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Department of Plant Pathology and Microbiology, Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, TX 77843-2132, USA
Correspondence
Won-Bo Shim
wbshim{at}tamu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of CPP1 is DQ924537.
A description of PP179 complementation using the YEC3 construct (section 1) and a description of how exogenous application of protein phosphatase inhibitor okadaic acid did not induce hyphal swelling in Fusarium verticillioides (section 2) are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Marasas, 2001; Minorsky, 2002; Missmer et al., 2006). Due to these serious concerns, the US Food and Drug Administration has established guidelines for the fumonisin level in feed and foodstuff (Park & Troxell, 2002). Significant progress has been made in elucidating the fumonisin biosynthetic pathway, and regulatory mechanisms associated with the toxin production. The biosynthetic gene cluster, including the polyketide synthase (PKS) gene FUM1 (Proctor et al., 1999, 2003; Seo et al., 2001), and a number of regulatory genes involved in fumonisin biosynthesis, namely PAC1, FCC1 and ZFR1, have been identified and characterized (Flaherty et al., 2003; Flaherty & Woloshuk, 2004; Shim & Woloshuk, 2001). However, these genes do not show clear epistatic relationships, and therefore it is conceivable that multiple signalling pathways are associated with fumonisin regulation. Moreover, a putative regulatory gene, FUM21, within the FUM cluster, has recently been identified and characterized, but it was shown that the deletion of the FUM21 gene did not completely block fumonisin biosynthesis (Brown et al., 2007). That report suggested that transcriptional regulation of FUM genes can also be affected by putative regulatory gene(s) outside the FUM cluster. Additionally, the complexity of fumonisin regulation is further enhanced by a variety of physiological and nutritional conditions, notably acidic pH and nitrogen stress, which are known to favour or perhaps trigger fumonisin biosynthesis (Flaherty et al., 2003; Shim & Woloshuk, 1999).
In an effort to identify additional genes associated with fumonisin regulation, Shim & Woloshuk (2001) constructed a subtractive suppression hybridization (SSH) cDNA library to screen genes that are differentially expressed during fumonisin biosynthesis in F. verticillioides wild-type (WT) and FT536 strains. FT536 harbours a mutation in the FCC1 gene, which encodes a type-C cyclin. Mutation of FCC1 results in reduced conidiation and drastic reduction in fumonisin production when grown on maize kernels (Shim & Woloshuk, 2001). Microarray analysis using F. verticillioides oligoarrays has further verified genes that are expressed concomitantly with fumonisin production (Pirttilä et al., 2004). A number of these genes have been selected for characterization of their role in fumonisin biosynthesis. For instance, cDNA encoding a putative zinc binuclear cluster-type transcription factor, later designated ZFR1, has been shown to be positively associated with fumonisin biosynthesis (Flaherty & Woloshuk, 2004). Deletion of ZFR1 in F. verticillioides has been shown to result in greater than 90 % reduction in fumonisin production when compared with the WT. Also, Sagaram et al. (2006) have recently demonstrated that GBP1, a gene originally identified in the FT536 SSH cDNA library, encodes a monomeric G-protein that is negatively associated with fumonisin biosynthesis.
In the FT536 SSH cDNA library, we isolated a 300 bp cDNA fragment (ft536_ 0_M14) that shows a high level of similarity to a Neurospora crassa gene that encodes a probable cell-shape-control protein phosphatase 2A catalytic subunit, PPE-1 (E value, 5e-45). Protein phosphatases catalyse the dephosphorylation of specific substrates that are important for processing various biological and cellular functions (Ceulemans & Bollen, 2004; Dickman & Yarden, 1999; Hunter, 1995), and this catalytic action is known to de-activate signalling pathways induced by a variety of protein kinases (Hunter, 1995). In Arabidopsis, for instance, protein phosphatase type 2C is involved in the negative regulation of the abscisic acid signalling pathway (Saez et al., 2004). Overall, we have limited understanding of the role of protein phosphatases in filamentous fungi, particularly in association with secondary metabolism. Moreover, protein phosphatases in F. verticillioides have not been functionally characterized to date.
