Recent Progress of Deubiquitinating Enzymes in Human and Plant Pathogenic Fungi

In eukaryotic cells, a large number of proteins are modified by ubiquitination, which leads to proteasomal degradation or change in protein function. The protein ubiquitination process can be reversed by a process called deubiquitination, which plays an important regulatory mechanism in cellular control. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs); the cysteine proteases specifically cleave off ubiquitin from ubiquitinated substrates or ubiquitin precursors. Over the past two decades, components of different DUB families have been found to play important roles in both human and plant pathogenic fungi. Given the importance of DUBs for fungal development and virulence, in this review, we concentrate on recent findings and new insights into the roles of DUBs in different fungal pathogens, with a focus on infection-related morphogenesis and virulence, as well as their roles in development and stress response. We also summarize the DUBs-mediated regulatory mechanisms during the above processes. These findings should allow us to develop novel inhibitors to control fungal pathogens.


Introduction
Ubiquitination is one of the most important post-translational modifications that binds ubiquitin proteins to target proteins [1]. This modification process is catalyzed sequentially by the action of three hierarchical enzymes, i.e., a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). During this cascade, there are hundreds of E3 ligases with different families for selective modification of the target proteins. Then, the carboxyl group of ubiquitin glycine and the ε-amino group of the substrate form an isopeptide bond [2]. There are seven internal lysine residues on ubiquitin that can be attached to target proteins, including K6, K11, K27, K29, K33, K48, and K63. A target protein can be attached with either a mono-or poly-ubiquitin chain [2]. The K48-linked ubiquitin chain attached on target proteins is the most common linkage type, which acts as a proteolysis marker, and leads to protein degradation by the proteasome system [3]. Other atypical ubiquitin chains that are attached on target proteins are usually involved in regulating protein localization, protein-protein interactions, and protein/enzyme activity [3]. Some other proteins, the so-called ubiquitin-like modifiers (UBLs), such as NEDD8, SUMO, and ISG15, can also modify target proteins. UBLs are also attached to target proteins by E1-E2-E3 enzyme cascades [4]. Neddylation and sumoylation are two commonly found ubiquitin-like modifications in eukaryotic cells [4].
Ubiquitination and ubiquitin-like modifications are reversible modifications that can be reversed by the deubiquitination process. A large number of deubiquitinating enzymes
Fine-tuning of the development, stress response, nutrient utilization, and infection processes requires a balance between the addition and removal of ubiquitin moieties in both human and plant pathogenic fungi. In this review, we introduce the basic knowledge of DUBs and summarize the roles and mechanisms of DUBs in control of the development, stress response, nutrient utilization, and infection processes in human and plant pathogenic fungi. In particular, we highlight the advances in mechanisms of fungal DUBs related to pathogenesis. The information in this review extends the body of knowledge regarding the roles that DUBs play in regulating fungal infection, and we also describe how targeting DUBs could be utilized to develop DUB inhibitors that could be applied to yield therapies for fungal diseases, both in humans and plants.

