International Journal of Molecular Sciences Article Protein Phosphatase Ppz1 Is Not Regulated by a Hal3-Like Protein in Plant Pathogen Ustilago maydis Chunyi Zhang 1 , Antonio de la Torre 2 , José Pérez-Martín 2 and Joaquín Ariño 1, * 1 Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain 2 Instituto de Biología Funcional y Genómica (CSIC), 37007 Salamanca, Spain * Correspondence: Joaquin.Arino@uab.es; Tel.: +34-935811315  Received: 27 June 2019; Accepted: 30 July 2019; Published: 5 August 2019  Abstract: Ppz enzymes are type-1 related Ser/Thr protein phosphatases that are restricted to fungi. In S. cerevisiae and other fungi, Ppz1 is involved in cation homeostasis and is regulated by two structurally-related inhibitory subunits, Hal3 and Vhs3, with Hal3 being the most physiologically relevant. Remarkably, Hal3 and Vhs3 have moonlighting properties, as they participate in an atypical heterotrimeric phosphopantothenoyl cysteine decarboxylase (PPCDC), a key enzyme for Coenzyme A biosynthesis. Here we identify and functionally characterize Ppz1 phosphatase (UmPpz1) and its presumed regulatory subunit (UmHal3) in the plant pathogen fungus Ustilago maydis. UmPpz1 is not an essential protein in U. maydis and, although possibly related to the cell wall integrity pathway, is not involved in monovalent cation homeostasis. The expression of UmPpz1 in S. cerevisiae Ppz1-deficient cells partially mimics the functions of the endogenous enzyme. In contrast to what was found in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. UmHal3, an unusually large protein, is the only functional PPCDC in U. maydis and, therefore, an essential protein. However, when overexpressed in U. maydis or S. cerevisiae, UmHal3 does not reproduce Ppz1-inhibitory phenotypes. Indeed, UmHal3 does not inhibit UmPpz1 in vitro (although ScHal3 does). Therefore, UmHal3 might not be a moonlighting protein. Keywords: protein phosphorylation; protein phosphatase; CoA biosynthesis; Ustilago maydis; fungi 1. Introduction Ppz1 defines a subfamily of Ser/Thr protein phosphatases (PPases) which are structurally related to type-1 (PP1c) enzymes. In contrast to the ubiquitous PP1c, Ppz1 phosphatases are restricted to fungi [1,2]. The enzyme has been extensively characterized in S. cerevisiae as a 692-residue protein whose C-terminal domain is similar to the sole PP1c (Glc7), and is preceded by a long N-terminal segment (~350 residues) unrelated to other proteins [3,4]. ScPpz1 plays a key role in monovalent cation homeostasis through regulation of the function of the high-affinity Trk transporters and the expression of the ENA1 gene, encoding a Na+ -ATPase [5–8]. S. cerevisiae ppz1 mutants are hypertolerant to Na+ and Li+ cations and, likely due to the increased influx of potassium, they are sensitive to agents affecting cell wall remodeling, such as caffeine or calcofluor white [4,9,10]. High levels of Ppz1 are toxic in S. cerevisiae, resulting in slow growth and cell cycle blockage at the G1 –S transition [9,11,12]. ScPpz1 is regulated by its inhibitory subunits Hal3 and Vhs3, which interact with a 1:1 stoichiometry to the catalytic domain of ScPpz1 [9,13–16]. Hal3 is more relevant in vivo than Vhs3 and, in contrast to ppz1 strains, hal3 mutants are sensitive to Na+ and Li+ , whereas its overexpression confers tolerance to high concentrations of these cations [13]. Homologs of ScPpz1 have been identified in other fungi, such as Schizosaccharomyces pombe, Neurospora crassa and Debaryomyces hansenii [17–19], although they have been characterized in less Int. J. Mol. Sci. 2019, 20, 3817; doi:10.3390/ijms20153817 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3817 2 of 19 detail. Recent work has pointed out a role in virulence for Ppz1 enzymes in certain pathogenic fungi, such as Candida albicans and Aspergillus fumigatus [20–23], although this does not seem to be the case in Cryptococcus neoformans [24]. From a structural point of view, Ppz enzymes display a highly conserved catalytic C-terminal half, but their N-terminal half is usually much shorter than that of S. cerevisiae and, although their similarity in sequence is relatively low, in all cases investigated so far, the N-terminus includes a likely myristoylable Gly2. In S. cerevisiae, change of Gly-2 to Ala results in a lack of myristoylation and the loss of complementation of salt tolerance [25]. As a rule, the heterologous expression of Ppz enzymes in S. cerevisiae can fulfill, at least partially, many of the functions of the native enzyme. The observation that, contrary to Ppz enzymes, orthologues of HAL3 are widely distributed in both prokaryotic and eukaryotic organisms is explained by the fact that ScHal3 (and ScVhs3) are moonlighting proteins that, in addition to regulate Ppz1, are involved in the biosynthesis of coenzyme A by contributing to the generation of an unusual heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme [26]. In most organisms, PPCDC is a homotrimer in which the three equivalent catalytic sites are formed at the interface of the monomers, and involve specific His, Asn (within a conserved PXMNXXMW motif) and Cys residues (i.e., His90 , Asn142 , and Cys175 in the Arabidopsis thaliana Hal3a protein, see Figure 1). In contrast, in S. cerevisiae PPCDC is a heterotrimer made up of an essential Cab3 subunit and two Hal3 or Vhs3 subunits (or one of each). Cab3 participates in this complex by providing the catalytic Cys478 and Asn443 residues (absent in Hal3 and Vhs3), whereas Hal3 or Vhs3 contribute with the catalytic His (His378 and His459 , respectively, see Figure 1 and Figure S7) [26]. S. cerevisiae Hal3 also differs from A. thaliana AtHal3 PPCDC (the prototype for PPCDC in most organisms) in that the conserved PPCDC central core is flanked by a relatively long N-terminal extension and by an unusually acidic C-terminal tail (Figure 1). Contrary to the relative homogeneity of fungal Ppz enzymes, Hal3-like proteins are much more varied. Thus, the C. albicans genome encodes two proteins whose structures are reminiscent of those of ScCab3 and ScHal3 (orf19.3260 and orf19.7378, respectively) [27]. They can replace the corresponding S. cerevisiae protein when tested for their ability to build up an heteromeric PPCDC, but only CaCab3 (and not CaHal3) exhibits Ppz1 phosphatase regulatory potential in vivo [27]. Even more striking is the case of the fission yeast S. pombe, where SpHal3 was shown to be the result of a gene fusion event composed of an N-terminal PPCDC core (including a short acidic tail), followed by a functional C-terminal thymidylate synthase [28]. Interestingly, SpHal3 was a functional PPCDC, but its capacity to inhibit ScPpz1 or its S. pombe homolog (Pzh1) in vitro was limited. Very recently, the Ppz/Hal3 system has also been investigated in C. neoformans [24]. This fungus encodes two Hal3-like proteins (CnHal3a and CnHal3b) which essentially consist of a PPCDC core and a non-acidic C-terminal tail. Interestingly, whereas both are functional PPCDC enzymes, none of them appears to regulate Ppz enzymes in vivo or in vitro. In conclusion, fungal Hal3-like proteins are very diverse and, whereas their PPCDC capacity can be reasonably inferred from their structure, in the absence of experimental data, it is not feasible to predict their ability to regulate Ppz phosphatases. monovalent cation homeostasis. UmHal3 does not inhibit in vitro UmPpz1 and does not behave as an in vivo regulator of the phosphatase, but it is the only functional PPCDC in U. maydis and an essential protein. Therefore, UmHal3 might lack moonlighting properties. In contrast to what has been reported for the phosphatase in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. Int. J. Mol. Sci. 2019, 20, 3817 3 of 19 Figure 1. Comparison of the structures of S. cerevisiae and U. maydis Ppz1 and Hal3 proteins. Panel A. Figureand ScPpz1 1. Comparison of the structures UmPpz1 structures. Note theof shorter S. cerevisiae and U. maydis N-terminal Ppz1inand extension Hal3 proteins. UmPpz1, PanelofA. the absence a Gly at position 2, and the presence of a Met followed by a Gly (red) at positions 9 and 10. Panelof ScPpz1 and UmPpz1 structures. Note the shorter N-terminal extension in UmPpz1, the absence B.a Comparison of A. thaliana Hal3a, S. cerevisiae Hal3 and Cab3 and U. maydis Hal3 (UmHal3). The HisB. Gly at position 2, and the presence of a Met followed by a Gly (red) at positions 9 and 10. Panel Comparison and of A. Cys residues thalianafor required Hal3a, PPCDC S. cerevisiae Hal3 activity are and Cab3 indicated and U. as blue and maydis Hal3 (UmHal3). red arrowheads, The His respectively. andred The Cyssection residues required for corresponds to PPCDC activity are the PXMNXXM indicated motif. as blue To note and redin the absence arrowheads, UmHal3 ofrespectively. the acidic The red C-tail section found corresponds in both S. cerevisiaetoproteins. the PXMNXXM motif. The length To note of each the absence protein in UmHal3 is indicated of the acidic in parentheses. ScalesC- tail found in both S. cerevisiae in panel A and B are different. proteins. The length of each protein is indicated in parentheses. Scales in panel A and B are different. Ustilago maydis, the causal agent of corn smut disease, is a plant pathogenic fungus that causes 2. Results tumors on all aerial parts of its host plant maize (Zea mays). Together with the human pathogen C. neoformans, it serves as model