Since cDNA ft536_ 0_M14 was identified among a collection of genes upregulated during fumonisin suppression, we hypothesize that this gene, designated CPP1 (probable cell-shape-control protein phosphatase), is negatively associated with fumonisin biosynthesis in F. verticillioides. To test this hypothesis, we generated a CPP1 gene deletion mutant, and investigated the role of CPP1 in F. verticillioides. In this study, we report that deletion of CPP1 results in elevated fumonisin production via de-repression (or upregulation) of FUM1 expression. We also show that mutation in CPP1 results in pleiotropic phenotypes, suggesting that CPP1 is associated with multiple signalling pathways that control fungal development and differentiation. Lastly, we demonstrate that N. crassa WT ppe-1 gene can complement F. verticillioides CPP1 gene deletion.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The medium (100 ml) was inoculated with 1x105 conidia, and incubated at 25 °C shaking at 150 r.p.m., under a 14 h light/10 h dark cycle. For the fumonisin assay and total RNA isolation, fungal strains were grown on cracked-corn medium, as previously described (Shim & Woloshuk, 1999). To study colony morphology and growth of fungal strains, we used diluted (0.2x) potato dextrose agar (PDA; Difco). For macroconidia and microconidia counts, agar blocks (0.5 cm diameter) were inoculated at the centre of V8 juice agar, and allowed to grow for 7 days. The conidia were harvested in 0.1 % Triton X-100, and counted using a haemocytometer.
Nucleic acid isolation and manipulation.
Bacterial plasmid DNA and fungal genomic DNA were extracted by using the Wizard miniprep DNA purification system (Promega) and the OmniPrep genomic DNA extraction kit (G Biosciences), respectively. Total RNA was prepared with Trizol reagent (Invitrogen), or with RNeasy plant mini kit (Qiagen). Southern and Northern analyses were performed as described previously (Sagaram et al., 2006; Sambrook & Russell, 2001). The probes used in all hybridization experiments were labelled with 32P using Prime-It II random primer labelling kit (Stratagene). DNA sequencing was performed at Gene Technologies Lab (Texas A&M University).
PCR and quantitative real-time PCR (qRT-PCR).
All primers used in this study are listed in Table 1. PCR amplifications were performed in a GeneAmp PCR system 9700 thermocycler (PE Applied Biosystems). PCR of DNA (except single-joint PCR) was performed in a 25 µl total volume with Taq DNA polymerase (Promega). The PCR conditions were 2 min of pre-denaturation at 94 °C, followed by 30 cycles of 45 s denaturation at 94 °C, 45 s annealing at 54–57 °C, and 2 min extension at 72 °C, unless specified otherwise. Single-joint PCR was performed using Expand Long Polymerase (Roche) using the manufacturer's suggested protocol.
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F. verticillioides transformation.
F. verticillioides protoplasts were generated using the protocol described by Shim & Woloshuk (2001), except that Mureinase (2 mg ml–1) was replaced with Drieselase (5 mg ml–1) (Sigma). The CPP1 gene disruption vector YEC2 was created by inserting 571 bp DNA from the 5' region of the CPP1 gene, and 529 bp DNA from the 3' region of the CPP1 gene, into pBP15 vector, which contains a hygromycin phosphotransferase (HPH) gene as a selectable marker (Fig. 1a) (Sagaram et al., 2006). Primers CPP1 A, CPP1-B, CPP1-C and CPP1-D were used to amplify the 5' and 3' regions, which were subsequently cloned into pBP15 vector (Fig. 1a). The vector YEC2 was linearized with NotI prior to protoplast transformation. Hygromycin-resistant transformants were selected on regeneration agar medium containing hygromycin (150 µg m–1), and screened for CPP1 deletion by PCR and Southern analysis. For PCR, primers (CPP1-che-F, CPP1-che-R and HPH-R2) that provide specific positive and negative amplification were used to detect homologous recombination events (Fig. 1a). For Southern analysis, fungal genomic DNA samples were digested with EcoRI before they were subjected to electrophoresis in a 1 % agarose gel. A 500 bp DNA fragment of YEC2, excised by double enzyme digestion (SpeI and SmaI), was labelled with 32P and used as a probe (Fig. 1a).