Summary of DUB Studies in Different Human and Plant Pathogenic Fungi
There are around 93 DUBs that have been identified in humans, which have been classified into five families, including 54 USPs/UBPs (ubiquitin-specific proteases), 16 OTUs (ovarian tumor proteases), 4 UCHs (ubiquitin C-terminal hydrolases), 4 MJDs (Machado-Joseph deubiquitinases), and 7 JAMMs (JAB1/MPN/MOV34 metalloproteases) [9]. These subclasses of DUBs are different in their catalytic domains and other domains. We used the EnsemblFungi genome database (http://fungi.ensembl.org/index.html (accessed on  -MGG_04031  UBP2  CNAG_07333  MGG_00877  UBP3  CNAG_06920  MGG_05996  UBP4  CNAG_00757  MGG_04957  UBP5  -UBP6  CNAG_03109  MGG_02970  UBP7  --UBP8  CNAG_06637  MGG_03527  UBP9  CNAG_02395  -UBP10  CNAG_00455  -UBP11  --UBP12  CNAG_07717  MGG_05666  UBP13  CNAG_02395  MGG_09907  UBP14  CNAG_05708  MGG_08270  UBP15  CNAG_05650  MGG_07468  UBP16  -MGG_04494  UCHs  YUH1  CNAG_00180  MGG_06319  MGG_01683  OTUs  OTU1  -MGG_03532  OTU2  CNAG_02004  MGG_11505  MINDY  MIY1  -MGG_03906  YGI2  -MGG_05274  JAMM  RPN11  CNAG_07028  MGG_16706  CNAG_04809  MGG_05274  CNAG_06563 MGG_01059 MGG_10653 The UBP (USPs in human) family represents the largest DUBs in fungal genomes that mainly process larger leaving groups. The catalytic domain of UBPs is well-conserved in this family, but distinct from other domains, including domains of Zn-finger in ubiquitinhydrolases and other protein (zf-UBP), ubiquitin-associated domain (UBA), repeated domain in UCH-protein (RPT), rhodanese, ubiquitin-like domain (UBL), domain present in ubiquitin-specific protease (DUSP), meprin and TRAF homology (MATH), ubiquitinspecific protease C-terminal (USP7_C2), and ICP0-binding domain of ubiquitin-specific protease 7 (USP7_ICP0_bdg) (Figure 2A) The UCH family of DUBs consists of only one protein (YUH1) in yeasts S. cerev and C. neoformans (CNAG_00180), but contains two proteins (MGG_06319 MGG_01683) in M. oryzae ( Figure 2B). Studies have suggested that the UCHs pref cleave small protein substrates from ubiquitin, and recycle ubiquitin inappropriately jugated to substrates (Amerik and Hochstrasser, 2004). UCHs may also be involv processing newly synthesized ubiquitin precursors, while the specific functions of U remain largely unknown. The OTU family of DUBs consists of two proteins (OTU1 OTU2) in S. cerevisiae, one protein in C. neoformans (CNAG_02004), and two pro (MGG_03532 and MGG_11505) in M. oryzae. The OTU family of DUBs is defined o basis of their homology to the ovarian tumor gene, which functions in the develop of the ovaries in fruit flies [14]. The JAMM family of DUBs contains one protein (RP in S. cerevisiae, three proteins in C. neoformans (CNAG_07028, CNAG_04809, CNAG_06563), and four proteins (MGG_16706, MGG_05274, MGG_01059, MGG_10653) in M. oryzae ( Figure 2B). The CSN5 protein homologs contain a metallo tease motif (JAMM) in the MPN domain and a CSN5_C domain, making them the cata center for the COP9 signalosome complex (CSN complex) [15]. The CSN complex tions as a deneddylation DUB to remove the UBL NEDD8 from cullin proteins [16]. I estingly, in S. cerevisiae, no CSN5 homologue is identified, but in C. neoformans an oryzae, they have one homologue (CNAG_04809 and MGG_05274), suggesting an e tionary difference in them. The MINDY family of DUBs contains two proteins (MIY1 YGI2) in S. cerevisiae, one protein in M. oryzae (MGG_03906), but is absent in C. neofor Some other predicted DUBs could also function toward moieties of UBLs, su NEDD8, SUMO, ISG15, and ATG8 [4]. For example, in S. cerevisiae, UBL ATG8 ca The UCH family of DUBs consists of only one protein (YUH1) in yeasts S. cerevisiae, and C. neoformans (CNAG_00180), but contains two proteins (MGG_06319 and MGG_01683) in M. oryzae ( Figure 2B). Studies have suggested that the UCHs prefer to cleave small protein substrates from ubiquitin, and recycle ubiquitin inappropriately conjugated to substrates (Amerik and Hochstrasser, 2004). UCHs may also be involved in processing newly synthesized ubiquitin precursors, while the specific functions of UCHs remain largely unknown. The OTU family of DUBs consists of two proteins (OTU1 and OTU2) in S. cerevisiae, one protein in C. neoformans (CNAG_02004), and two proteins (MGG_03532 and MGG_11505) in M. oryzae. The OTU family of DUBs is defined on the basis of their homology to the ovarian tumor gene, which functions in the development of the ovaries in fruit flies [14]. The JAMM family of DUBs contains one protein (RPN11) in S. cerevisiae, three proteins in C. neoformans (CNAG_07028, CNAG_04809, and CNAG_06563), and four proteins (MGG_16706, MGG_05274, MGG_01059, and MGG_10653) in M. oryzae ( Figure 2B). The CSN5 protein homologs contain a metalloprotease motif (JAMM) in the MPN domain and a CSN5_C domain, making them the catalytic center for the COP9 signalosome complex (CSN complex) [15]. The CSN complex functions as a deneddylation DUB to remove the UBL NEDD8 from cullin proteins [16]. Interestingly, in S. cerevisiae, no CSN5 homologue is identified, but in C. neoformans and M. oryzae, they have one homologue (CNAG_04809 and MGG_05274), suggesting an evolutionary difference in them. The MINDY family of DUBs contains two proteins (MIY1 and YGI2) in S. cerevisiae, one protein in M. oryzae (MGG_03906), but is absent in C. neoformans.
Some other predicted DUBs could also function toward moieties of UBLs, such as NEDD8, SUMO, ISG15, and ATG8 [4]. For example, in S. cerevisiae, UBL ATG8 can be processed by the protease ATG4. Likewise, in S. cerevisiae, UBL Smt3 (SUMO) can be processed by the cysteine proteases Ulp1 and Ulp2.