system of choice for pathogenic basidiomycetes [29]. The fungus has a 2.1. Identification dimorphic life cycleof Ustilago maydis Ppz1 with a yeast-like and Hal3-Like saprophytic phaseProteins switching to filamentous, pathogenic growth upon Blastp hyphalanalysis fusion. of The amenability for reverse-genetics, fungal databases using the S. cerevisiae as well Ppz1,asHal3 its intricate and Cab3 life style,sequences protein is very connected revealed with cell cycle a putative regulation, ortholog for making PPZ1 in U.Ustilago maydis a model maydis,system for both encoding the fungal cell biology hypothetical as protein well as the study of biotrophic plant interaction [30]. Analysis of the UMAG_04827.1, and a single putative ortholog for Hal3 and Cab3 (hypothetical protein U. maydis genome revealed a single gene (umag_04827,As UMAG_05916.1). namedshown here in UmPpz1) which likely encodes Figure 1, UMAG_04827.1 (fromanow Ppz1on phosphatase, UmPpz1) is and a 497a second -residue gene (umag_05916) which encodes a sequence that is compatible with a putative protein that shows an overall 38.2% identity (46.5% similarity) with ScPpz1. The C-terminal section PPCDC, denoted here as UmHal3. of UmPpz1, Remarkably, containing the latter was the characteristic an unusually traits of a Ser/Thr large protein protein (1430-residue) phosphatase, showsin high which the identity PPCDC domain is restricted to the last 240 amino acids and is not followed with the C-terminal phosphatase domain of ScPpz1 (67.9%, 77.6% similarity from residue 151 to the by an acidic C-terminal tail. The fact residue). C-terminal that Ppz phosphatases Therefore, thehave never been N-terminal investigated extension of UmPpz1 in plant fungal is about pathogens half the size ofand the ScPpz1 abnormal structure of the sole Hal3-like protein found in Ustilago (Figure 1 and (Figure 1A). Inspection of the very N-terminal region of this ORF shows that the initiating Met is notFigure S7) prompted us tofollowed explore the by anature Gly, butof theby aPpz/Hal3 system in Phe, indicating thatthis fungus. this proteinOur investigations cannot reveal that by N-terminally UmPpz1 is myristolylated not an essential (Figure 1A). protein in U. maydis, and that it might be functionally related to the cell wall integrity pathway, The UMAG_05916.1 protein (from now on, UmHal3) is a very large polypeptide of does but that it is not involved in maintenance of monovalent cation homeostasis. UmHal3 1430 not inhibit in vitro UmPpz1 and does not behave as an in vivo regulator residues. The last 240 amino acids show significant identity with known PPCDCs, such as AtHal3a of the phosphatase, but it is(34.2% the only functional PPCDC in U. maydis and an essential protein. Therefore, identity, 48.9% similarity), as well as with the PPCDC core of both ScHal3 (24.1%/39.7%) and UmHal3 might lack moonlighting properties. In contrast to what has been reported for the phosphatase in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. Int. J. Mol. Sci. 2019, 20, 3817 4 of 19 2. Results 2.1. Identification of Ustilago maydis Ppz1 and Hal3-Like Proteins Blastp analysis of fungal databases using the S. cerevisiae Ppz1, Hal3 and Cab3 protein sequences revealed a putative ortholog for PPZ1 in Ustilago maydis, encoding the hypothetical protein UMAG_04827.1, and a single putative ortholog for Hal3 and Cab3 (hypothetical protein UMAG_05916.1). As shown in Figure 1, UMAG_04827.1 (from now on UmPpz1) is a 497-residue protein that shows an overall 38.2% identity (46.5% similarity) with ScPpz1. The C-terminal section of UmPpz1, containing the characteristic traits of a Ser/Thr protein phosphatase, shows high identity with the C-terminal phosphatase domain of ScPpz1 (67.9%, 77.6% similarity from residue 151 to the C-terminal residue). Therefore, the N-terminal extension of UmPpz1 is about half the size of ScPpz1 (Figure 1A). Inspection of the very N-terminal region of this ORF shows that the initiating Met is not followed by a Gly, but by a Phe, indicating that this protein cannot by N-terminally myristolylated (Figure 1A). The UMAG_05916.1 protein (from now on, UmHal3) is a very large polypeptide of 1430 residues. The last 240 amino acids show significant identity with known PPCDCs, such as AtHal3a (34.2% identity, 48.9% similarity), as well as with the PPCDC core of both ScHal3 (24.1%/39.7%) and ScCab3 (26.8%/40.5%), although the Ustilago protein lacks the characteristic C-terminal acidic tail found in S. cerevisiae (Figure 1B) and C. albicans. Therefore, UmHal3 has a very long N-terminal extension whose similarity to the one present in Hal3 or Cab3 is only marginal (Figure S1). To note, UmHal3 presents at positions 777–894 a Vps9-like sequence. In fact, the entire Vps9 can be aligned with UmHal3 (from residue 570 to 1096), albeit with relatively low identity (19.4%, 35.5% similarity). In S. cerevisiae, Vps9 acts as a guanine nucleotide exchange factor (GEF) for Vps21, and is needed for the transport of proteins from biosynthetic and endocytic pathways into the vacuole. 2.2. Expression and Functional Characterization of UmPpz1 in S. cerevisiae To this end, the UmPpz1 open reading frame (ORF) was expressed from the ScPPZ1 gene promoter in both centromeric (low-copy number) and episomal (high-copy) vectors. As shown in Figure 2A, expression of UmPpz1 from low or high-copy number vectors was unable to decrease the abnormally high tolerance of the ppz1 mutant to LiCl. The mutation of PPZ1 yields S. cerevisiae cells which are hypersensitive to caffeine; the sensitivity to the drug is normalized when a centromeric copy of ScPpz1 is reintroduced in the cells. Transformation of ppz1 cells with centromeric UmPpz1 increased the tolerance to caffeine only marginally, but high-copy expression of UmPpz1 restored wild-type tolerance (Figure 2A). The fact that cells expressing UmPpz1 from the episomal vector grow better in the presence of caffeine than cells expressing ScPpz1 can be explained by previous observations that high-copy expression of ScPpz1 from its own promoter has a negative effect on cell growth [9]. A modest beneficial effect of the expression of UmPpz1 was also observed when the experiment was carried out in a slt2 mutant, which is defective in cell-wall remodeling and highly sensitive to caffeine. Int. J. Mol. Sci. 2019, 20, 3817 5 of 19 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 20 Figure 2. Phenotypic analysis of the expression of UmPpz1 in S. cerevisiae. (A) The wild type BY4741 Figure 2. Phenotypic analysis of the expression of UmPpz1 in S. cerevisiae. (A) The wild type BY4741 strain (WT) and strain (WT) ppz1 itsand ppz1slt2 itsand andderivatives were slt2 derivatives weretransformed transformed with withYCplac111 YCplac111 LEU2 LEU2 (centromeric, (centromeric, marker), marker), or YEplac181 (episomal, or YEplac181 LEU2 (episomal, marker) LEU2 marker) plasmids carryingS.S. plasmids carrying cerevisiae cerevisiae (ScPpz1) (ScPpz1) or U. or U. maydis maydis (UmPpz1) phosphatases. (UmPpz1) Cultures phosphatases. Cultureswere spotted were spottedatatOD OD660 = 0.05 660 =0.05 andand at 1/5 at 1/5 dilutions dilutions on synthetic on synthetic medium medium plates lacking plates lacking leucine leucine and and containing containing the theindicated indicated amounts amountsofof LiCl or caffeine. LiCl PlatesPlates or caffeine. were were grown forgrown for 4Ø, 4 days. days. emptyØ , empty plasmid. plasmid. (B)(B) Wildtype Wild typeandand ppz1 ppz1 strains strainswere were transformed transformedwith with emptyempty pYES2 plasmid (pYES2-Ø ) or the same plasmid expressing ScPpz1-HA or UmPpz1-HA from the pYES2 plasmid (pYES2-Ø) or the same plasmid expressing ScPpz1-HA or UmPpz1-HA from the GAL1 GAL1 promoter. Cells were grown on synthetic medium lacking uracil supplemented with 2% glucose promoter.andCells were grown on synthetic medium lacking uracil supplemented with 2% glucose and spotted on the same plates containing either 2% glucose or 2% galactose. Growth was monitored spotted on the same after 2 days. (C)plates containing The same either strains were 2% inglucose grown or 2% galactose. liquid medium Growth with 2% galactose. was monitored Samples were after 2 days. taken(C) afterThe 6 h same strainsfor and processed were SDSgrown in liquidgelmedium -polyacrylamide with 2% electrophoresis galactose. (SDS-PAGE) Samples were as described. 80 6µg taken after h of protein and were electrophoresed processed in 10% SDS -polyacrylamide for SDS-polyacrylamide gel electrophoresis gels, transferred (SDS-PAGE) to membranes as described. 