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; Yu et al., 2004). Primers CPP1-LC-F and CPP1-LC-R amplified the complete CPP1 gene plus the 850 bp 5' untranslated region (UTR) and the 450 bp 3' UTR. This amplicon was fused with the GEN marker, and the final YEC4 construct was amplified with primers Gene-F and CPP1-LC-R2, using Expand Long Polymerase (Roche). We then transformed YEC4 into PP179 protoplasts, and screened for colonies resistant to geneticin and hygromycin (Sagaram et al., 2006). The introduction of YEC4 in geneticin-resistant strains was verified by PCR and Southern blot analyses.
Fumonisin B1 analysis.
F. verticillioides strains were grown on cracked-corn medium for 10 days for fumonisin production analysis. FB1, the major fumonisin produced by F. verticillioides, was extracted and analysed by HPLC, following the method described by Shim & Woloshuk (1999). In addition to FB1 HPLC analysis, qRT-PCR was used to investigate the relative expression of FUM1 and other selected PKS genes (Kroken et al., 2003) in the WT and mutant strains. Total RNA samples were prepared from the WT, PP87, PP179 and PPC4 grown on cracked-corn medium for 10 days, and qRT-PCR analysis was performed with SYBR-Green as the fluorescent reporter, using gene-specific primers (FUM1-F and FUM1-R2) for the FUM1 gene. The expression of each gene was normalized to endogenous TUB2 gene expression. The gene expression was calibrated using 
method (Ct, threshold cycle) (Livak & Schmittgen, 2001); the range of expression was calibrated using 
, where s is the standard deviation of the 
Ct value. Subsequently, the total RNA samples of the WT and PP179 strains were subjected to qRT-PCR analysis using gene-specific primers for FUM1, PKS1 (PKS1-F and PKS1-R), PKS2 (PKS2-F and PKS2-R), and PKS4 (PKS4-F and PKS4-R). Again, the gene expression was calibrated using the 
method, with TUB2 as the endogenous control.
Microscopy.
Microscopic observations were made using an Olympus BX51 microscope (Olympus America). A detailed description of features used for imaging from this microscope has been given (Shaw & Upadhyay, 2005). Imaging of hyphal growth phenotypes was performed using an Olympus DP70 camera and DP70-BSW software (version 01.01). Nuclei were stained with Hoechst 33258 dye, as described previously (Shaw & Upadhyay, 2005).
Complementation of PP179 strain with N. crassa ppe-1.
N. crassa ppe-1 (GenBank accession no. XM_951629) encodes a probable cell-shape-control protein phosphatase. The complementation construct YEC5 was generated by single-joint PCR strategy (Fig. 1a
) (Yu et al., 2004). WT ppe-1 (1.43 kb ppe-1 gene plus 1.6 kb 5' UTR and 500 bp 3' UTR) was amplified from the N. crassa genomic DNA using the primers PPE1-com-F1 and PPE1-com-R1. These amplicons were fused to GEN by joint-PCR using PPE1-com-R2 and Gene-F primers. The final construct, YEC5 was introduced into the protoplasts of PP179, and the transformants were screened for geneticin resistance. The selected isolates were further analysed by PCR, using primers PPE1-che-F1 and PPE1-com-R2 to determine the presence of YEC5 in the genome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Sequence analysis of Cpp1 revealed a type 2A protein phosphatase catalytic domain (PP2Ac) between amino acids 32 and 385. This domain is present among a large family of serine/threonine phosphatases. Likewise, Cpp1 displays a high similarity to the PP2Ac domains in a number of eukaryotes, particularly those present in filamentous fungi (Fig. 2). Namely, Cpp1 shares significant similarity with PPE-1 protein, which is a probable cell-shape-control PP2Ac in N. crassa (E value, 0.0) (unpublished; GenBank accession no. XM_951629), and with SitA, which is a PP2Ac involved in the TOR pathway of Aspergillus nidulans (E value, 8e-165) (Fitzgibbon et al., 2005; GenBank accession no. CAG30555). Significantly, these PP2Ac proteins of filamentous fungi are orthologous to the Schizosaccharomyces pombe protein Ppe1, which plays a role in cell morphogenesis and mitosis (Shimanuki et al., 1993). We determined via Northern blot analysis that there is no significant differential expression of CPP1 during the course of fungal growth in DL culture, thereby concluding that CPP1 is a constitutively expressed gene in F. verticillioides (data not shown).