Overview of Roles of Ubiquitination and DUBs in Different Pathogenic Fungi
The functions of ubiquitination or ubiquitin-like modification have been widely revealed in both human fungal pathogens (C. albicans and C. neoformans) and plant fungal pathogens (M. oryzae, Fusarium oxysporum, and Cryphonectria parasitica) [17][18][19][20][21][22][23][24][25][26][27]. In fungal pathogens, the role of ubiquitination has been studied by analyzing ubiquitin-related genes, and it was found that ubiquitination played important roles in fungal development, stress resistance, nutrient utilization, and fungal virulence [19][20][21][22]25,27]. For example, in C. albicans, deletion of the ubiquitin gene UBI4 affects the cell cycle, morphology, resistance to thermal stress, oxidative stress, and cell wall perturbing stresses, and results in catabolism block and virulence reduction [19][20][21]. In C. neoformans, deletion of the ubiquitin gene UBI1 leads to defects in vegetative growth, reduction in melanin synthesis, and attenuation in virulence during infection [22]. Likewise, in the plant pathogen M. oryzae, UBI4 deletion also results in defects of growth and loss of virulence, and it also leads to significant reductions in sporulation, spore germination, as well as appressorium formation [25]. The ubiquitin gene UBI4 has also been found to be important for fungal development, stress response, and virulence in other plant pathogenic fungi, such as Cryphonectria parasitica [27].
Genes encoding E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases have also been studied in human fungal pathogens and plant fungal pathogens. Explaining the dynamics of E2-E3 profiling may help to decode which E2:E3 pairs are true partners. In humans, it is very important to determine specific E2-E3 pairing in different disease states, because an accurate description of E2-E3 is crucial in the development of therapeutic drugs. Future research on E2-E3 coordination selectivity will contribute to early diagnosis and the development of cancer therapeutics [28]. Disruption of these E2 and E3 encoding genes also affects fungal development, stress response, and virulence. C. albicans Rad6 is a ubiquitin conjugating enzyme, which positively regulates UV damage repair but negatively regulates hyphal growth [29]. While, in M. oryzae, Rad6 plays key roles in development and infection of plant hosts [30]. It has been widely reported that homologues of F-box proteins were associated with virulence of important fungal pathogens [31], including GRR1 in C. albicans [32], GRR1 in Gibberella zeae [33], CDC4 in M. oryzae [34], and C. albicans [35,36]. For example, in C. neoformans, the GRR1 homolog FBP1 is essential for mycelial growth, sexual reproduction, and virulence [24,37]. In M. oryzae, two homologs of GRR1 are required for fungal pathogenicity to rice [34]. The roles of subunits of the SCF complex (E3 ligase component) in pathogenic fungi, such as cullin CDC53 in C. albicans [38], FBX15 in A. fumigatus [39], FRP1 in F. oxysporum [40], MoFWD1, MoCDC4, and MoFBX15 in M. oryzae [34] have also been reported. In C. albicans, another SCF complex component CDC4 is also essential for morphogenesis and biofilm formation [35,36]. F-box proteins also play key roles in human cancer initiation and progression, such as FBXL8 and FBXO43 [41,42]. Therefore, the roles of ubiquitination revealed by ubiquitin encoding genes and E2/E3 encoding genes in both human and plant pathogenic fungi demonstrate key roles in various biological functions.
Although the function of ubiquitination has been well revealed [17,18], deubiquitination mediated by DUBs has not been well summarized in pathogenic fungi. On the one hand, similar to ubiquitination, the biological functions of DUBs are quite different. On the other hand, DUBs are highly redundant in function, and it is hard to specify physiological functions to each of the DUBs. A comparative investigation of the single DUB gene to determine phenotypic differences is required. Several studies have addressed this issue in yeast, human, and plant pathogenic fungi ( Table 2). For example, 16 UBP genes identified in S. cerevisae were found to be important for growth, nutrient utilization, stress response, energy metabolism, and sexual reproduction [43][44][45]. Disruption of S. cerevisiae DOA4, UBP6, UBP8, UBP10, and UBP14 have all resulted in increased ubiquitin levels [46]. More specifically, DOA4 and UBP10 are required for carbon utilization and nitrogen utilization, respectively [44]; UBP14 is required for adaptation to the presence of glucose to mediate the degradation of enzymes involved in gluconeogenesis [47]; and UBP15 plays key roles in stress response [48].  [58] In C. neoformans, all 19 DUB genes are not essential, whose mutants have been described and have shown normal growth under standard conditions [54,59]. According to a phenotypic analysis, it was found that DUBs, UBP5, DOA4, UBP13, and UBP14 were required for pigmentation, and UBP5 could be the major DUB required for stress response with roles in virulence, capsule formation, and sporulation [54]. In M. oryzae, all of the UBP genes have been systematically analyzed [49].
In M. oryzae, 11 putative UBP genes have been characterized (Table 2) [49]. The results have shown that UBP genes play various roles in hyphal growth, conidium formation, stress response, nutrient utilization, and infection-related structure formation [49]. Evaluation of the expression profiles of these UBP genes has demonstrated that UBP3, UBP6, UBP1,2 and UBP14 are constantly expressed at high levels during all tested developmental stages, while UBP1, UBP13, and UBP15 are expressed at low levels. Four genes in mycelia, seven genes in conidia and appressoria, and five genes in invasive hyphae have high expression levels [49]. These data suggest that UBP genes play various biological functions during different development and infection stages. Among these, M. oryzae UBP genes, i.e., UBP1, UBP3, UBP4, UBP8, and UBP14 are required for colony growth, nearly all UBP genes are required for conidia production, and most of them are required for full virulence (except for UBP2 and UBP16) [49]. Accordingly, except for UBP2 and UBP16, all other UBP genes are required for oxidative stress response, but they are differently required for responses to other stresses [49]. Detailed phenotypic roles of these DUBs in human and plant pathogenic fungi are discussed below.

