Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle.

Although most of the early events of the hepatitis C virus (HCV) life cycle are well characterized, our understanding of HCV egress is still unclear. Some reports implicate the conventional endoplasmic reticulum (ER)-Golgi route, while some propose noncanonical secretory routes. Initially, the envelopment of HCV nucleocapsid occurs by budding into the ER lumen. Subsequently, the HCV particle exit from the ER is assumed to be mediated by coat protein complex II (COPII) vesicles. COPII vesicle biogenesis also involves the recruitment of cargo to the site of vesicle biogenesis via interaction with COPII inner coat proteins. We investigated the modulation and the specific role of the individual components of the early secretory pathway in HCV egress. We observed that HCV inhibits cellular protein secretion and triggers the reorganization of the ER exit sites and ER-Golgi intermediate compartments (ERGIC). Gene-specific knockdown of the components of this pathway such as SEC16A, TFG, ERGIC-53, and COPII coat proteins demonstrated the functional significance of these components and the distinct role played by these proteins in various aspects of the HCV life cycle. SEC16A is essential for multiple steps in the HCV life cycle, whereas TFG is specifically involved in HCV egress and ERGIC-53 is crucial for HCV entry. Overall, our study establishes that the components of the early secretory pathway are essential for HCV propagation and emphasize the importance of the ER-Golgi secretory route in this process. Surprisingly, these components are also required for the early stages of the HCV life cycle due to their role in overall intracellular trafficking and homeostasis of the cellular endomembrane system. IMPORTANCE The virus life cycle involves entry into the host, replication of the genome, assembly of infectious progeny, and their subsequent release. Different aspects of the HCV life cycle, including entry, genome replication, and assembly, are well characterized; however, our understanding of the HCV release is still not clear and subject to debate due to varied findings. Here, we attempted to address this controversy and enhance our understanding of HCV egress by evaluating the role of the different components of the early secretory pathway in the HCV life cycle. To our surprise, we found that the components of the early secretory pathway are not only essential for HCV release but also contribute to many other earlier events of the HCV life cycle. This study emphasizes the importance of the early secretory pathway for the establishment of productive HCV infection in hepatocytes.

Hanging the coat on a collar: Same function but different localization and mechanism for COPII.

An entirely different mechanism and localization were recently proposed for the COPII coat complex, challenging its well-accepted function to select and concentrate cargo into small COPII-coated spherical transport vesicles. Instead, the COPII complex is suggested to form a dynamic yet stationary collar that forms a boundary between the ER and the ER export membrane domain. This membrane domain, the ER exit site (ERES), is the site of COPII-mediated sorting and concentration of transport competent proteins. Subsequently, the ERES is implicated to mature and bud to form a sizeable pleiomorphic transport carrier that translocate on microtubules to fuse with the Golgi apparatus. Despite this drastic mechanistic dogma shift, most of the underlying protein-protein and protein-membrane interactions remain unchanged. Here, we attempt to provide a detailed description of the newly proposed model of how ER to Golgi transport works by describing the role of several essential proteins of the transport machinery.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Golgi Complex form and Function: A Potential Hub Role Also in Skeletal Muscle Pathologies?

A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC's form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC's form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.

COPII-mediated trafficking at the ER/ERGIC interface.

Coat proteins play multiple roles in the life cycle of a membrane-bound transport intermediate, functioning in lipid bilayer remodeling, cargo selection and targeting to an acceptor compartment. The Coat Protein complex II (COPII) coat is known to act in each of these capacities, but recent work highlights the necessity for numerous accessory factors at all stages of transport carrier existence. Here, we review recent findings that highlight the roles of COPII and its regulators in the biogenesis of tubular COPII-coated carriers in mammalian cells that enable cargo transport between the endoplasmic reticulum and ER-Golgi intermediate compartments, the first step in a series of trafficking events that ultimately allows for the distribution of biosynthetic secretory cargoes throughout the entire endomembrane system.

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

Understanding the Contribution of Zinc Transporters in the Function of the Early Secretory Pathway.

More than one-third of newly synthesized proteins are targeted to the early secretory pathway, which is comprised of the endoplasmic reticulum (ER), Golgi apparatus, and other intermediate compartments. The early secretory pathway plays a key role in controlling the folding, assembly, maturation, modification, trafficking, and degradation of such proteins. A considerable proportion of the secretome requires zinc as an essential factor for its structural and catalytic functions, and recent findings reveal that zinc plays a pivotal role in the function of the early secretory pathway. Hence, a disruption of zinc homeostasis and metabolism involving the early secretory pathway will lead to pathway dysregulation, resulting in various defects, including an exacerbation of homeostatic ER stress. The accumulated evidence indicates that specific members of the family of Zn transporters (ZNTs) and Zrt- and Irt-like proteins (ZIPs), which operate in the early secretory pathway, play indispensable roles in maintaining zinc homeostasis by regulating the influx and efflux of zinc. In this review, the biological functions of these transporters are discussed, focusing on recent aspects of their roles. In particular, we discuss in depth how specific ZNT transporters are employed in the activation of zinc-requiring ectoenzymes. The means by which early secretory pathway functions are controlled by zinc, mediated by specific ZNT and ZIP transporters, are also subjects of this review.