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). We selected PP179 for further molecular characterization. Southern blot analysis further confirmed that the HPH gene replaced 419 bp DNA within the CPP1 gene. The 571 bp 32P-labelled DNA probe hybridized to a 1.3 kb band in the WT and PP87 strains, whereas it hybridized to a 0.8 kb band in the PP179 strain (Fig. 1c). Northern blot analysis was performed using a 571 bp CPP1 DNA fragment probe, which hybridized to the expected 1.35 kb transcript band in the WT and PP87, but did not hybridize in PP179 (Fig. 1d).
Measurement of the radial growth of F. verticillioides strains on 0.2x PDA plates revealed that PP179 had approximately a 40 % reduction in growth rate when compared with the WT progenitor and the PP87 strain (Fig. 3). However, no significant difference in fungal mass (fresh wet weight) was observed when strains were grown in 0.2x PDB and YPD broth. Subsequently WT, PP87 and PP179 strains were grown on cracked-corn medium and DL medium to test FB1 production. TLC and HPLC analysis revealed that the WT and PP87 did not differ in FB1 production in either cracked-corn medium or DL medium; however, we observed a significant increase in FB1 production in PP179. Specifically, FB1 production on inoculated cracked-corn medium was more than four times greater for PP179 than for the WT (Fig. 4a).
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). Following transformation, complemented strains were selected by testing growth on 0.2x PDA containing hygromycin and geneticin. The putative complemented strain containing YEC4 was designated PPC4. We determined via PCR that PPC4 contained the introduced CPP1 gene (from YEC4) and the HYG-replaced mutant locus (Fig. 1b). Southern analysis verified the PCR data (Fig. 1c). Northern blot analysis of PPC4 showed that CPP1 gene expression was restored in PPC4 (Fig. 1d). Growth rate reduction and increased FB1 reduction in PP179 were restored to the level of the WT in the PPC4 strain, thus providing further evidence for roles of CPP1 in growth rate and FB1 production (Fig. 3 and 4a).
Increased FB1 production in PP179 is due to upregulation of FUM1, the FB1-specific PKS gene
FUM1 encodes a PKS that is critical to FB1 biosynthesis (Proctor et al., 1999). Here, we asked the question whether the drastic increase in FB1 production in PP179 is due to altered FUM1 expression. Total RNA was harvested from WT, PP87, PP179 and PPC4 strains grown on cracked-corn medium, the culture condition used for FB1 production. qRT-PCR analysis of FUM1 revealed at least 11-fold higher FUM1 expression in PP179 than in other strains tested (Fig. 4b). Analysis of variance (ANOVA) of the gene expression data from all four strains (P<0.01) suggested that FUM1 expression levels in the WT, PP87 and PPC4 strains were significantly different from that of PP179 strain. When the test was limited to the WT, PP87 and PPC4, the resulting P value was 0.862, suggesting that the complementation of PP179 with the CPP1 gene restored the WT level of FUM1 expression. The data provide molecular evidence that CPP1 serves as a negative regulator of FB1 biosynthetic genes in F. verticillioides.
As there are over 15 PKS genes in F. verticillioides (Kroken et al., 2003), we next investigated the specificity of CPP1 to regulation of FUM1. Kroken et al. (2003) classified 15 type-I PKS genes present in F. verticillioides into major clades or subclades. To test the impact of CPP1 deletion on the expression of PKS genes, we selected four PKS genes from different subclades: PKS1 (reducing PKS clade II), PKS2 (reducing PKS clade I), PKS4 (non-reducing PKS clade I) and PKS11 (FUM1, reducing PKS clade III). qRT-PCR analysis of the four PKS genes revealed that only FUM1 expression was upregulated significantly in PP179, whereas PKS1, PKS2 and PKS4 were either downregulated or unchanged (Fig. 4c). The t tests for PKS1, PKS2 and PKS4 expression confirmed no significant difference in expression between the WT and PP179. Our results showed that the major impact of CPP1 deletion was on FUM1 expression, suggesting that CPP1 may have a specific function to regulate FUM1 in F. verticillioides.