80 and probed with anti-HA antibodies to detect the tagged proteins (arrowheads). µg of protein were electrophoresed in 10% SDS-polyacrylamide gels, transferred to membranes Ponceau staining of and the membranes was carried out (lower panel) to monitor for correct loading and transfer. probed with anti-HA antibodies to detect the tagged proteins (arrowheads). Ponceau staining of the membranes was carriedstrong As mentioned, out (lower panel) to monitor overexpression of Ppz1 has forharmful correct loading effects on and transfer. cell growth. For S. cerevisiae instance, the expression of the phosphatase in a pYES2 vector from the powerful GAL1-10 promoter Although has been a shown Gly istonot found fully as aproliferation block cell second residue at the ([25] and defined Figure N-terminal 2B). However, region expression of UmPpz1, of UmPpz1 residue number nine is a Met followed by a Gly (Figure 1A). In spite of the fact that computational under the same conditions did not affect growth (Figure 2B). The absence of effect was not due to analysis of the residues insufficient following expression levels,this Glyimmunoblot since does not predict this residue experiments showed being myristoylated that HA-tagged UmPpz1[31], we accumulates considered even more the possibility thatabundantly the actual than ScPpz1may protein (Figure 2C). Therefore, initiate UmPpz1 at this second doesand, Met not reproduce therefore, we the toxic effects of ScPpz1. The same result was obtained by overexpression of UmPpz1 -s (not expressed a C-terminally HA-tagged shorter version (UmPpz1-s-HA) in S. cerevisiae. However, as shown). shown in Supplementary Figure S1, this version not only failed to decrease the tolerance of a ppz1 mutant to + cations but, in contrast to UmPpz1-HA, was totally ineffective in restoring normal 2.3.LiFunctional Characterization of UmHal3 in S. cerevisiae tolerance to caffeine. Immunoblot experiments failed to detect tagged UmPpz1-s-HA, suggesting that the protein was very poorly expressed or readily degraded (not shown). Therefore, the first few amino acids in UmPpz1 might be important for protein stability. As mentioned, strong overexpression of Ppz1 has harmful effects on S. cerevisiae cell growth. For instance, the expression of the phosphatase in a pYES2 vector from the powerful GAL1-10 promoter has been shown to fully block cell proliferation ([25] and Figure 2B). However, expression of UmPpz1 under the same conditions did not affect growth (Figure 2B). The absence of effect was not due to insufficient Int. J. Mol. Sci. 2019, 20, 3817 6 of 19 expression levels, since immunoblot experiments showed that HA-tagged UmPpz1 accumulates even more abundantly than ScPpz1 (Figure 2C). Therefore, UmPpz1 does not reproduce the toxic effects of ScPpz1. The same result was obtained by overexpression of UmPpz1-s (not shown). 2.3. Functional Characterization of UmHal3 in S. cerevisiae Expression of UmHal3 in S. cerevisiae was accomplished by cloning the entire ORF in the high-copy Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 20 plasmid pWS93. In this way, the protein is expressed from the ADH1 promoter as a N-terminally 3xHA epitope-tagged Expression of polypeptide. UmHal3 in S. In addition, cerevisiae was we cloned in by accomplished thecloning same thevector the entire ORFC-terminal in the high- region of copy plasmid pWS93. In this way, the protein is expressed from the UmHal3, corresponding to the PPCDC domain (UmPD region) and this same region followed by the ADH1 promoter as a N-terminally 3xHA epitope-tagged highly acidic C-terminal tail polypeptide. found inIn addition, ScHal3 we cloned in the(Figure (UmPD_ScCtD) same vector theFigure 1 and C-terminal S8).region As shown in of UmHal3, corresponding to the PPCDC domain (UmPD region) and this same region followed by Figure 3A, expression of UmHal3 or its PD region in a hal3 strain did not improve tolerance to LiCl the highly acidic C-terminal tail found in ScHal3 (UmPD_ScCtD) (Figure 1 and Figure S8). As shown or NaCl,in which Figure 3A, is aexpression typical effect of ScHal3 of UmHal3 overexpression. or its PD Interestingly, region in a hal3 strain expression did not improve tolerance of to UmPD_ScCt LiCl increased or NaCl, which is a typical effect of ScHal3 overexpression. Interestingly, expression of UmPD_ScCt to the tolerance to LiCl somewhat. In contrast, neither version restored wild-type tolerance toxic cation spermine increased tolerance or to toLiCl caffeine. In addition, somewhat. overexpression In contrast, of any of neither version restored the UmHal3 wild-type tolerance variants to the had a toxic cation spermine or to caffeine. In addition, overexpression of negative effect on the growth of a slt2 mutant (Figure S2). We considered whether the virtualany of the UmHal3 variants had a lack of negative effect on the growth of a slt2 mutant (Figure S2). We considered whether the virtual lack of effects derived from the introduction in S. cerevisiae of the UmHal3 protein and its variants could be effects derived from the introduction in S. cerevisiae of the UmHal3 protein and its variants could be attributed to defective expression. To test this, we monitored the expression levels of these proteins attributed to defective expression. To test this, we monitored the expression levels of these proteins taking advantage taking advantage of the of N-terminal the N-terminal3xHA3xHA tag. tag. However, as observed However, as observed in in Figure Figure3B,3B,allallUmHal3 UmHal3variants, including variants, including the very large, full-length UmHal3, were expressed at considerableTherefore, the very large, full-length UmHal3, were expressed at considerable levels. levels. the absenceTherefore, of phenotypic effects the absence must be attributed of phenotypic effects mustto be theattributed inherenttoproperties the inherent ofproperties these proteins. of theseBecause proteins. Because the phenotypes tested thearephenotypes functionally tested are functionally related to the rolerelated to the role of ScHal3 of ScHal3 as as inhibitor ofinhibitor Ppz1, our of results Ppz1, our results suggest that UmHal3 is not able to inhibit Ppz1 in S. cerevisiae. suggest that UmHal3 is not able to inhibit Ppz1 in S. cerevisiae. 3. Effects Figure Figure of the expression of UmHal3 in S. cerevisiae on the tolerance to diverse stress conditions. 3. Effects of the expression of UmHal3 in S. cerevisiae on the tolerance to diverse stress (A) Wild type BY4741 (WT) conditions. (A) Wild typeand its hal3 BY4741 (WT)derivative and its hal3 were transformed derivative with plasmid were transformed pWS93 with plasmid (episomal, pWS93 URA3 marker) and the same plasmid expressing ScHal3, full-length UmHal3, its (episomal, URA3 marker) and the same plasmid expressing ScHal3, full-length UmHal3, its PPCDC PPCDC domain (PD) and thisdomain domain followed (PD) and this by the acidic domain C-terminal followed tailC-terminal by the acidic form ScHal3 (PD_ScCt). tail form Cells wereCells ScHal3 (PD_ScCt). spotted on syntheticwere spotted on medium synthetic plates medium lacking plates uracil lacking uracil supplemented supplemented with the indicated with the indicated compounds. compounds. Growth was Growth was recorded after 48 h. (B) Protein extracts from cultures of the strains described in (A) were recorded after 48 h. (B) Protein extracts from cultures of the strains described in (A) were prepared, prepared, subjected to SDS-PAGE, and proteins were transferred to membranes. The different 3xHA- subjected to SDS-PAGE, and proteins were transferred to membranes. The different 3xHA-tagged tagged proteins were detected with anti-HA antibodies (indicated by arrowheads). Ponceau staining proteins were detected with anti-HA antibodies (indicated by arrowheads). Ponceau staining of the of the membranes is shown. membranes is shown. 2.4. Characterization of Recombinant UmPpz1 and UmHal3 Proteins Int. Int.J.J.Mol. Mol.Sci. 2019,20, Sci.2019, 20,3817 x FOR PEER REVIEW 7 of 19 7 of 20 Int. J. Mol. Sci. 2019, 20, x FOR PEER R EVIEW 7 of 20 To this end, UmPpz1 was expressed in E. coli as a fusion protein with glutathione S-transferase To this end, UmPpz1 2.4. Characterization was expressed of Recombinant UmPpz1 in and E. coli as a fusion UmHal3 protein with glutathione S -transferase Proteins (GST), purified by glutathione agarose affinity chromatography and the GST moiety removed by (GST), purified by glutathione agarose affinity chromatography and the GST moiety removed by treatment Towith this end, UmPpz1protease. PreScission was expressed in E. coli as a enzyme The recombinant fusion protein (as wellwithasglutathione UmPpz1-s)S-transferase showed treatment with PreScission protease. The recombinant enzyme (as well as UmPpz1 -s) showed (GST), purified phosphatase activitybyagainst glutathione agarose substrate the synthetic affinity chromatography pNPP which was and the GST similar moiety to that removed observed for by phosphatase activity against the synthetic substrate pNPP which was similar to that observed for treatment ScPpz1 or its with PreScission C-terminal protease. catalytic domainThe recombinant Our (ScPpz1-Cter). enzyme (as well attempts as UmPpz1-s) to express showed and purify ScPpz1 or its C-terminal catalytic domain (ScPpz1-Cter). Our attempts to express and purify phosphatase significant amounts activity against the of full-length synthetic UmHal3 in E.substrate coli were pNPP which was unsuccessful, similar possibly duetotothat observed the large size for significant amounts of full-length UmHal3 in E. coli were unsuccessful, possibly due to the large size ScPpz1 of the or its protein. InC-terminal contrast, we catalytic were abledomain (ScPpz1-Cter). to purify both the UmPDOur attempts to express polypeptides. and UmPD_ScCt and purify significant We of the protein. In contrast, we were able to purify both the UmPD and UmPD_S cCt polypeptides. We firstamounts of full-length set an assay to test theUmHal3 inhibitory E. coli wereof in properties unsuccessful, the Ustilagopossibly due to the Hal3 proteins. large size of As presented inthe protein. Figure first set an assay to test the inhibitory properties of the Ustilago Hal3 proteins. As presented in Figure In contrast, 4, neither UmPD we