Glutaredoxin-like proteins were elicited by α-sarcin and/or BE27, indicating that toxins incubation promoted the expression of defence-related enzymes to modulate redox homeostasis.
    Another stress-response protein, downregulated following treatments with α-sarcin and BE27, was the MFS monosaccharide transporter. MFS transporters are involved in virulence by regulating the secretion of host-specific toxins or providing protection against plant defence components [31]. Together with ABC transporters, these carriers are the most important efflux pumps involved in fungal protection against fungicides [32][33]. More importantly, they were also reported to increase resistance to fungicides through their ability to transport a wide variety of compounds, such as toxic products [22].
    BE27 treatment alone also downregulate the expression level of the small cysteine-rich protein Antifungal protein AFP, which is endowed with a specific antifungal activity [34][35]. P. digitatum genome sequencing allowed the identification of several potential AFP-like proteins [36]. Although AFP constitutive expression in filamentous fungi has a negative impact on their growth and virulence, the biological role of endogenous proteins remains unclear. In other filamentous fungi such as A. niger and A. giganteus, it was suggested that AFPs are molecules that are important for survival under nutrient limitation [37]. In order to trigger a strong defence response, they were also hypothesized to act as sensors, signalling or effector molecules for plasma membrane destabilization and subsequent cell lysis [37].

    References

    1. I. Talibi; H. Boubaker; E.H. Boudyach; A. Ait Ben Aoumar; Alternative methods for the control of postharvest citrus diseases. Journal of Applied Microbiology 2014, 117, 1-17, 10.1111/jam.12495.
    2. S. Tian; R. Torres; A-R. Ballester; B. Li; L. Vilanova; L. González-Candelas; Molecular aspects in pathogen-fruit interactions: Virulence and resistance. Postharvest Biology and Technology 2016, 122, 11-21, 10.1016/j.postharvbio.2016.04.018.
    3. Yulin Cheng; Yunlong Lin; Haohao Cao; Zhengguo Li; Citrus Postharvest Green Mold: Recent Advances in Fungal Pathogenicity and Fruit Resistance. Microorganisms 2020, 8, 449, 10.3390/microorganisms8030449.
    4. Lucas, J.A.; Hawkins, N.J.; Fraaije, B.A. . The Evolution of Fungicide Resistance. In Advances in Applied Microbiology; Sariaslani, S., Gadd, G.M., Eds.; Academic Press: Cambridge, MA, USA, 2015; pp. Vol 90, pp. 29-92.
    5. Ren-Shui Liu; Hu Huang; Qiang Yang; Wang-Yi Liu; Purification of α-Sarcin and an Antifungal Protein from Mold (Aspergillus giganteus) by Chitin Affinity Chromatography. Protein Expression and Purification 2002, 25, 50-58, 10.1006/prep.2001.1608.
    6. T. Theis; M. Wedde; V. Meyer; U. Stahl; The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization. Antimicrobial Agents and Chemotherapy 2003, 47, 588-593, 10.1128/aac.47.2.588-593.2003.
    7. Henrietta Szappanos; Gyula Péter Szigeti; Balázs Pál; Zoltán Rusznák; Géza Szűcs; Éva Rajnavölgyi; József Balla; György Balla; Emőke Nagy; Éva Leiter; et al. The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells. Peptides 2006, 27, 1717-1725, 10.1016/j.peptides.2006.01.009.
    8. Ana Beatriz Moreno; Álvaro Martínez del Pozo; Marisé Borja; Blanca San Segundo; Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea. Phytopathology® 2003, 93, 1344-1353, 10.1094/phyto.2003.93.11.1344.
    9. Lucía Citores; Rosario Iglesias; Sara Ragucci; Antimo Di Maro; José M. Ferreras; Antifungal Activity of α-Sarcin against Penicillium digitatum: Proposal of a New Role for Fungal Ribotoxins. ACS Chemical Biology 2018, 13, 1978-1982, 10.1021/acschembio.8b00410.
    10. Feng Zhu; Yang-Kai Zhou; Zhao-Lin Ji; Xiao-Ren Chen; The Plant Ribosome-Inactivating Proteins Play Important Roles in Defense against Pathogens and Insect Pest Attacks. Frontiers in Plant Science 2018, 9, 146, 10.3389/fpls.2018.00146.