CPP1 is required for hyphal polarity maintenance in F. verticillioides
In contrast to the WT and PP87, PP179 showed a distinguishable hyphal swelling when grown in liquid medium (Fig. 5a). This phenotype in PP179 became more apparent in the cultures incubated longer than 7 days (data not shown). Nucleus staining was utilized to investigate whether the hyphal-swelling phenotype in PP179 was linked to defective cell-cycle progression. Swollen hyphae of PP179 contained multiple nuclei, whereas nuclei in the WT were uniformly distributed along hyphae (Fig. 5b). This phenotype of PP179 suggested that cell-cycle progression was not affected, but maintenance of hyphal polarity was impaired. Notably, suppression of hyphal swelling in PP179 occurred with the addition of sucrose (1 M) to the medium (Fig. 5c), and this suppression was maintained for up to 10 days. PPC4 showed complete recovery from hyphal swelling demonstrating that this phenotype was due to CPP1 deletion (Fig. 5a).
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; Chant et al., 1995; Sagot et al., 2002; Sheu et al., 1998). Homologues of these genes in filamentous fungi are known to be associated with cell-polarity maintenance and cytokinesis (Harris et al., 1997; Virag & Harris, 2006; Wendland, 2003). We isolated corresponding F. verticillioides homologues FVEG_04885.3, FVEG_06243.3, FVEG_11303.3 and FVEG_00700.3, and tested the transcriptional levels in the WT and PP179 strains. qRT-PCR, using gene-specific primers (Table 1), showed no significant difference between the WT and PP179 in expression levels of these genes (data not shown).
CPP1 is involved in microconidia–macroconidia equilibrium and conidia germination in F. verticillioides
One striking feature of PP179 was the high percentage of macroconidia production on V8 agar medium. In F. verticillioides, the formation of macroconidia has been shown under a few select conditions, such as UV exposure (Nelson et al., 1983). The WT and PP87 strains produced microconidia, but not macroconidia, on V8 agar. In contrast, approximately 44 % of the conidia harvested from PP179 on V8 agar were macroconidia (Fig. 6a). The conidiation profile was the same as the WT in PPC4. We also observed significant reduction of the conidia-germination rate in PP179. WT and PP87 conidia (1x105), when resuspended in DL medium, successfully germinated after 24 h incubation (Fig. 6b). However, under the same conditions, PP179 germination rate was only 30 % of that of the WT and PP87 strains (Fig. 6b). The germination deficiency in PP179 was only partially recovered with the complementation by CPP1 in PPC4 strain (Fig. 6b).
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), was transformed into PP179 protoplasts, and the transformants were selected by geneticin and hygromycin resistance. Isolates were further tested by PCR to demonstrate the presence of WT ppe-1. No amplicon of ppe-1 was detected in the WT and PP179 strains, whereas amplicons of the expected size (1.5 kb) were observed in the YEC5-complemented isolates (data not shown). One isolate was selected, and designated ‘NC-com’ strain, and further analysed. Interestingly, the complementation of PP179 with YEC5 resulted in restorations of WT morphology, and the reduced growth rate of PP179 was restored to WT level in NC-com (Fig. 3). Notably, the hyphal swelling phenotypes of PP179 were less apparent in NC-com (Fig. 5d). Macroconidia production by PP179 was completely eliminated in NC-com, and germination efficiency was restored to that of WT progenitor (data not shown). Elevated FB1 production in PP179 was significantly reduced in NC-com similar to the WT level (data not shown). These results strongly support the hypothesis that N. crassa PPE-1 and F. verticillioides Cpp1 are functionally conserved across the two species. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It has been suggested that about one-third of the eukaryotic proteins are regulated by reversible phosphorylations of specific serine, threonine and/or tyrosine residues (Ceulemans & Bollen, 2004). To date, the major group of serine/threonine protein phosphatases are encompassed by two different structural families. PP1, PP2A, PP2B and PP5 are classified as members of the protein phosphatase P family, while PP2C is a member of Mg2+-dependent protein phosphatase M family (Dickman & Yarden, 1999). Each protein phosphatase has specific or overlapping roles in regards to regulating cellular functions. Despite their central roles in regulating a wide variety of cellular function, including signal transduction and gene expression, limited information is available on the functions of protein phosphatases in filamentous fungi. In general, fungi have a larger number of protein kinases than protein phosphatases in their genome. For instance, Sac. cerevisiae has approximately 6000 genes, of which 113 encode protein kinase genes, but only 31 encode protein phosphatases (Dickman & Yarden, 1999; Sakumoto et al., 1999; Stark, 1996). Considering the deviation in prevalence between protein kinases and protein phosphatases, it is likely that Cpp1 has broad substrate specificity, and interacts with multiple protein kinases in several signalling pathways. Further complicating the process, a 36 kDa PP2A catalytic subunit combines with a 65 kDa regulatory subunit to form a PP2A holoenzyme dimer, with a third variable subunit, known as the B subunit, responsible for substrate specificity (Stark, 1996; Mayer-Jaekel & Hemmings, 1994). Since CPP1 encodes a putative catalytic subunit of PP2A, the broad specificity of substrates of CPP1 may be narrowed with additional protein phosphatase subunit components (Mayer-Jaekel & Hemmings, 1994).
In this study, our main hypothesis was that CPP1 serves as a putative FB1 regulatory gene, perhaps in a negative or suppressive manner. Consistent with our hypothesis, we detected elevated FB1 production in PP179 via TLC and HPLC analyses (Fig. 4a), and subsequent qRT-PCR analysis suggested that this increased FB1 production is a result of an elevated FUM1 expression (Fig. 4b). PKS1 and PKS2 were classified, along with FUM1, as reducing PKSs, whereas PKS4 resided in a separate group of non-reducing PKSs (Kroken et al., 2003). Interestingly, deletion of CPP1 did not have a drastic effect on the expression of other PKS genes, suggesting that the regulatory role of CPP1 is specific to FUM genes and FB1 biosynthesis (Fig. 4c). An expanded transcriptional profiling study is necessary to help us better understand the role of CPP1 in F. verticillioides secondary metabolism pathways. Of particular interest is whether CPP1 affects other fumonisin biosynthesis regulatory genes, particularly the genes FUM21, ZFR1 and GBB1 that are positively associated with fumonisin biosynthesis (Brown et al., 2007; Flaherty & Woloshuk, 2004; Sagaram & Shim, 2007). Meanwhile, it is well recognized that type 2A protein phosphatases are associated with cell differentiation and development in eukaryotes, including filamentous fungi (Dickman & Yarden, 1999; Hunter, 1995; Mayer-Jaekel & Hemmings, 1994; Shenolikar, 1994; Yatzkan et al., 1998), and multiple PP179 phenotypes suggested that CPP1 is also involved in multiple downstream signalling pathways in F. verticillioides. Strikingly, CPP1 deletion led to a hyphal-swelling phenotype, thereby suggesting the functional role of CPP1 in morphogenesis. We hypothesized that hyphal swelling in PP179 is caused by random deposition of cell wall components, leading to impaired hyphal polarity. In the yeast Sac. cerevisiae, mutation in genes BNI1, BUD3, BUD6 and SPA2, which regulate polarity and proper cell development, triggers cell-polarity phenotypes (Amberg et al., 1997; Chant et al., 1995; Sagot et al., 2002; Sheu et al., 1998). While there is no experimental evidence that homologues of these yeast genes are involved in F. verticillioides cell-polarity maintenance, studies in other filamentous fungi provide support to our hypothesis. For instance Asp. nidulans sepA, the BNI1 homologue, plays a critical role in cytokinesis, and is required for maintenance of polarity during hyphal growth (Harris et al., 1997). In Ashbya gossypii, deletion of AgBUD3, the BUD3 homologue, led to a change in actin ring localization, and subsequent aberrant chitin accumulation, ultimately generating delocalized septa (Wendland, 2003). However, our results suggest that hyphal swelling in PP179 is not due to transcriptional defect in F. verticillioides BNI1, BUD3, BUD6 and SPA2-like genes. We are currently investigating putative signalling pathways downstream of CPP1 involved in maintaining hyphal polarity and growth in F. verticillioides.