DUBs in Fungal Growth
For fungi, hyphal growth is relevant to cell division, cell cycle, nutrient metabolism, and energy metabolism. In S. cerevisiae, deubiquitination is an important process to regulate energy uptake and the cell cycle for growth [60]. In human pathogen C. neoformans, UBP5 deletion mutant is temperature sensitive, it grows slower at 37 • C and loses growth ability at 39 • C [54]. The mutant still grows slower at 30 • C in a rich medium. This phenotype may be due to the functions of UBP5 in nutritional utilization and cell cycle regulation [54]. Similarly, the orthologs of CnUBP5 in S. cerevisiae (UBP15) and in C. gattii (CgUBP5) are also closely relevant to their proliferation or propagation [54].
In M. oryzae, UBP1, UBP3, UBP4, UBP8, and UBP14 deletions have all been reported to lead to defects in vegetative growth (Table 2) [49]. The growth defect of ∆MoUBP14 was much more severe than other UBP mutants. The ∆MoUBP14 mutant colony was much whiter, suggesting a regulatory role of UBP14 in mycelial melanin-related pigmentation [49,53]. The cell cycle of ∆MoUBP14 was affected, demonstrated by a shorter hyphal tip cell [53]. Another reason why ∆MoUBP14 grows slower is that UBP14 is involved in nutrient utilization. Similar phenotypes in growth have also been found in the ∆MoUBP3 mutant, with an evident cell cycle defect in the hyphal tip cell [50]. The ∆MoUBP4 mutants have shown reduced colony diameter and compact mycelium mass when incubated in liquid complete medium (CM) [51]. The ∆MoUBP8 mutant is slightly reduced in both mycelial growth and melanin synthesis [52]. In S. scitamineum, the DUB gene SsCI33130 is not relevant to growth [57].

DUBs in Fungal Spore Production
Many fungi can produce asexual spores, called conidia, which germinate to form mycelium in water and adequate nutrition conditions, or to form infection structures on host surfaces. The formation of asexual spores is very important for the life cycle of plant pathogenic fungi, and conidiogenesis is a key step in the infection process. In the plant pathogenic fungus M. oryzae, most of the identified UBP genes (except UBP2 and UBP15) have been found to be required for conidiogenesis [49]. The UBP1, UBP3, UBP4, UBP6, UBP8, UBP12, UBP13, UBP14, and UBP16 deletion mutants are evidently decreased in conidiation. Only a few visible spores could be found on culture plates of the ∆UBP8 and ∆UBP14 mutants. Accordingly, conidiophore formation of these UBP mutants was also much more sparse as compared with the wild type.
In A. alternata, the AaCSN5 gene has been reported to be required for conidiation. The ∆AaCSN5 mutant is severely reduced in conidia formation [58]. In another plant pathogenic fungus Pestalotiopsis fici [61], and in non-plant pathogenic fungus N. crassa, deletion of CSN5 homolog genes also severely affects conidia production [62,63]. Interestingly, the homologous gene of CSN5 in A. nidulans seems not to be involved in asexual spore production, but is required for the sexual developmental cycle [64].

DUBs in Fungal Sexual Reproduction
Sexual reproduction of fungi, similar to that of other organisms, includes a combination of two compatible nuclei. Through this method, fungi can create a diverse genetic background, which is conducive to their adaptation to the environment and host. It has been reported that in the human pathogen C. neoformans, UBP5 is essential for sexual reproduction and helps to produce the main infectious propagules (basidiospores) of human cryptococcosis [54]. The mating between WT and the UBP5∆ mutant cannot produce any filaments, due to blockage at the cell fusion point. This phenotype of UBP5∆ may be partially caused by blocking MFa pheromone gene expression [54].
For the plant pathogenic fungus S. scitamineum, sexual mating is closely related to its pathogenicity. The OTU1 deubiquitinase gene SsCI33130 regulates the expression of genes involved in the synthesis of cAMP and tryptophol [57]. The cAMP and tryptophol are small molecule signaling substances required for the sexual mating of S. scitamineum. CSN5 in A. nidulans also seems to be required for the sexual developmental cycle [64].