Positive feedback between Golgi membranes, microtubules and ER exit sites directs de novo biogenesis of the Golgi.

The Golgi complex is the central organelle of the secretory pathway. It undergoes dynamic changes during the cell cycle, but how it acquires and maintains its complex structure is unclear. To address this question, we have used laser nanosurgery to deplete BSC1 cells of the Golgi complex and have monitored its biogenesis by quantitative time-lapse microscopy and correlative electron microscopy. After Golgi depletion, endoplasmic reticulum (ER) export is inhibited and the number of ER exit sites (ERES) is reduced and does not increase for several hours. Occasional fusion of small post-ER carriers to form the first larger structures triggers a rapid and drastic growth of Golgi precursors, due to the capacity of these structures to attract more carriers by microtubule nucleation and to stimulate ERES biogenesis. Increasing the chances of post-ER carrier fusion close to ERES by depolymerizing microtubules results in the acceleration of Golgi and ERES biogenesis. Taken together, on the basis of our results, we propose a self-organizing principle of the early secretory pathway that integrates Golgi biogenesis, ERES biogenesis and the organization of the microtubule network by positive-feedback loops.

Scyl1 scaffolds class II Arfs to specific subcomplexes of coatomer through the γ-COP appendage domain.

Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO(-) motif to interact directly with the appendage domain of coatomer subunit γ-2 (also known as COPG2 or γ2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to γ2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-Golgi, similar to that observed following the loss of Arf and Arf-guanine-nucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.

Activation of zinc-requiring ectoenzymes by ZnT transporters during the secretory process: Biochemical and molecular aspects.

In humans, about 1000 enzymes are estimated to bind zinc. In most of these enzymes, zinc is present at the active site; thus, these enzymes are functional as "zinc-requiring enzymes". Of these zinc-requiring enzymes, zinc-requiring ectoenzymes (defined as secretory, membrane-bound, and organelle-resident enzymes) have received much attention because of their important physiological functions, involvement in a number of diseases, and potential applications as therapeutic targets for diseases. Zinc-requiring ectoenzymes may become active by coordinating zinc at their active site during the secretory process, which requires elaborate control of zinc mobilization from the extracellular milieu to the cytosol and then lumen in the early secretory pathway. Therefore, zinc transporters should properly maintain the process at systemic, cellular, and subcellular levels by mobilizing zinc across biological membranes. However, few studies have examined the mechanisms underlying this process. In this review, current knowledge of the activation process of zinc-requiring ectoenzymes by ZnT zinc transporters in the early secretory pathway is briefly reviewed at the molecular level, with a focus on tissue-nonspecific alkaline phosphatase. Moreover, we also discuss whether zinc-chaperone proteins function during the activation of these enzymes.

Imaging ER-to-Golgi transport: towards a systems view.

Proteins synthesised at the endoplasmic reticulum (ER) have to undergo a number of consecutive and coordinated steps to reach the Golgi complex. To understand the dynamic complexity of ER-to-Golgi transport at the structural and molecular level, light microscopy approaches are fundamental tools that allow in vivo observations of protein dynamics and interactions of fluorescent proteins in living cells. Imaging protein and organelle dynamics close to the ultra-structural level became possible by combining light microscopy with electron microscopy analyses or super-resolution light microscopy methods. Besides, increasing evidence suggests that the early secretory pathway is tightly connected to other cellular processes, such as signal transduction, and quantitative information at the systems level is fundamental to achieve a comprehensive molecular understanding of these connections. High-throughput microscopy in fixed and living cells in combination with systematic perturbation of gene expression by, e.g. RNA interference, will open new avenues to gain such an understanding of the early secretory pathway at the systems level. In this Commentary, we first outline examples that revealed the dynamic organisation of ER-to-Golgi transport in living cells. Next, we discuss the use of advanced imaging methods in studying ER-to-Golgi transport and, finally, delineate the efforts in understanding ER-to-Golgi transport at the systems level.

Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway.

The Wnt signaling pathway is crucial for development and disease. The regulation of Wnt protein trafficking is one of the pivotal issues in the Wnt research field. Here we performed a genetic screen in Drosophila melanogaster for genes involved in Wingless/Wnt secretion, and identified the p24 protein family members Baiser, CHOp24, Eclair and a v-SNARE protein Sec22, which are involved in the early secretory pathway of Wingless/Wnt. We provided genetic evidence demonstrating that loss of p24 proteins or Sec22 impedes Wingless (Wg) secretion in Drosophila wing imaginal discs. We found that Baiser cannot replace other p24 proteins (CHOp24 or Eclair) in escorting Wg, and only Baiser and CHOp24 interact with Wg. Moreover, we showed that the v-SNARE protein Sec22 and Wg are packaged together with p24 proteins. Taken together, our data provide important insights into the early secretory pathway of Wg/Wnt.

Novel Components of the Stress Assembly Sec Body Identified by Proximity Labeling.