In Sac. cerevisiae, PP2A-related genes have been identified, and designated PPH21, PPH22, PPH3, SIT4 and PPG1 (Zabrocki et al., 2002). A phylogenic tree containing the well-characterized protein phosphatases in Sac. cerevisiae clearly indicates that Cpp1 has the highest homology with Sit4 (76 % identity, 84 % similarity). Sit4 is a component of the conserved TOR (the target of rapamycin) signalling pathway (Di Como & Arndt, 1996) that interacts with Tap42, the phosphatase regulatory subunit of the TOR pathway, in response to nutrient stress (Cutler et al., 2001). Considering the similarity between Sit4 and Cpp1, we hypothesized that hyphal swelling in PP179 may be caused by the defect in nutritional stress response, and perhaps this process is mediated by F. verticillioides Tap42 homologue (FVEG_06413.3) (Fig. 5a, b). The hyphal swelling phenotype in PP179 was particularly apparent in the later stages of growth, perhaps when the fungus is under nutritional stress. In contrast, the hyphal swelling could be reversed in PP179 by osmotic remediation with sucrose (Fig. 5c). In Sac. cerevisiae, the protein phosphatase catalytic subunit Sit4 is implicated in G1 to S transition (Jiang, 2006), and this leads us to presume that growth and germination defects in PP179 are the consequence of a defect in cell-cycle progression (Fig. 3 and 6b). In Asp. nidulans, an amino acid change in PP2A protein (PphA) led to a growth defect, lack of germ tube, and mitotic defect (Kosmidou et al., 2001). In N. crassa, the transcript levels of ppp-1 were increased during hyphal germination and elongation (Zeke et al., 2003). We also speculate that the germination defect in PP179 may be linked to hyphal swelling. The hyphal polarity defect caused by CPP1 deletion may have initially affected germination, and later led to hyphal swelling.
A report by Yatzkan & Yarden (1999) has suggested a link between PP2A regulatory subunit rgb-1 and macroconidiation in N. crassa: inactivation of rgb-1 leads to a failure in the formation of mature macroconidia. In contrast, we observed that macroconidiation was activated in PP179 (Fig. 6a). Since macroconidia production occurs rarely in F. verticillioides under laboratory conditions (Nelson et al., 1983), it is reasonable to presume that CPP1 plays a role in suppressing macroconidia production in the WT strain. We identified the homologue of rbg-1 in F. verticillioides genome (FVEG_01508.3), and we are currently investigating its role. Moreover, recent characterization of F. verticillioides FvVE1 revealed that deletion of this gene results in macroconidiation and growth defect phenotypes similar to those of PP179 (Li et al., 2006). However, the molecular genetic linkage between CPP1 and FvVE1 has not been determined to date. Taken as a whole, we anticipate that additional proteins, such as other protein phosphatase subunit components and putative Cpp1-interacting proteins, are involved in regulating secondary metabolism and fungal development in F. verticillioides. Identification and functional characterization of these proteins will help us unravel the complex regulatory mechanisms associated with Cpp1.
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ACKNOWLEDGEMENTS |
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Edited by: J.-R. Xu
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Received 9 July 2007;
revised 11 September 2007;
accepted 13 September 2007.
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method, with TUB2 as the endogenous control. Data represent the fold differences in gene expression. Three biological replications were performed to obtain standard deviations. (c) Expression analysis of select PKS genes in the WT and PP179. Total RNA samples were prepared from fungal strains grown in cracked-corn medium for 10 days, and qRT-PCR analysis was performed with SYBR-Green as the fluorescent reporter. The levels of transcription were evaluated using the 
method, with TUB2 as the endogenous control. Data represent the fold differences in gene expression. Three biological replications were performed to obtain standard deviations. 
, WT; 
, PP179.