    11. Lucía Citores; Rosario Iglesias; José Ferreras; Antiviral Activity of Ribosome-Inactivating Proteins. Toxins 2021, 13, 80, 10.3390/toxins13020080.
    12. Lucía Citores; Rosario Iglesias; Carolina Gay; José Miguel Ferreras; Antifungal activity of the ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) against the green mouldPenicillium digitatum. Molecular Plant Pathology 2015, 17, 261-271, 10.1111/mpp.12278.
    13. Yongchun Li; Meirong Zhao; Zhi Zhang; Quantitative proteomics reveals the antifungal effect of canthin-6-one isolated from Ailanthus altissima against Fusarium oxysporum f. sp. cucumerinum in vitro. PLOS ONE 2021, 16, e0250712, 10.1371/journal.pone.0250712.
    14. Thomas Aumer; Sébastien N. Voisin; Thomas Knobloch; Céline Landon; Philippe Bulet; Impact of an Antifungal Insect Defensin on the Proteome of the Phytopathogenic Fungus Botrytis cinerea. Journal of Proteome Research 2020, 19, 1131-1146, 10.1021/acs.jproteome.9b00638.
    15. Liming Shi; Beibei Ge; Jinzi Wang; Binghua Liu; Jinjin Ma; Qiuhe Wei; Kecheng Zhang; iTRAQ-based proteomic analysis reveals the mechanisms of Botrytis cinerea controlled with Wuyiencin. BMC Microbiology 2019, 19, 1-14, 10.1186/s12866-019-1675-4.
    16. Mingyan Li; Chi Chen; Xiaoshuang Xia; Betchem Garba; Linlin Shang; Yun Wang; Proteomic analysis of the inhibitory effect of chitosan on Penicillium expansum. Food Science and Technology 2020, 40, 250-257, 10.1590/fst.40418.
    17. Isabel Martins; Adélia Varela; Luís M. T. Frija; Mónica A. S. Estevão; Sébastien Planchon; Jenny Renaut; Carlos A. M. Afonso; Cristina Silva Pereira; Proteomic Insights on the Metabolism of Penicillium janczewskii during the Biotransformation of the Plant Terpenoid Labdanolic Acid. Frontiers in Bioengineering and Biotechnology 2017, 5, 45, 10.3389/fbioe.2017.00045.
    18. Ting Zhou; Bishun Ye; Zhiqian Yan; Xiaohong Wang; Tongfei Lai; Uncovering proteomics changes of Penicillium expansum spores in response to decanal treatment by iTRAQ. Journal of Plant Pathology 2020, 102, 721-730, 10.1007/s42161-020-00486-6.
    19. Shu-Hua Lin; Pan Luo; En Yuan; Xiangdong Zhu; Bin Zhang; Xiaoyu Wu; Physiological and Proteomic Analysis of Penicillium digitatum in Response to X33 Antifungal Extract Treatment. Frontiers in Microbiology 2020, 11, 584331, 10.3389/fmicb.2020.584331.
    20. Yujun Zhou; Jianqiang Xu; Yuanye Zhu; Yabing Duan; Mingguo Zhou; Mechanism of Action of the Benzimidazole Fungicide on Fusarium graminearum: Interfering with Polymerization of Monomeric Tubulin But Not Polymerized Microtubule. Phytopathology 2016, 106, 807-813, 10.1094/phyto-08-15-0186-r.
    21. Di Pietro, A.; Roncero, M.I.G.; Roldán, M.C.R.. From Tools of Survival to Weapons of Destruction: The Role of Cell Wall-Degrading Enzymes in Plant Infection. In Plant Relationships. The Mycota (A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research); Deising, H.B., Eds.; Springer: Berlin, Heidelberg, 2009; pp. Volume 5.
    22. Mario López-Pérez; Ana-Rosa Ballester; Luis González-Candelas; Identification and functional analysis ofPenicillium digitatumgenes putatively involved in virulence towards citrus fruit. Molecular Plant Pathology 2014, 16, 262-275, 10.1111/mpp.12179.
    23. Qiya Yang; Xin Qian; Solairaj Dhanasekaran; Nana Adwoa Serwah Boateng; Xueli Yan; Huimin Zhu; Fangtao He; Hongyin Zhang; Study on the Infection Mechanism of Penicillium Digitatum on Postharvest Citrus (Citrus Reticulata Blanco) Based on Transcriptomics. Microorganisms 2019, 7, 672, 10.3390/microorganisms7120672.