DUBs in Fungal Stress Response
Various extracellular or intracellular stresses, such as high temperature, oxidative damage, high salt, and antifungal drugs, lead to the accumulation of misfolded or damaged proteins and cause cell damage. Removal of these misfolded or damaged proteins through ubiquitin-dependent proteolysis can prevent cell damage. Ubiquitination is an essential element during stress response, which is required for resistance to heat stress, oxidative stress, and other stresses [2,3]. Conversely, deubiquitination mediated by DUBs is required for maintaining ubiquitin homeostasis through processing of the ubiquitin precursors or proofreading of the ubiquitin-conjugated proteins [2,3,6], and therefore, is also important for stress response in fungi. Ubiquitination and deubiquitination may be important modification mechanisms of fungal stress response-related signaling pathways.
In C. neoformans, many genes encoding E2 are significantly upregulated in the HOG pathway mutants. In addition, some genes encoding components of Ca2+/calcineurin signaling and MAPK signaling pathways also change expression in the deletion mutant of CnUBP5 [54]. UBP5 could be the major DUB to regulate resistance to stresses in C. neoformans. During infection, C. neoformans needs to encounter temperature stress of the host cells, as well as other stresses including oxidative and nitrosative stress, salt stress, and antifungal agents. Interestingly, the UBP5∆ mutant is sensitive to all the abovementioned stresses, as well as cell wall and cell membrane perturbing agents [54], reflecting a major role of CnUBP5 in stress adaptation. CnUBP5 may coordinate with UBI4 to keep a balance of cellular ubiquitin under stresses. In addition to CnUBP5, C. neoformans DOA4 is also required for response to stresses of high sodium ions and cell wall perturbing agents SDS and caffeine [54]. CnDOA4 and CnUBP13 are also required for responses to antifungal drugs such as fluconazole and caspofungin [54].
In C. gattii, the DUB UBP5 also plays a key role in stress response. The deletion mutant of UBP5 is sensitive to various stressors, including high temperature stress, oxidative stress, NO stress, salt stress, antifungal drugs, etc. [55]. It is predicted that Cryptococcus spp. UBP5 could regulate substrates in different stress responsive signaling pathways to encounter various cellular and environmental stresses.
In M. oryzae, most of the UBP genes are responsive to different stresses, positively or negatively [49]. For instance, positive UBP regulators include UBP1, UBP3, UBP8, UBP12, and UBP14, while UBP1, UBP2, and UBP4 are negative UBP regulators in oxidative stress response, and UBP2 and UBP4 are negative UBP regulators in cell wall stress response, among which, MoUBP14 could be a main regulator in stress response. The ∆UBP14 mutant is more sensitive to various stresses, such as oxidative stress, osmotic stress, salt stress, cell wall perturbing agents, alkaline pH stress, as well as high temperature stress [49,53].
All of the above studies suggest that DUBs, especially UBPs, are very important for stress adaptation in human and plant pathogenic fungi, which can help fungi to encounter harsh host and environmental conditions for survival and infection.

DUBs in Fungal Nutrient Utilization
In fungi, nutrients provide energy and substrates for biosynthesis and catabolism, as well as play important roles in cellular regulation. Whether UBP genes are required for carbon source utilization has been evaluated in M. oryzae. On minimal medium (MM) plates supplemented with each of glucose, NaAc, ethanol, and glycerol as the sole carbon sources, growth of the ∆UBP4 and ∆UBP14 mutants were significantly reduced [49,51,53]. This result suggests that UBP4 and UBP14 are required for usage of all the tested carbon sources, therefore, acting as positive regulators of carbon source utilization. On the contrary, UBP1 and UBP8 function as negative regulators of carbon source utilization [49], demonstrating a complex regulatory mechanism in plant pathogenic fungi. In another plant pathogenic fungus A. alternata, AaCSN5 plays a role in nitrogen metabolism, and several amino acid metabolism pathways are positively regulated by it [58].