nor wereUmPD_ScCt able to purify bothable were the UmPD andrecombinant to inhibit UmPD_ScCt ScPpz1, polypeptides. We first or ScPpz1-Cter, set even an assay 4, neither UmPD nor UmPD_ScCt were able to inhibit recombinant ScPpz1, ScPpz1 -Cter, or even UmPpz1. to test In thecontrast, inhibitory ScHal3 was fully properties effective of the Ustilago asHal3 an inproteins. vitro inhibitor of UmPpz1. As presented This4,implies in Figure neitherthat UmPD UmPpz1. In contrast, ScHal3 was fully effective as an in vitro inhibitor of UmPpz1. This implies that UmPpz1 retains the were nor UmPD_ScCt structural able tofeatures required to be inhibit recombinant inhibited ScPpz1, by functional ScPpz1-Cter, or evenHal3 proteins, UmPpz1. and In contrast, UmPpz1 retains the structural features required to be inhibited by functional Hal3 proteins , and suggests ScHal3 that wasat fully least the UmPD effective as and an inUmPD_ScCt vitro inhibitor variants of UmHal3 of UmPpz1. Thisare devoid implies thatofUmPpz1 Ppz1 inhibitory retains the suggests that at least the UmPD and UmPD_ScCt variants of UmHal3 are devoid of Ppz1 inhibitory properties. structural features required to be inhibited by functional Hal3 proteins, and suggests that at least the properties. UmPD and UmPD_ScCt variants of UmHal3 are devoid of Ppz1 inhibitory properties. Figure 4. Inhibitory Figure capacity 4. Inhibitory of different capacity UmHal3 of different UmHal3versions on ScPpz1 versions and and on ScPpz1 UmPpz1. UmPpz1. FiveFive to ten topmols ten pmols Figure 4. Inhibitory capacity of different UmHal3 versions on ScPpz1 and ◦ UmPpz1. Five to ten pmols of ScPpz1, ScPpz1-Cter, of ScPpz1, or UmPpz1 ScPpz1-Cter, or UmPpz1 werewere pre-incubated for 5for pre-incubated min at 30°C 5 min at 30with different C with amounts different of of amounts of ScPpz1, ScPpz1-Cter, or UmPpz1 were pre-incubated for 5 min at 30°C with different amounts of ScHal3 (--), ScHal3 UmHal3_PD (--), UmHal3_PD (--) or UmHal3-ScCt (--) or UmHal3-ScCt ( (--), and ), and thethe assay assay was was initiated initiated byby addition addition of the ScHal3 (--), UmHal3_PD (--) or UmHal3-ScCt (--), and the assay was initiated by addition of the of the substrate. substrate. DataData are are meansmeans ± S.E.± S.E. fromfrom 4 to 74different to 7 different assaysassays and correspond and correspond to the to the ratio ratio of of phosphatase substrate. Data are means ± S.E. from 4 to 7 different assays and correspond to the ratio of phosphatase activity inactivity phosphatase the presence in the and the absence presence andofthe inhibitor, absence expressed as percentage. of inhibitor, expressedTwo as different percentage.preparations Two activity in the presence and the absence of inhibitor, expressed as percentage. Two different of thepreparations different phosphatases ofand the the inhibitors were phosphatases tested. and the inhibitors were tested. preparations of the phosphatases and the inhibitors were tested. TheThe inability inability of UmHal3 of UmHal3 proteins proteins to inhibit to inhibit ScPpz1 ScPpz1 or UmPpz1 or UmPpz1 could could be due be due to the to the inability inability of of The inability of UmHal3 proteins to inhibit ScPpz1 or UmPpz1 could be due to the inability of these these proteins proteins to effectively to effectively interactwith interact withthethephosphatases. phosphatases. To To test test this, this, we wesetseta apull-down pull-down assay assayusing these as proteins baits the to effectively recombinant interact phosphatases GST-tagged with the phosphatases. bound to Toglutathione the test this, we set that resin a pull-down were assay incubated using as baits the recombinant GST-tagged phosphatases bound to the glutathione resin that were using as baits the recombinant GST-tagged phosphatases bound to the glutathione resin that were with S. with incubated cerevisiae extractsextracts S. cerevisiae containing equivalent containing amountsamounts equivalent of HA-tagged ScHal3, UmPD of HA-tagged ScHal3,orUmPD UmPD_ScCt or incubated with S. cerevisiae extracts containing equivalent amounts of HA-tagged ScHal3, UmPD or (Figure S3). UmPD_ScCt As shown (Figure S3). Asinshown Figurein5A, ScHal3 Figure 5A,effectively interactsinteracts ScHal3 effectively with UmPpz1, whereaswhereas with UmPpz1, full-length UmPD_ScCt (Figure S3). As shown in Figure 5A, ScHal3 effectively interacts with UmPpz1, whereas UmHal3UmHal3 full-length and UmPD andare barelyare UmPD visible barely(seevisible Figure(see S4 for an overloaded Figure S4 for an version overloadedof the experiment) version of theand full-length UmPD_ScCt UmHal3 and UmPD are barely visible (see Figure S4 for an overloaded version of the experiment) and cannot UmPD_ScCtbe detected. cannotOn the other On be detected. hand the(Figure 5B), full-length other hand (Figure 5B), UmHal3 weakly full-length interacts UmHal3 experiment) and UmPD_ScCt cannot be detected. On the other hand (Figure 5B), full-length UmHal3 withinteracts weakly both ScPPz1with and bothScPpz1-Cter. ScPPz1 and Weak signals Weak ScPpz1-Cter. can besignals also observed for UmPD can be also observed andfor UmPD_ScCt UmPD weakly when interacts ScPpz1 is withasboth used bait,ScPPz1 but theyandare ScPpz1-Cter. not detected Weak the when signals can bewith interaction also ScPpz1-Cter observed forisUmPDtested and UmPD_ScCt when ScPpz1 is used as bait, but they are not detected when the interaction with and UmPD_ScCt when ScPpz1 is used as bait, but they are not detected when the interaction with (even if these ScPpz1-Cter lanes (even is tested are overloaded, as shown if these lanes in Figure 5B). are overloaded, as Collectively, shown in Figure our results indicate that our 5B). Collectively, ScHal3 ScPpz1-Cter isbind tested (even if these lanes are overloaded, ascan shown in Figure 5B). Collectively, our results indicate that ScHal3 can effectively bind to and inhibit UmPpz1, and that UmHal3 canwith can effectively to and inhibit UmPpz1, and that UmHal3 (although very weakly) interact results indicate that ScHal3 can effectively bind to and inhibit UmPpz1, and that UmHal3 can ScPpz1very (although and ScPpz1-Cter, weakly) interact but that withitScPpz1 is completely unable to inhibit and ScPpz1-Cter, theiritphosphatase but that is completely activity. unable to (although very weakly) interact with ScPpz1 and ScPpz1-Cter, but that it is completely unable to inhibit their phosphatase activity. inhibit their phosphatase activity. Int. J.Sci. Int. J. Mol. Mol. Sci. 20, 2019, 2019, 20, x FOR PEER R EVIEW 3817 88of of19 20 Figure 5. Evaluation of the interaction between UmHal3 variants with ScPpz1 and UmPpz1. Glutathione Figure 5. Evaluation of the interaction between UmHal3 variants with ScPpz1 and UmPpz1. beads containing 4 µg of the GST-tagged UmPpz1 (panel A) or 8 µg of ScPpz1 and ScPpz1-Cter (panel B) Glutathione were beadsoncontaining immobilized g ofand glutathione4beads themixed GST-tagged UmPpz1 with extracts from(panel 8 g cells A) or hal3) IM021 (ppz1 of ScPpz1 and expressing ScPpz1-Cter (panel B) were immobilized on glutathione beads and mixed with extracts from IM021 3HA-tagged ScHal3, UmHal3, UmHal3_PD (PD) or UmHal3_PD_ScCter (PD_ScCt) from the pWS93 (ppz1 hal3) plasmid. cellswere Beads expressing washed,3HA-tagged resuspended ScHal3, in 100 UmHal3, UmHal3_PD µL of 2x sample (PD)processed buffer and or UmHal3_PD_ScCter for SDS-PAGE (PD_ScCt) from the pWS93 plasmid. Beads were washed, resuspended (6% gels for UmHal3 and 10% for PD and PD_ScCter). Typically, 25 µL of the samplesin 100 l of was 2x sample buffer used, except and processed for SDS-PAGE (6% gels for UmHal3 and 10% for PD and PD_ScCter). in lanes marked with an asterisk, where 50 µL was employed. Proteins were immunoblotted using Typically, 25 l of the samples anti-HA was as antibodies used, except in described in Methods. lanes marked with staining Ponceau an asterisk, where of the 50 l was membranes is employed. Proteins shown to evaluate for correct loading and transfer (ScPpz1-Cter was not seen in the left blot in panel B because dueof were immunoblotted using anti-HA antibodies as described in Methods. Ponceau staining to the its membranes is shown to evaluate for correct loading and relatively low molecular mass run with the front of the 6% gel).transfer (ScPpz1-Cter was not seen in the left blot in panel B because due to its relatively low molecular mass run with the front of the 6% gel). 