Sec bodies are membraneless stress-induced assemblies that form by the coalescence of endoplasmic reticulum exit sites (ERES). Through APEX2 tagging of Sec24AB, we biotinylated and identified the full complement of Sec body proteins. In the presence of biotin-phenol and H2O2 (APEX on), APEX2 facilitates the transfer of a biotin moiety to nearby interactors of chimeric Sec24AB. Using this unbiased approach comparing APEX on and off (-H2O2) conditions, we identified 52 proteins specifically enriched in Sec bodies. These include a large proportion of ER and Golgi proteins, packaged without defined stoichiometry, which we could selectively verify by imaging. Interestingly, Sec body components are neither transcriptionally nor translationally regulated under the conditions that induce Sec body formation, suggesting that incorporation of these proteins into granules may be driven instead by the aggregation of nucleating proteins with a high content of intrinsically disordered regions. This reinforces the notion that Sec bodies may act as storage for ERES, ER and Golgi components during stress.

Structural and thermodynamic analyses of human TMED1 (p24γ1) Golgi dynamics.

The transmembrane emp24 domain-containing (TMED) proteins, also called p24 proteins, are members of a family of sorting receptors present in all representatives of the Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMED's structure across different subfamilies. Besides, although the presence of a TMED homo-oligomerization was suggested previously based on crystallographic contacts observed for the isolated Golgi Dynamics (GOLD) domain, no further analyses of its presence in solution were done. Here, we describe the first high-resolution structure of a TMED1 GOLD representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers in solution even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane, are also presented.

Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System.

The early secretory pathway encompasses the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC) organelles. The ERGIC is now understood to be a complex cargo sorting hub involved in a variety of cellular and tissue processes, however the traffic pathways to and from the ERGIC are still unclear.Classical methods employed for the analysis of a cargo 's journey along the secretory pathway rely on reversible traffic blocks leading to cargo accumulation in the ER . Although these methods were key to characterize Golgi and post-Golgi traffic routes, their poor specificity to the cargo of interest and limited spatiotemporal resolution make them inadequate for the fine characterization of cargo traffic in the early secretory pathway.In this chapter, we describe a protocol to study the traffic of cargo proteins in the early secretory pathway using the Retention Using Selective Hook (RUSH ) system, a highly specific and sensitive tracking system with a high spatiotemporal resolution. Taking GLUT4 and GLUT1 as examples of unconventionally and conventionally secreted cargo respectively, we describe the steps to clone the cargoes in the RUSH vector and follow and quantify their traffic along the early secretory pathway. This RUSH method can also be used to study the traffic of other cargo proteins in the early secretory pathway.

Early Secretory Pathway-Associated Proteins SsEmp24 and SsErv25 Are Involved in Morphogenesis and Pathogenicity in a Filamentous Phytopathogenic Fungus.

Proper protein secretion is critical for fungal development and pathogenesis. However, the potential roles of proteins involved in the early secretory pathway are largely undescribed in filamentous fungi. p24 proteins are cargo receptors that cycle between the endoplasmic reticulum (ER) and Golgi apparatus in the early secretory pathway and recruit cargo proteins to nascent vesicles. This study characterized the function of two p24 family proteins (SsEmp24 and SsErv25) in a phytopathogenic fungus, Sclerotinia sclerotiorum. Both SsEmp24 and SsErv25 were upregulated during the early stages of S. sclerotiorum infection. ΔSsEmp24 mutant and ΔSsErv25 mutant displayed abnormal vegetative growth and sclerotium formation, were defective in infection cushion formation, and showed lower virulence on host plants. ΔSsEmp24 mutant had a more severe abnormal phenotype than ΔSsErv25 mutant, implying that SsEmp24 could play a central role in the early secretory pathway. Similar to their Saccharomyces cerevisiae counterparts, SsEmp24 interacted with SsErv25 and predominantly colocalized in the ER or nuclear envelope. The absence of SsEmp24 or SsErv25 led to defective in protein secretion in S. sclerotiorum, including the pathogenicity-related extracellular hydrolytic enzymes and effectors. It is proposed that SsEmp24 and SsErv25, components in the early secretory pathway, are involved in modulating morphogenesis and pathogenicity in S. sclerotiorum by mediating protein secretion. IMPORTANCE Understanding the reproduction and pathogenesis mechanism of phytopathogens could provide new opinions to effectively control fungal diseases. Although it has been known that effectors and extracellular hydrolytic enzymes secreted by phytopathogenic fungi play important roles in fungus-host interactions, the secretion system for the delivery of virulence factors to the host is still largely undescribed. Although the role of the early secretory pathway-associated p24 proteins in S. cerevisiae has been well characterized, the function of these proteins in filamentous fungi was scarcely known prior to this study. The present research provides evidence that p24 proteins participate in the reproduction and pathogenesis of phytopathogenic fungi through the mediation of protein secretion. This research advances our understanding of p24 proteins in filamentous phytopathogenic fungi. In addition, the candidate cargos of the two p24 proteins, SsEmp24 and SsErv25, were screened out by comparative proteomics, which could aid the identification of novel development and virulence-associated factors in phytopathogenic fungi.