DUBs in Fungal Virulence
Many filamentous fungi are pathogenic to humans or plants. Diseases caused by pathogenic fungi threaten both plant health and human health.
The human pathogenic fungus C. neoformans arranges multiple pathogenic factors to overcome host cell stresses and to cause infection of the host. Extracellular polysaccharide capsule, melanin, and hydrolytic enzyme are key pathogenic factors in C. neoformans, which are essential for pathogenicity [65]. CnUBP5 deletion leads to a defect in capsule attachment. UBP5 probably affects the capsule by involvement in cell wall polysaccharide assembly but not in the capsule synthesis or secretion [54]. Another important virulence-related DUB in C. neoformans is DOA4, whose deletion mutant is dramatically attenuated in virulence in the macrophage assay [54]. Thermotolerance in host cells is an important virulence factor that is regulated by both UBP5 and DOA4 [54].
In another human pathogenic fungus C. gattii, the CgUBP5∆ mutant is significantly attenuated in virulence in a mammalian host [55]. Interestingly, this mutant shows a slightly enhanced capsule and melanin production, which is different to that of the CnUBP5∆ mutant. Thermotolerance is also reduced in the CgUBP5∆ mutant [55], which is part of the factor that leads to the reduced survival rate of mutants in macrophages. Therefore, CgUBP5 is necessary for the survival of C. gattii in the lung space and its extrapulmonary transmission.
For plant pathogenic fungi, their pathogenicity is closely associated with infectionrelated morphogenesis and form special structures for host infection [66]. The rice blast fungus M. oryzae forms a special structure called appressorium, which forms a penetrating peg and penetrates the host plant epidermis through the physical force provided by the swelling pressure in the appressorium [66]. Whether each UBP gene in M. oryzae is involved in regulating virulence and infection processes has been evaluated [49]. The results have demonstrated that UBP1, UBP3, UBP4, UBP6, UBP8, and UBP14 are relevant to the virulence of M. oryzae, while UBP2 and UBP16 are not [49]. The deletion mutants of those virulence-related genes are normal in appressorium formation, however, ∆UBP14 was severely reduced in appressorium-mediated penetration. The ∆UBP1, ∆UBP3, ∆UBP4, ∆UBP6, and ∆UBp8 mutants, as well as ∆UBP14, are normal in penetration, but are blocked in infection hyphae formation and extension [49]. All of the virulence-related genes are required for eliminating ROS accumulation of the host plant cells, suggesting that these UBP genes are important for host cellular oxidative stress resistance, as one of the roles in regulating invasive growth.
The ∆UBP14 mutant totally loses its virulence due to several reasons. First, the ∆UBP14 mutant is blocked in appressorium-mediated penetration, which is due to poor utilization of glycogen and lipid storage, defect in cell wall integrity, and a reduction in appressorial turgor [53]. Second, the mutant is also retarded in invasive growth, which is partly attributed to the sensitivity to distinct stresses (including oxidative stress), and a reduction in nutrient utilization [53]. The severe reduction of virulence in the ∆UBP8 mutant is synthetically attributed to defects in melanin synthesis, appressorium turgor pressure, and ROS eliminating, as well as adaptation to other stresses such as salt stress, osmotic stress, and cell wall perturbing stresses [52]. Expressions of two pigment synthesis genes (MoBUF1 and MoRSY1) are significantly reduced in the ∆UBP8 mutant [52], suggesting a regulatory mechanism of UBP8 in melanin synthesis. The ∆UBP4 mutant is delayed in conidial glycogen and lipid droplets utilization, but forms normal appressorium turgor [51]. Therefore, the reduced virulence in the ∆UBP4 mutant could mainly be due to defect in stress response.
MoATG4 deletion, a gene encoding protease of Ubl Atg8 in M. oryzae, resulted in reduction of appressorium formation and maturation [67]. The ∆MoATG4 mutant accumulates lower appressorium turgor pressure and fails to penetrate rice and barley [67].
In plant pathogenic fungus A. alternata, deletion of DUB gene AaCSN5 significantly reduces its virulence and necrotic lesions on citrus leaves [58]. The reduction of the virulence is due to activation of the host immune response and a decrease in toxin production [58]. However, AaCSN5 is not relevant to oxidative stress tolerance and host ROS scavenging.
In S. scitamineum, DUB gene SsCI33130 deletion also results in a severe reduction in the pathogenicity to host plants [57]. Interestingly, it seems that SsCI33130 does not affect the fungal growth and development, stress response, and other phenotypes of S. scitamineum. The pathogenicity reduction of the deletion mutant is only attributed to sexual mating [57], which is the primary condition for dikaryotic mycelia formation and pathogenicity development in S. scitamineum.

Regulatory Mechanisms Mediated by DUBs in Different Pathogenic Fungi
DUBs are part of the ubiquitin-proteasome system, which are involved in some cellular key processes such as protein quality control, DNA damage repair, and cell cycle regulation, etc. [5]. Recent studies in pathogenic fungi have revealed some regulatory mechanisms mediated by DUBs, including appressorium associated signaling pathways, autophagy, nutrient utilization, histone modification, endocytosis, proteasome function, and circadian clock.

DUBs and Cellular Signaling Pathways
As a central nutrient signaling pathway, the target of rapamycin (TOR) signaling pathway negatively regulates macro-autophagic processes [68]. This signaling pathway works in parallel with the Ras/PKA signaling pathway to regulate appressorium formation of plant pathogenic fungi such as M. oryzae [69]. It has been reported that in M. oryzae, UBP3-mediated ribophagy may be regulated by the TOR signaling pathway [50]. The ∆UBP3 mutant is blocked in ribophagy and is very sensitive to rapamycin. This phenotype is consistent with a study in S. cerevisiae, in which the TOR signaling pathway activated UBP3p-mediated ribophagy processes [70]. However, it is still unknown whether UBP3mediated ribophagy plays important roles in appressorium formation and maturation.
In general, the TOR signaling pathway is antagonistic with the Ras/PKA signaling pathway, and increased sensitivity to rapamycin usually leads to overactivation of the Ras/PKA signaling pathway [68,69]. Consistent with this, UBP3 deletion has resulted in overactivation of the RAS2/PKA signaling pathway in M. oryzae [50], which was important for appressorium formation. This regulatory mechanism is achieved by UBP3-mediated deubiquitination of Smo1, which is a GTPase-activating protein (RasGAP) involved in regulating the balance of Ras proteins in M. oryzae [71]. Moreover, UBP3-mediated deubiquitination of Smo1 also regulates the Pmk1 MAPK signaling pathway, another main signaling pathway regulating appressorium formation. As a result, the ∆UBP3 mutant is delayed in appressorium formation and maturation, fails to form enough appressorial turgor for penetration, and is reduced in virulence [50]. Therefore, UBP3-mediated deubiquitination of Smo1 is a molecular switch for Ras signaling. During appressodium formation, UBP3 expresses at a low level to promote ubiquitination of Smo1, then increases the protein level of GTP-bound active RAS2. Subsequently, the cAMP-PKA and Pmk1-MAPK signaling pathways are activated to promote appressorium formation (Figure 3) [50]. ure 3) [50].
In S. scitamineum, another DUB gene SsCI33130 has also been found to be involv the cAMP signaling pathway. SsCI33130 deletion leads to a defect in sexual mating scitamineum, while exogenous addition of cAMP restores the sexual mating of the mu [57]. Expression of two cAMP synthesis-related genes, ARO8 and UAC1, are regulate SsCI33130, suggesting a potential regulatory mechanism of the cAMP signaling path [57].