2.5. UmHal3 Is a Functional PPCDC Enzyme 2.5. UmHal3 Is a Functional PPCDC Enzyme UV-visible scanning of purified recombinant UmPD preparations showed peaks at 382 and 452 nm, UV-visible characteristic scanning of the presence of of purified recombinant an oxidized UmPD flavin, which is apreparations showed cofactor involved peaks in the PPCDCat 382catalytic and 452 nm, characteristic process (Figure S5). of the presence In addition, of ancontains UmHal3 oxidizedallflavin, which isdeterminants the structural a cofactor involved necessary in the PPCDC for PPCDC catalytic activity process (i.e., (Figure catalytic His, S5). AsnIn addition, and UmHal3 Cys residues, seecontains Figure 1all the and structural Figure S7). Todeterminants confirm the necessary possible PPCDC for PPCDCfunction of UmHal3, activity we set (i.e., catalytic outAsn His, twoand functional tests. First, Cys residues, we transformed see Figure the E. coli strain 1 and Supplementary File A1). Towith BW369 confirm the possible pGEX-derived PPCDC vectors functionUmHal3, containing of UmHal3, UmPD we set andout two functional UmPD_ScCt. Thistests. strainFirst, we carries transformed the the E. coli strain temperature-sensitive dfp-707 ts mutation BW369 with pGEX-derived that affects PPCDC vectors containing function; UmHal3,cells consequently, UmPD and cannot grow at 37 ◦ C [32,33]. UmPD_ScCt. This strain carriesthis In parallel, thestrain temperature-sensitive was transformed with dfp-707 ts mutation AtHal3 (positivethat affects control) or PPCDC ScHal3 (negative function;control). As shown consequently, cellsincannot Figuregrow 6A, all at three 37°C versions [32,33]. In ofparallel, UmHal3this were able to strain wasrestore growthwith transformed of AtHal3 the BW369 (positive ◦ strain atcontrol) or ScHal3that 37 C, suggesting (negative control). all three are ableAs to shown provideinPPCDC Figureactivity 6A, all three (even versions if UmHal3 of isUmHal3 presumablywere expressed at very able to restore low of growth levels, see above). the BW369 strainInata37°C, second test, UmHal3 suggesting derivatives that all three are ablewereto provide PPCDC introduced activity in diploid (even if strains S. cerevisiae UmHal3 is presumably heterozygous for expressed the cab3 or at thevery hal3low vhs3levels , see above). mutations, and theIn a second test, UmHal3 derivatives were introduced in diploid S. cerevisiae strains heterozygous for Int. J. Mol. Sci. 2019, 20, x FOR PEER R EVIEW 9 of 20 Int. J. Mol. Sci. 2019, 20, 3817 9 of 19 the cab3 or the hal3 vhs3 mutations, and the ability of the Ustilago proteins to rescue the deletion mutants was monitored by random spore analysis and/or tetrad analysis. As observed in Figure 6B, ability of the Ustilago proteins to rescue the deletion mutants was monitored by random spore analysis the expression of all three Ustilago variants allowed growth of the mutant haploid strains, as deduced and/or tetrad analysis. As observed in Figure 6B, the expression of all three Ustilago variants allowed from the germination of all four spores in the tetrads. Therefore, full-length UmHal3 can substitute growth of the mutant haploid strains, as deduced from the germination of all four spores in the tetrads. either Cab3 and Hal3 (and Vhs3) in S. cerevisiae and provide PPCDC activity. As expected, this Therefore, full-length UmHal3 can substitute either Cab3 and Hal3 (and Vhs3) in S. cerevisiae and property can be assigned to the most C-terminal section of the protein. provide PPCDC activity. As expected, this property can be assigned to the most C-terminal section of the protein. Figure 6. Evaluation of UmHal3 as functional PPCDC. (A) The empty pGEX6P-1 plasmid (∅) and Figure 6. Evaluation of UmHal3 as functional PPCDC. (A) The empty pGEX6P-1 plasmid () and the the same vector bearing the Arabidopsis AtHal3 (as positive control), ScHal3 (as negative control), same vector bearing the Arabidopsis AtHal3 (as positive control), ScHal3 (as negative control), full- full-length UmHal3, and the indicated UmHal3 variants were introduced into the E. coli strain BW369, length UmHal3, and the indicated UmHal3 variants were introduced into the E. coli strain BW369, which carries the dfp-707ts mutation that abolish PPCDC activity when cells are grown at 37 ◦ C. which carries the dfp-707ts mutation that abolish PPCDC activity when cells are grown at 37 °C. Transformants were grown at 30 ◦ C, diluted till OD600 = 0.05, transferred to the permissive (30 ◦ C) or Transformants were grown at 30 °C, diluted till OD600 = 0.05, transferred to the permissive (30 °C) or 37 ◦ C (non-permissive) temperatures for 6 h, and then plated at the indicated temperatures. Growth 37 °C (non-permissive) temperatures for 6 h, and then plated at the indicated temperatures. Growth at 37 ◦ C indicate complementation of the ts PPCDC mutation. (B) The heterozygous diploid strains at 37 °C indicate complementation of the ts PPCDC mutation. (B) The heterozygous diploid strains MAR25 (CAB3/cab3∆) and AGS04 (HAL3/hal3∆ VHS3/vhs3∆) were transformed with the indicated MAR25 (CAB3/cab3Δ) and AGS04 (HAL3/hal3Δ VHS3/vhs3Δ) were transformed with the indicated plasmids, sporulation was induced, and spores from diverse asci (9 to 26 per transformation) were plasmids, sporulation was induced, and spores from diverse asci (9 to 26 per transformation) were isolated with a micromanipulator and incubated for 3 days to form colonies. Note that U. maydis isolated with a micromanipulator and incubated for 3 days to form colonies. Note that U. maydis full- full-length UmHal3, as well as of its variants containing the PD domain, rescue the lethal phenotype of thelength UmHal3, cab3 and as mutations hal3 vhs3 well as of its variants containing in haploidy, as denotedthe by PD domain, and germination rescue the lethal growth of all phenotype four spores of the cab3 and hal3 vhs3 mutations in haploidy, as denoted by germination and growth of all four spores in the tetrad. in the tetrad. 2.6. Phenotypic Characterization of UmPpz1 and UmHal3 Deletion Mutants 2.6. Phenotypic Characterization of UmPpz1 and UmHal3 Deletion Mutants U. maydis clones carrying a disruption of the gene encoding UmPpz1 were obtained as described U. maydis in Materials clones carrying and Methods a disruption by replacing of the one allele gene in the encoding diploid FBD11UmPpz1 wereaobtained strain with null alleleascarrying described in Materialsresistance a hygromycin and Methods (HygR)bymarker. replacing onesporulation, After allele in the wediploid FBD11 analyzed strain progeny the meiotic with a null allele of these carrying strains; a hygromycin roughly resistance half of the cells (HygR)ofmarker. in the progeny After the diploid sporulation, strain carrying theweppz1∆ analyzed allele the weremeiotic Hyg progenyindicating resistant, of these strains; that the roughly gene washalf notof the cellsWe essential. in the thenprogeny of mutant tested the the diploid strain strain carrying the for phenotypes ppz1Δ that allele were Hyg are characteristic of resistant, indicating the deletion of PPZ1 that the geneyeast. in budding was not As essential. shown in We then7,tested Figure the U.the mutant maydis mutant (in contrast to the S. cerevisiae ppz1 mutant) did not display altered tolerance to NaCl or LiCl,in strain for phenotypes that are characteristic of the deletion of PPZ1 in budding yeast. As shown Figure and was 7, the not U. maydis sensitive to mutant heat or (in contrast oxidative to the stress (HS. 2 Ocerevisiae ppz1cells 2 ). U. maydis mutant) did not deficient display altered in UmPpz1 also tolerance to NaCl or LiCl, and was not sensitive to heat or oxidative stress (H O displayed normal tolerance to calcium ions or chlorpromazine, an activator of Cell Wall Integrity (CWI) 2 2 ). U. maydis cells deficient pathway inin UmPpz1 this fungusalso[34]displayed normal (not shown). tolerance In contrast, thetomutant calciumwas ionsmore or chlorpromazine, sensitive to caffeine an activator and of Cell Wall Integrity (CWI) pathway in this fungus [34] (not shown). In contrast, the mutant was Int. J. Mol. Sci. 2019, 20, 3817 10 of 19 Int. J. Mol. Sci. 2019, 20, x FOR PEER R EVIEW 10 of 20 more sensitive calcofluor totwo white, caffeine and calcofluor compounds knownwhite, two compounds to interfere with normalknown to interfere cell wall with normal construction. cell As shown wall construction. As shown above, such sensitivity is characteristic above, such sensitivity is characteristic of S. cerevisiae ppz1 mutants. of S. cerevisiae ppz1 mutants. Figure7.7.Phenotypic Figure Phenotypicanalysis of the analysis U. maydis of the UmPpz1 U. maydis mutant. UmPpz1 Wild type mutant. Wild(WT) typeand UmPpz1-deficient (WT) and UmPpz1- cells (ppz1∆) deficient cellswere grown (ppz1Δ) in grown were YPD and ten-fold in YPD and dilutions of this culture ten-fold dilutions of this were spotted culture on CMD were spotted onplates CMD with the indicated chemicals. Cells were incubated for two days at 28 ◦ C. For temperature sensitivity, plates with the indicated chemicals. Cells were incubated for two days at 28°C. For temperature CMD platesCMD sensitivity, were incubated plates werefor two daysfor incubated at two the indicated temperature. days at the indicated temperature. InIn budding budding yeast, yeast, overexpression overexpression of of Hal3 Hal3 yields yields phenotypes phenotypes similar similar to to those those observed observed for for the the ppz1 ppz1mutant mutant(i.e., (i.e., sensitivity sensitivityto tocaffeine caffeineor orcalcofluor calcofluorwhite). white). ToTo test test whether whether this this was was the the case case forfor UmHal3, the entire protein, the C-terminal domain (residues 1187 to the stop UmHal3, the entire protein, the C-terminal domain (residues 1187 to the stop codon, equivalent to codon, equivalent to the PPCDC the PPCDC domain) and the domain) andlarge N-terminal the large fragment N-terminal were cloned fragment upstream were cloned the cgr1 the upstream promoter, which is cgr1 promoter, activated by arabinose. which is activated As shown As by arabinose. in Figure shown8,innone Figureof these proteins 8, none of theseresulted proteinsin altered resulted tolerance in altered to caffeine or calcofluor white when overexpressed, suggesting that they did not tolerance to caffeine or calcofluor white when overexpressed, suggesting that they did not have