    24. Congyi Zhu; Yuying Wang; Xu Hu; Mengying Lei; Mingshuang Wang; Jiwu Zeng; Hongye-Ye Li; Zheyu Liu; Ting Zhou; Dongliang Yu; et al. Involvement of LaeA in the regulation of conidia production and stress responses in Penicillium digitatum. Journal of Basic Microbiology 2019, 60, 82-88, 10.1002/jobm.201900367.
    25. Mitchell Mutz; Terry Roemer; The GPI anchor pathway: a promising antifungal target?. Future Medicinal Chemistry 2016, 8, 1387-1391, 10.4155/fmc-2016-0110.
    26. Ruoxin Ruan; Mingshuang Wang; Xin Liu; Xuepeng Sun; Kuang-Ren Chung; Hongye Li; Functional analysis of two sterol regulatory element binding proteins in Penicillium digitatum. PLOS ONE 2017, 12, e0176485, 10.1371/journal.pone.0176485.
    27. Sara J. Blosser; Brittney Merriman; Nora Grahl; Dawoon Chung; Robert A. Cramer; Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus. Microbiology 2014, 160, 2492-2506, 10.1099/mic.0.080440-0.
    28. Jonas Henrique Costa; Jaqueline Moraes Bazioli; João Guilherme De Moraes Pontes; Taícia Pacheco Fill; Penicillium digitatum infection mechanisms in citrus: What do we know so far?. Fungal Biology 2019, 123, 584-593, 10.1016/j.funbio.2019.05.004.
    29. D. Macarisin; L. Cohen; A. Eick; G. Rafael; E. Belausov; Michael Wisniewski; S. Droby; Penicillium digitatum Suppresses Production of Hydrogen Peroxide in Host Tissue During Infection of Citrus Fruit. Phytopathology® 2007, 97, 1491-1500, 10.1094/phyto-97-11-1491.
    30. Atiar Rahman; Suresh G. Kumar; Sang Woo Kim; Hye Jin Hwang; Yu Mi Baek; Sung Hak Lee; Hee Sun Hwang; Yun Hee Shon; Kyung Soo Nam; Jong Won Yun; et al. Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte differentiation. PROTEOMICS 2008, 8, 569-581, 10.1002/pmic.200700888.
    31. Ioannis Stergiopoulos; Lute-Harm Zwiers; Maarten A. De Waard; Secretion of Natural and Synthetic Toxic Compounds from Filamentous Fungi by Membrane Transporters of the ATP-binding Cassette and Major Facilitator Superfamily. Netherlands Journal of Plant Pathology 2002, 108, 719-734, 10.1023/a:1020604716500.
    32. Paloma Sánchez-Torres; Molecular Mechanisms Underlying Fungicide Resistance in Citrus Postharvest Green Mold. Journal of Fungi 2021, 7, 783, 10.3390/jof7090783.
    33. Marta de Ramón-Carbonell; Paloma Sánchez-Torres; Penicillium digitatum MFS transporters can display different roles during pathogen-fruit interaction. International Journal of Food Microbiology 2020, 337, 108918, 10.1016/j.ijfoodmicro.2020.108918.
    34. Sandra Garrigues; Mónica Gandía; Attila Borics; Florentine Marx; Paloma Manzanares; Jose F. Marcos; Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum. Frontiers in Microbiology 2017, 8, 592, 10.3389/fmicb.2017.00592.
    35. Sandra Garrigues; Mónica Gandía; Crina Popa; Attila Borics; Florentine Marx; María Coca; Jose F. Marcos; Paloma Manzanares; Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium digitatum. Scientific Reports 2017, 7, 1-13, 10.1038/s41598-017-15277-w.
    36. Marina Marcet-Houben; Ana-Rosa Ballester; Beatriz de la Fuente; Eleonora Harries; Jose F Marcos; Luis González-Candelas; Toni Gabaldón; Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics 2012, 13, 646-646, 10.1186/1471-2164-13-646.
    37. Vera Meyer; Sascha Jung; Antifungal Peptides of the AFP Family Revisited: Are These Cannibal Toxins?. Microorganisms 2018, 6, 50, 10.3390/microorganisms6020050.
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