DUBs and Autophagy
Autophagy plays a key role in infection of the plant pathogenic fungi, for exam in appressorium formation of the rice blast fungus M. oryzae. There are around 20 genes related to the autophagy process in M. oryzae, among which, Atg8 and Atg1 UbLs [72]. The DUB of Atg8 is Atg4 as reported in S. cerevisiae [73]. M. oryzae Atg complement ScAtg4 in yeast, and interact with MoAtg8 and cleave it in vitro [67] MoATG4 gene is induced by starvation and controls autophagic cell death during ap sorium formation (Figure 3) [67].
Under nitrogen starvation, ribosomes are rapidly degraded in S. cerevisiae, wh mediated by a selective autophagy process, so called ribophagy [74]. A similar proc also found in M. oryzae (Figure 3) [50]. The ribosome marker protein Rpl25 is degr during the ribophagy process, which is UBP3 dependent and induced by rapamycin ment and nitrogen starvation conditions [50]. In the yeast S. cerevisiae, full functio UBP3p requires BRE5p to form a complex. Studies have also found that the UBP3/B complex not only regulated ribophagy, but was also related to the regulation of mit agy [69]. The detailed mechanism and function of the UBP3/BRE5 complex-mediate tophagic processes in pathogenic fungi requires further study.

DUBs and Fungal Carbon Source Utilization
Nutrient utilization is essential for fungal development and formation of infec associated structures, such as appressorium and invasive hyphae in plant pathog fungi. We have mentioned above that M. oryzae UBP14 regulates carbon source utiliza In S. scitamineum, another DUB gene SsCI33130 has also been found to be involved in the cAMP signaling pathway. SsCI33130 deletion leads to a defect in sexual mating in S. scitamineum, while exogenous addition of cAMP restores the sexual mating of the mutant [57]. Expression of two cAMP synthesis-related genes, ARO8 and UAC1, are regulated by SsCI33130, suggesting a potential regulatory mechanism of the cAMP signaling pathway [57].

DUBs and Autophagy
Autophagy plays a key role in infection of the plant pathogenic fungi, for example, in appressorium formation of the rice blast fungus M. oryzae. There are around 20 ATG genes related to the autophagy process in M. oryzae, among which, Atg8 and Atg12 are UbLs [72]. The DUB of Atg8 is Atg4 as reported in S. cerevisiae [73]. M. oryzae Atg4 can complement ScAtg4 in yeast, and interact with MoAtg8 and cleave it in vitro [67]. The MoATG4 gene is induced by starvation and controls autophagic cell death during appressorium formation ( Figure 3) [67].
Under nitrogen starvation, ribosomes are rapidly degraded in S. cerevisiae, which is mediated by a selective autophagy process, so called ribophagy [74]. A similar process is also found in M. oryzae (Figure 3) [50]. The ribosome marker protein Rpl25 is degraded during the ribophagy process, which is UBP3 dependent and induced by rapamycin treatment and nitrogen starvation conditions [50]. In the yeast S. cerevisiae, full function of UBP3p requires BRE5p to form a complex. Studies have also found that the UBP3/BRE5 complex not only regulated ribophagy, but was also related to the regulation of mitophagy [69]. The detailed mechanism and function of the UBP3/BRE5 complex-mediated autophagic processes in pathogenic fungi requires further study.

DUBs and Fungal Carbon Source Utilization
Nutrient utilization is essential for fungal development and formation of infectionassociated structures, such as appressorium and invasive hyphae in plant pathogenic fungi. We have mentioned above that M. oryzae UBP14 regulates carbon source utilization. This regulation is at least achieved by UBP14-mediated deubiquitination of FBP1 and PCK1 (Figure 3). FBP1 and PCK1 are two key enzymes that catalyze gluconeogenesis. After the addition of glucose, FBP1 and PCK1 are subjected to protein degradation, by which they are rapidly degraded through the ubiquitin proteasome system. When glucose was supplied to M. oryzae growing in a low glucose medium, FBP1 and PCK1 were degraded in a short time (∼12 h) [53]. Therefore, UPS-mediated degradation of FBP1 and PCK1 may be related to coordinating gluconeogenesis and glycolysis balance, thereby facilitating the fungi to utilize nutrients during infection.
MoUBP8 is also involved in carbon utilization, through a regulatory mechanism different from MoUBP14 [52]. MoUBP8 is mainly associated with the pentose catabolite pathway and carbon catabolite repression (CCR) (Figure 3). In the pentose catabolic pathway, D-xylose is converted to xylitol and D-xylulose-5-phosphate for further utilization, however, utilization of D-xylose is blocked in the ∆MoUBP8 mutant. In addition, the ∆MoUBP8 mutant also shows a defect in CCR repression, demonstrated by more sensitivity to AA but resistance to 2-DG [52].