the have the expected effect on UmPpz1 expected function. effect The factfunction. on UmPpz1 that UmHal3 is thethat The fact onlyUmHal3 protein with is theaonly likelyprotein PPCDCwith domain in Ustilago a likely PPCDC led us to predict that the gene might be essential. To test this, we disrupted domain in Ustilago led us to predict that the gene might be essential. To test this, we disrupted one one allele in the diploid FBD11 alleleInt.instrain with the HygR marker. After analysis of 50After spores, we didofnot 50 find anywe Hygdidresistant J.the Mol.diploid Sci. 2019,FBD11 strain 20, x FOR PEER with the HygR R EVIEW marker. analysis spores, not11 find of 20 cells among any Hyg the haploid resistant progeny. cells among theThese results haploid confirmed progeny. Thesethat HAL3 results is an essential confirmed gene in that HAL3 U.essential is an maydis. gene in U. maydis. Figure 8. Growth of U. maydis strains overexpressing full-length UmHal3 and its N-terminal and Figure 8. Growth of U. maydis strains overexpressing full-length UmHal3 and its N-terminal and C- C-terminal regions. Cells were grown in YPD and ten-fold dilutions of this culture were spotted on terminal regions. Cells were grown in YPD and ten-fold dilutions of this culture were spotted on CMD CMD (2% glucose, repressing conditions) or CMA (2% arabinose, de-repressing conditions) plates with (2% glucose, repressing conditions) or CMA (2% arabinose, de-repressing conditions) plates with the the indicated chemicals. Pictures were taken after two days incubation at 28 ◦ C. indicated chemicals. Pictures were taken after two days incubation at 28°C. 2.7. Ppz1 Is Dispensable for Virulence in U. maydis Since U. maydis is a plant pathogenic fungus, we were curious about the effects of a lack of Ppz1 function in the ability of this fungus to infect plants. The virulence of U. maydis is dependent on mating; therefore, first we examined the behavior of haploid strains deleted for ppz1 with respect to Figure 8. Growth of U. maydis strains overexpressing full-length UmHal3 and its N-terminal and C- terminal regions. Cells were grown in YPD and ten-fold dilutions of this culture were spotted on CMD (2% glucose, repressing conditions) or CMA (2% arabinose, de-repressing conditions) plates with the Int. J.indicated Mol. Sci. 2019, 20, 3817 Pictures were taken after two days incubation at 28°C. chemicals. 11 of 19 2.7. Ppz1 Is Dispensable for Virulence in U. maydis 2.7. Ppz1 Is Dispensable for Virulence in U. maydis Since U. maydis is a plant pathogenic fungus, we were curious about the effects of a lack of Ppz1 Since U. maydis is a plant pathogenic fungus, we were curious about the effects of a lack of Ppz1 function in the ability of this fungus to infect plants. The virulence of U. maydis is dependent on function in the ability of this fungus to infect plants. The virulence of U. maydis is dependent on mating; mating; therefore, first we examined the behavior of haploid strains deleted for ppz1 with respect to therefore, first we examined the behavior of haploid strains deleted for ppz1 with respect to the ability to the ability to mate and to produce dikaryotic filament, which can be observed as a white-appearing mate and to produce dikaryotic filament, which can be observed as a white-appearing mycelial growth mycelial growth (fuzzy phenotype) [35]. We crossed compatible wild-type and ppz1Δ strains on (fuzzy phenotype) [35]. We crossed compatible wild-type and ppz1∆ strains on charcoal-containing charcoal-containing plates. Wild-type crosses led to a typical fuzzy colony appearance. In contrast, plates. Wild-type crosses led to a typical fuzzy colony appearance. In contrast, ppz1∆ strains mutants ppz1Δ strains mutants were slightly attenuated in filament formation (Figure 9A). were slightly attenuated in filament formation (Figure 9A). Figure 9. Analysis of virulence of mutants lacking Ppz1 function. (A) A ppz1∆ mutant is able to mate. Figure 9. Analysis of virulence of mutants lacking Ppz1 function. (A) A ppz1Δ mutant is able to mate. Crosses of control as well as ppz1∆ mutant strains carrying compatible mating types (a1 b1 and a2 b2) Crosses of control as well as ppz1Δ mutant strains carrying compatible mating types (a1 b1 and a2 b2) in charcoal-containing agar plates. Positive fuzzy phenotype can be detected as a white-appearance in charcoal-containing agar plates. Positive fuzzy phenotype can be detected as a white-appearance mycelial growth. Note that mutant combinations were slightly affected in the ability to produce mycelial growth. Note that mutant combinations◦were slightly affected in the ability to produce fuzzy fuzzy phenotype. Plates were incubated at 22 C for two days. (B) ppz1∆ mutant strains are able phenotype. Plates were incubated at 22°C for two days. (B) ppz1Δ mutant strains are able to infect to infect plants. Disease symptoms caused by crosses of wild-type and ppz1∆ mutant strains. The plants. Disease symptoms caused by crosses of wild-type and ppz1Δ mutant strains. The symptoms symptoms were scored 14 days after infection. Two independent experiments were carried out and were scored 14 days after infection. Two independent experiments were carried out and the average the average values are expressed as percentage of the total number of infected plants (n: 30 plants in values are expressed as percentage of the total number of infected plants (n: 30 plants in each each experiment). experiment). Following these results, we analyzed the ability of ppz1∆ strains to infect and cause symptoms in plants. We inoculated by stem injection maize plants with mixtures of wild type and mutant strains. U. maydis infection of maize results in anthocyanin pigment production by the plant and the formation of tumors that are filled with proliferating fungal cells that eventually differentiate into black teliospores [36]. We found that the ppz1∆ strains were able to induce pigmentation and tumors on the stems and leaves of infected plants at similar ratio as wild-type (Figure 9B), indicating a minor, if any, role of Ppz1 during the infection process. 3. Discussion The expression of UmPpz1 in S. cerevisiae shows that this phosphatase could partially replace endogenous Ppz1 in S. cerevisiae, suggesting that the Ustilago enzyme has the capacity to impact on specific intracellular targets of the Saccharomyces phosphatase. Thus, the introduction of UmPpz1 in S. cerevisiae ppz1 or slt2 mutants improves tolerance to caffeine. However, and in contrast to what has been found for Ppz enzymes from other organisms [17,18,20,22,24,28] UmPpz1 does not complement at all the ppz1 mutation with respect to tolerance to Li+ cations. It could be hypothesized that the lack of the arginine-rich conserved motif (SxRSxRxxS) in the N-terminal half of UmPpz1, which was found to be important for cation tolerance in D. hansenii [19], could be the reason for such failure. However, this motif is also absent in the N-terminal region of CnPpz1, and expression of this enzyme in S. cerevisiae partially normalizes Li+ tolerance [24]. Besides, a CaPpz1 mutated version lacking this conserved motif retains the ability to restore normal Li+ tolerance in vivo [37]. A second commonly Int. J. Mol. Sci. 2019, 20, 3817 12 of 19 lacking structural feature in UmPpz1 is the N-terminal myristoylable Gly. Previous work has shown that mutation of this Gly to Ala in ScPpz1 greatly affects the role of the Ppz1 on Li+ tolerance, but does not influence the tolerance to caffeine [25]. Consistently, the overexpression of a version of CaPpz1 carrying the same mutation restores normal caffeine tolerance but does not normalize growth in the presence of Li+ [37]. Myristoylation of Ppz1 has been found to be important for proper localization of the phosphatase to the plasma membrane ([38] and our own unpublished results) and for the role of the enzyme in methionine-triggered Mup1 endocytosis [38]. We observe here that overexpression of UmPpz1 in S. cerevisiae does not mimic the toxic effect of the endogenous enzyme, even if the enzyme is expressed at substantial levels. However, this lack of toxicity in UmPpz1 is no necessarily due to the absence of a terminal Gly because, although mutation of this residue decreases the toxicity to some extent (our unpublished results), the expression of other putatively myristoylable Ppz1 enzymes, such as DhPpz1 or CnPpz1, is not toxic for the cell [19,24]. Therefore, putting our results within the context of the literature, it appears that myristoylation of Ppz1 is necessary for some, but not all, the functions of the phosphatase, and that the actual protein expressed in U. maydis is not myristoylated. We show here that deletion of Ppz1 in U. maydis does not affect tolerance to Li+ or Na+ cations, oxidants such as hydrogen peroxide or high temperature. In contrast, and similarly to budding yeast, mutant cells are sensitive to caffeine and calcofluor white. These results suggest that in U. maydis UmPpz1 is potentially involved in the CWI pathway but not in the maintenance of monovalent cation homeostasis. It has been postulated that in S. cerevisiae, the link between Ppz phosphatases and the Slt2/Mpk1 pathway is based on the regulatory role of the phosphatase on potassium uptake [8,10]. However, ionic homeostasis mechanisms have been shown to be different in U. maydis and S. cerevisiae [39]. Thus, in contrast to budding yeast, the internal Na+ /K+ ratio in U. maydis can be > 2 without detrimental effects. In addition, U. maydis has two ENA ATPases, UmEna1 and UmEna2. UmEna1 is a typical ENA ATPase, although it shows low-Na+ /K+ discrimination, whereas UmEna2 shows a different endosomal/plasma membrane distribution, and its function is still unclear [39]. In addition, it has been demonstrated that U. maydis escapes from G2 phase by activating the CWI pathway [34], which contrasts with the G2 cell cycle arrest that occurs in S. cerevisiae in response to CWI pathway activation [40,41]. Therefore, even if the phosphatases in both organisms are functionally related to the CWI, the underlying mechanisms are likely different. Saccharomyces cerevisiae Hal3/Vhs3/Cab3 proteins can be taken as a prototype of what is found, almost without exception, in the genome of Saccharomycetes. In contrast, in other ascomycota classes, such as Eurotiomycetes, Dothideomycetes or Sordariomycetes, the protein is reduced to the PD domain and lacks the acidic tail. U. maydis Hal3 is an exceptionally large protein in which the PD domain is located at the very C-terminus and lacks the acidic tail. Our results show that UmPpz1 interacts much better with ScHal3 in vitro than with full-length UmHal3, the PD domain or the PD_ScCter version, suggesting that UmPpz1 retains the ability to be regulated by Hal3-like proteins. Indeed, ScHal3 is a powerful inhibitor of UmPpz1 phosphatase activity. In contrast, none of the versions of UmHal3 assayed had any inhibitory effect on ScPpz1 or UmPpz1. Therefore, despite the ability to interact up to some extent with ScPpz1 or UmPpz1, the Ustilago Hal3-like proteins are completely ineffective as phosphatase inhibitors in vitro. This behavior, also observed for the C. neoformans Hal3a and Hal3b proteins, provides additional support to the idea that in Hal3-like proteins, the structural determinants responsible for binding and inhibition of Ppz1 are different [24,42,43]. The absence of any effect on caffeine or calcofluor white when UmHal3 is overexpressed in U. maydis is in agreement with the failure of UmHal3 to inhibit UmPpz1 in vitro, and strongly suggests that in U. maydis, UmHal3 is not a regulatory subunit of the Ppz1 phosphatase. This raises the question of how the activity of this phosphatase is regulated in U. maydis. As suggested for C. neoformans, one possibility would be through phosphorylation [24]. Indeed, two conserved phosphorylated residues (S683 and S690) have been identified near the C-terminus in S. cerevisiae ([44,45] and our own unpublished data). The equivalent residues (S497 and S506) can be also phosphorylated in CnPpz1 [46]. Although, as far as we know, no phosphoproteomic data has been deposited for U. maydis, it is worth Int. J. Mol. Sci. 2019, 20, 3817 13 of 19 noting that the C-terminal tail of CnPpz1 and UmPpz1 is rather conserved, and the mentioned Ser residues are also found in the Ustilago phosphatase (Ser487 and Ser496). In fact, analysis of potential phosphorylatable sites using the NetPhosYeast 1.0 server [47] reveals that it is likely that these residues (score 0.918 and 0.772, respectively) will be phosphorylated. Our results also show that UmHal3 acts as a fully functional PPCDC in S. cerevisiae and E. coli, and that this ability is retained by its PD domain, suggesting that all assayed version are able to form productive homotrimeric enzymes and that the very long N-terminal extension is not necessary for UmHal3 PPCDC function. Therefore, our data indicates that, as found for CnHal3b and CnHal3a in C. neoformans, the biological role of UmHal3 may be restricted to the participation in the biosynthesis of CoA, and, in this case, it would be the unique PPCDC encoded in the U. maydis genome. This would fit with our finding that UmHal3 is an essential gene. From these results, it could be concluded that UmHal3 would not be a moonlighting protein. It is worth noting, however, that the long N-terminal extension of UmHal3 contains a putative VPS9 domain. Vps9 is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport [48]. A similar combination of VPS9 domain and PD domain in a Hal3-like protein can be also found in other species of the order Ustilaginales, such as the maize pathogenic fungus Sporisorium scitamineum and the sugarcane pathogenic fungus Sporisorium reilianum, but not in other fungi, even in the closely-related order of Malasseziales. The function of this specifically conserved region is unknown, and we observed that its overexpression devoid of the PD domain does not affect growth or tolerance to certain stresses. Therefore, although it the hypothesis that the UmHal3-like protein may have the potential to perform as a moonlighting protein cannot be fully discarded, in this case, the non-PPCDC function would be different than in S. cerevisiae and C. albicans. In any case, it must be noted that UmHal3 is not likely the bona fide Vps9 in Ustilago, since a closer protein (UMAG_05369.1) is encoded in the genome of this fungus. Our results demonstrate that, in contrast with what has been found in C. albicans and A. fumigatus, Ppz1 is not a virulence determinant in U. maydis. Since we recently showed that Ppz1 is not relevant for virulence in C. neoformans, it must be concluded that a role of Ppz1 phosphatases in fungal virulence is not a general feature, and must be investigated for each specific case. It is worth mentioning that both U. maydis and C. neoformans belongs to basidiomycetes, and it is possible that in this phylogenetic branch, the roles in virulence of Ppz1 phosphatases could be assumed by other phosphatases. Recent crystallization and elucidation of the PD domain of the C. neoformans CnHal3b isoform [24] showed the presence of an extra α-helix in the CnHal3b structure not present in AtHal3 (nor in human PPCDC), due to the 17-residue sequence insertion from Val86 to Gly102 . Similarly, in comparison with AtHal3 and human PPCDC, UmHal3 has exactly in this same region an even larger extra insert (residues 1256–1289, Figure S6). Because of the essential nature of PPCDC function, the presence of this structural feature in basidiomycete fungi might serve as basis for the development of selective drugs for human beings and antifungal treatments for maize, targeting Coenzyme A biosynthesis by interfering with PPCDC catalytic site or trimerization interface. Drugs targeting this step in the pathway have not yet been reported [49]. In this regard, the presence of subtle structural differences in the essential U. maydis protein kinase GSK3 compared to the human enzyme have been utilized for developing specific kinase inhibitors as potential antifungals [50]. 4. Materials and Methods 4.1. Yeast Strains and Growth Conditions U. maydis cells were grown on YPD (10 g L−1 yeast extract, 20 g L−1 peptone and 20 g L−1 dextrose) or Complete Medium (CM) at 28 ◦ C, unless otherwise stated [51]. S. cerevisiae cells were grown at 28 ◦ C in YPD medium or, when containing plasmids, in synthetic (SD) medium lacking the appropriate selection agents [52]. Sporulation of S. cerevisiae diploid strains was accomplished by transferring cells to a liquid medium containing 10 g L−1 potassium acetate, 1 g L−1 yeast extract and 0.5 g L−1 glucose (pH 7.2) for several days. Int. J. Mol. Sci. 2019, 20, 3817 14 of 19 4.2. Generation of Fungal Deletion Mutant Strains Deletions of the genes encoding U. maydis UmPpz1 and UmHal3 was made as follows. Deletion cassettes were generated using the Golden Gate cloning technology [53]. Briefly, upstream flank (UF) and downstream flank (DF) of hal3 (umag_05916) or ppz1 (umag_04827) were generated by PCR on FB1 genomic DNA using oligonucleotide combinations. To construct Hal3 deletion cassette the following oligos were used: HAL3-2/HAL3-3 (UF fragment, 1004 bp) and HAL3-7/HAL3-8 (DF, 889 bp). To construct the ppz1 deletion cassette the following oligos were used: PPZ1-2/PPZ1-3 (UF, 1068 bp) and PPZ1-4/PPZ1-5 (DF, 1085 bp). PCR products and pUMa1507 (storage vector I for simple knockout with HygR) were cut and ligated into pUMa1467 (destination vector) in a one-pot BsaI restriction/ligation reaction. To construct the different strains, transformation of U. maydis diploid strain FBD11 protoplasts with the indicated constructions was performed as described previously [54]. Integration of the disruption cassette into the corresponding loci was verified in each case by diagnostic PCR. U. maydis DNA isolation was performed as previously described [54]. Plant infections were performed with the maize cultivar Early Golden Bantam (Old Seeds, Madison, WI, USA) as described previously [55]. For the isolation of progeny and meiotic studies, spores from tumors were collected 10–14 days after inoculation and plated on plates containing 2% water agar. After 2 days, microcolonies (with about 200 cells) were removed and resuspended in YPD and dilutions plated onto YPD plates with and without hygromycin as selective agent. Fungal strains used in this work are described in Table 1. Table 1. List of fungal strains used in this work and their genotypes. Organism Strain Genotype Source/Reference U. maydis FB1 a1 b1 [35] FB2 a2 b2 [35] FBD11 a1a2 b1b2 [35] UMP341 a1a2 b1b2 hal3∆::HygR/hal3 This work UMP342 a1a2 b1b2 ppz1∆::HygR/ppz1 This work UMP351 a1 b1 ppz1∆::HygR This work UMP352 a2 b2 ppz1∆::HygR This work S. cerevisiae BY4741 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 Euroscarf ppz1 BY4741 