DUBs and Histone Modification
In S. cerevisiae, the transcriptional activation process can be regulated by UBP8mediated deubiquitination [75]. The UBP8p is a component of the DUB module, which is composed of UBP8, SGF11, SUS1, and SGF73 [76]. The UBP8 DUB module is responsible for H2B deubiquitination, which coordinates with the histone SAGA complex (Spt-Ada-Gcn5acetyltransferase complex) to activate gene expression in Candida albicans (Figure 3) [76,77]. Another DUB, UBP10, is also required for deubiquitination of H2B in yeast [76], however, the downstream regulatory mechanism, either UBP8 or UBP10, has not been revealed in the pathogenic fungi and needs to be uncovered in the future.

DUBs and Endocytosis
DUBs participate in the endocytic pathway and are also involved in other intracellular traffic types. In S. cerevisiae, the DUB DOA4/UBP4 plays a role in recycling ubiquitin at late endosome. Disruption of S. cerevisiae DOA4 results in defects of many ubiquitinrelated processes [78]. The endosomal sorting complex required for transport III (ESCRT-III) proteins BRO1 and SNF7 can activate the deubiquitinating activity of DOA4 by recruiting it to the endosomes [79]. Although a yeast hybrid assay has confirmed the interaction between MoUBP4 and MoSNF7/MoBRO1 in M. oryzae [51], the biological functions and regulatory mechanisms of the MoUBP4-MoBRO1-MoSNF7 module are still unknown and require exploration.

DUBs and Circadian Clock
The DUB protein CSN5 is one component of the COP9 signalosome (CSN), which contains eight subunits and functions as a deneddylation enzyme to remove NEDD8 from tagged proteins [16]. The cullin subunit of the SCF-FWD1 complex is tagged with a NEDD8 molecule while it is part of the SCF complex [16]. NEDD8 can tag to the cullin subunit of the SCF-FWD1 complex to be part of the SCF complex as a ubiquitin E3 ligase [16]. CSN5mediated cullin deneddylation can regulate SCF complex disassembly, which is required for FRQ ubiquitination [62,63]. In the CSN mutants, FRQ is accumulated and rhythmicity is lost [62,63]. However, the CSN5-mediated regulatory mechanism in circadian clock requires more attention.

DUBs and Drug Resistance
Drug resistance is very important for adaptation and survival of human and plant pathogenic fungi. In C. glabrata, BRE5 and UBP3-mediated deubiquitination of PDR1 play key roles in drug resistance [56]. UBP3 is co-purified with BRE5 and PDR1 [56]. The deubiquitinating enzyme UBP3 has been found to function together with BRE5 for its activity in S. cerevisiae [80]. This UBP3:BRE5 deubiquitinase complex deubiquitinates the drug resistance transcription factor PDR1, and negatively regulates its activity to regulate drug resistance (Figure 3).

Conclusions
The past 10 years have yielded much new knowledge regarding DUBs in human and plant pathogenic fungi. Here, we demonstrated the physiological roles of DUBs in fungal growth, development, asexual spore reproduction, sexual mating, stress response, nutrient utilization, and infection processes. These studies have emphasized key roles of DUBs in virulence of both human and plant pathogenic fungi, especially in the human fungal pathogen C. neoformans and the plant fungal pathogen M. oryzae. In this article, we have primarily discussed the mechanisms of the regulatory roles of DUBs in cellular signaling pathways, autophagy, carbon source utilization, histone modification, endocytosis, and circadian clock.
As is evident from the brevity of this review, the potential roles of deubiquitinases in human and plant pathogenic fungi remains largely unexplored. Much work also remains to be done with the DUBs in regulating pathogenicity, in addition to the human fungal pathogen C. neoformans and the plant fungal pathogen M. oryzae. The substrates of the enzyme and the molecular mechanisms of DUBs in virulence remain largely unknown. Future investigations will focus on high-throughput identification of each DUB targets by ubiquitination proteomics, which would lead to a comprehensive understanding of the regulatory mechanism. Investigation of the upstream regulation of DUBs, DUB coordination, and interaction with other pathways or complexes are also required. In addition, the regulatory mechanisms of DUBs in transcription, translation, epigenetics, and post-translational modifications would be of interest.
At present, small molecule inhibitors have been identified to suppress DUB targets to control cancers and virus infection in humans [81,82]. Considering that DUBs play key roles in the pathogenicity of fungal pathogens, it is possible to identify potential antimicrobial drugs or fungicides in the future.