ppz1::kanMx4 Euroscarf hal3 BY4741 hal3::kanMx4 Euroscarf slt2 BY4741 slt2::kanMx4 Euroscarf JA100 (MATa, ura3-52 leu2-3,112 trp1-1 IM21 [42] his4 can-1r) ppz1::kanMx4 hal3::LEU2 1788 (a/α, ura3-52 leu2-3,112 trp1-1 his4 AGS04 can-1r) VHS3/vhs3::kanMx4 [15] HAL3/hal3::LEU2 1788 (a/α, ura3-52 leu2-3,112 trp1-1 his4 MAR25 [25] can-1r) CAB3/cab3::kanMx4 4.3. Recombinant DNA Techniques and Plasmid Construction The Escherichia coli DH5α strain was used as plasmid DNA host and grown in Luria-Bertani (LB) medium broth at 37 ◦ C supplemented, if needed, with ampicillin (100 µg/mL). Recombinant techniques, and bacterial and yeast transformations were carried out by standard methods. To overexpress Umhal3 and derivatives in U. maydis, DNA fragments carrying the desired sequences were amplified by PCR, and cloned into the pRU11 plasmid [56] under the control of crg1 promoter as NdeI-EcoRI fragments. The oligonucleotide combinations were: UmHal3 full-length (HAL3-10/HAL3-13, 4300 bp), UmHal3 1-1186 (HAL3-10/HAL3-11, 3574 bp) and UmHal3 1187-1430 (HAL3-12/HAL3-13, 751 bp). These plasmids were inserted by homologous recombination into the cbx locus as described in [56]. Int. J. Mol. Sci. 2019, 20, 3817 15 of 19 For expression in S. cerevisiae, UmPpz1 was cloned into different vectors. Cloning into YCplac111 (low-copy vector) was as follows. Plasmids YCp111-CaPpz1 [20] was introduced into GM2163 (a dam- E. coli strain). The CaPpz1 ORF was removed by digestion with XbaI and HindIII, and replaced with the UmPpz1 ORF, amplified with oligonucleotides UmPpz1_XbaI and UmPpz1_HindIII from U. maydis genomic DNA, to yield YCp-UmPpz1. Then, the PPZ1 promoter followed by the UmPpz1 ORF was released by digestion with EcoRI and HindIII and introduced into YEplac181 (high-copy vector) linearized with the same restriction enzymes. A C-terminally HA tagged version of UmPpz1 was constructed by PCR amplification from YCp-UmPpz1 using oligonucleotides UmPpz1_XbaI and UmPpz1_HA_HindIII and cloning into XbaI/HindIII digested YCp-UmPpz1 to yield YCp-UmPpz1-HA. Constructs containing the short version (1-8∆) of UmPpz1 (from now on UmPpz1-s) were made in the same way except that the UmPpz1 ORF was amplified with oligonucleotides UmPpz1_short_XbaI as forward primer. An HA-tagged version of UmPpz1 expressed from plasmid pYES2 (2-micron, GAL1 promoter, URA3 marker) was made by PCR amplification of the tagged ORF, using YCp-UmPpz1-HA as template, with oligonucleotides UmPpz1_ATG_BamHI (or UmPpz1_short_ATG_BamHI, for UmPpz1-s) and UmPpz1_HA_EcoRI, following by cloning into the pYES2 vector at BamHI and EcoRI restriction sites. Similarly, the HA-tagged version of ScPpz1 was amplified from plasmid pCM190-PPZ1-HA (our laboratory, unpublished data) with oligonucleotides ScPpz1-HA-NotI and ScPpz1_in_PacI and cloned into pYES2 upon NotI and PacI digestion to yield pYES2-Ppz1-HA. To overexpress UmHal3-like proteins with an N-terminal 3x-HA tag in S. cerevisiae and under the control of the ADH1 promoter, the UmHal3 ORF (4995 bp) was amplified with oligonucleotides UmHal3_EcoRI and UmHal3_BamHI; then, it was inserted into the vector pWS93 [57] after digestion with EcoRI and BamHI. In addition, the C-terminal region (UmHal3_PD, corresponding to the putative PPCDC domain) was amplified with oligonucleotides UmHal3_PD_EcoRI and UmHal3_BamHI, and was then inserted into these sites of pWS93 as for UmHal3 ORF. The UmHal3_PDScCter version was obtained from plasmid pGEX-UmHal3_ScCter (see below) by digestion with EcoRI and XhoI and cloning into the plasmid pWS93 linearized by EcoRI and SalI (Figure S8). For expression in E. coli, the UmPpz1 ORF was amplified from U. maydis FB1 genomic DNA with oligonucleotides UmPpz1_BamHI (UmPpz1_short_BamHI for UmPpz1-s) and UmPpz1_EcoRI. Then, the fragment was digested and inserted into the vector pGEX-6p-1 (GE Healthcare, Chicago, Il, USA) into the BamHI and EcoRI restriction sites. The UmHal3 and UmHal3_PD ORFs were amplified with the forward oligonucleotides UmHal3_EcoRI and UmHal3_PD_EcoRI, respectively, and the common reverse primer UmHal3_NotI. The fragments were then inserted into pGEX-6p-1 at the restriction sites EcoRI and NotI. The acidic tail of ScHal3 was added to UmHal3_PD by amplification of the UmHal3_PD sequence with oligonucleotides UmHal3_PD_EcoRI and UmHal3_ScCtD_RV using pWS-UmHal3 as template; the downstream C-terminal tail was generated with oligonucleotides UmHal3_ScCtD_FW and Reverse HAL3-XhoI using pWS-ScHal3 as template. The fusion fragment was amplified using UmHal3_PD_EcoRI and Reverse HAL3-XhoI and then inserted into pGEX-6p-1 by digestion with EcoRI and XhoI. Oligonucleotide sequences can be found in Table S1, and a list of plasmids used in this work is found in Table S2. 4.4. Phenotypic Analysis To test the sensitivity of U. maydis strains to different stress factors, cells were grown in YPD and ten-fold dilutions of this culture were spotted on CMD plates with the indicated chemicals. Cells were incubated for two days at 28 ◦ C. For temperature sensitivity, CMD plates were incubated for two days at the indicated temperature. The sensitivity of S. cerevisiae cells was evaluated by spotting serial dilutions of starting cultures (OD600 of 0.05) on SD medium lacking uracil (for pWS93-based vectors) or leucine (for strains bearing YCplac111 or YEplac181 plasmids) and containing a range of concentrations of diverse compounds. Growth of strains overexpressing ScPpz1 or UmPpz1 (or their Int. J. Mol. Sci. 2019, 20, 3817 16 of 19 HA-tagged versions) from the GAL1-10 promoter (pYES2-based vectors) was evaluated using synthetic medium lacking uracil and containing 2% galactose as carbon source. Plates were then incubated at 28 ◦ C. 4.5. Expression and Purification of Recombinant Proteins GST-ScPpz1, GST-ScPpz1-Cter, GST-UmPpz1, UmPpz1-s, GST-ScHal3, GST-UmHal3, GST-UmHal3_PD and GST-UmHal3_PD_ScCter were expressed using BL21 (DE3) RIL E. coli cells and purified as reported [58] with specific modifications [24]. The GST-tag was removed by PreScission protease (GE Healthcare), as described. The recombinant proteins were analyzed by SDS-PAGE followed by Coomassie Blue-staining and gels were scanned with an Epson Perfection V500 Photo scanner apparatus. Estimation of the amount of full length proteins was done with Gel Analyze 2010a software (http://www.gelanalyzer.com/) in comparison with bovine serum albumin (BSA) standards. 4.6. Yeasts Protein Extraction and Immunoblot Analysis Yeast extracts were prepared as previously described [24,58]. Following SDS-PAGE, proteins were transferred to Immobilon-P membranes (Millipore, Burlington, MA, USA). and membranes were blocked and incubated with anti-HA antibodies (1:1000 dilution, Covance, Princeton, NJ, USA) followed by anti-mouse peroxidase antibodies (1:10000 dilution, GE Healthcare). Immunoreactive proteins were visualized using ECL western blotting detection kit (GE Healthcare) and chemiluminescence was detected in a Versadoc Image System 4000 MP (Bio-Rad Hercules, CA, USA). 4.7. In Vitro Interaction Experiments The interaction between the Ppz phosphatases and the U. maydis proteins was determined by in vitro binding assays performed as previously described [58]. Aliquots of glutathione agarose beads containing 4 µg of the GST-tagged UmPpz1 (panel A) or 8 µg of ScPpz1 and ScPpz1-Cter were incubated with protein extracts (0.6–1.0 µg of proteins) prepared from strain IM021 (ppz1∆ hal3∆) expressing the diverse 3HA-tagged Hal3 versions. The amount of protein extracts used was determined after detection and quantification by immunoblot of the expression levels of the relevant proteins (which were quite similar). The beads were finally resuspended in 100 µL of 2×DS sample buffer and, after boiling for 5 min, 25 or 50 µL of the samples were analyzed by SDS-PAGE and immunodetection. 4.8. Other Techniques The S. cerevisiae and U. maydis Ppz1 phosphatase activity was assayed as previously described [58], using 5 to 10 pmol/assay and different amounts (0.5 to 4-fold molar excess) of the inhibitors. Phosphatase activity was measured using 10 mM of p-nitrophenylphosphate (Sigma Chem. Co, St. Louis, MO, USA) as substrate. The visible-UV absorption profile of recombinant UmHal3_PD devoid of the GST tag was obtained in a Nanodrop ND1000. Tetrad analysis were performed as in [15,27], respectively. Modeling of the PD region of UmHal3 was done at the Swiss-Model server (https: //www.swissmodel.expasy.org/) [59]. Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/15/3817/s1. Author Contributions: Conceptualization, J.A.; formal analysis, J.P.-M. and J.A.; funding acquisition, J.P.-M. and J.A.; investigation, C.Z., A.d.l.T., J.P.-M. and J.A.; methodology, C.Z. and A.d.l.T.; project administration, J.A.; supervision, J.P.-M. and J.A.; writing—original draft, J.A.; writing—review & editing, J.P.-M. and J.A. Funding: This research was funded by grant BFU2014-54591-C2-1-P and BFU2017-82574-P to JA and BIO2017-88938-R to JP-M (Ministerio de Industria, Economía y Competitividad, Spain). C.Z. was recipient of a PhD fellowship from the China Scholarship Council. Acknowledgments: The technical assistance of Montserrat Robledo is acknowledged. Thanks are given to D. Reverter (IBB, UAB) for help in UmHal3 modeling. Int. J. Mol. Sci. 2019, 20, 3817 17 of 19 Conflicts of Interest: The authors declare no conflict of interest. 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