Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence
2007; Springer Nature; Volume: 26; Issue: 15 Linguagem: Inglês
10.1038/sj.emboj.7601795
ISSN1460-2075
AutoresRichard A. Wilson, Joanna M. Jenkinson, Robert P. Gibson, Jennifer A. Littlechild, Zheng‐Yi Wang, Nicholas J. Talbot,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle19 July 2007free access Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence Richard A Wilson Richard A Wilson School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Joanna M Jenkinson Joanna M Jenkinson School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Robert P Gibson Robert P Gibson School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Jennifer A Littlechild Jennifer A Littlechild School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Zheng-Yi Wang Zheng-Yi Wang School of Biosciences, University of Exeter, Exeter, UKPresent address: Biotechnology Institute, Zhejiang University, Hangzhou 310029, China Search for more papers by this author Nicholas J Talbot Corresponding Author Nicholas J Talbot School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Richard A Wilson Richard A Wilson School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Joanna M Jenkinson Joanna M Jenkinson School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Robert P Gibson Robert P Gibson School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Jennifer A Littlechild Jennifer A Littlechild School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Zheng-Yi Wang Zheng-Yi Wang School of Biosciences, University of Exeter, Exeter, UKPresent address: Biotechnology Institute, Zhejiang University, Hangzhou 310029, China Search for more papers by this author Nicholas J Talbot Corresponding Author Nicholas J Talbot School of Biosciences, University of Exeter, Exeter, UK Search for more papers by this author Author Information Richard A Wilson1, Joanna M Jenkinson1, Robert P Gibson1, Jennifer A Littlechild1, Zheng-Yi Wang1 and Nicholas J Talbot 1 1School of Biosciences, University of Exeter, Exeter, UK *Corresponding author. School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK. Tel.: +44 1392 264673; Fax: +44 1392 264668; E-mail: [email protected] The EMBO Journal (2007)26:3673-3685https://doi.org/10.1038/sj.emboj.7601795 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Trehalose fulfils a wide variety of functions in cells, acting as a stress protectant, storage carbohydrate and compatible solute. Recent evidence, however, indicates that trehalose metabolism may exert important regulatory roles in the development of multicellular eukaryotes. Here, we show that in the plant pathogenic fungus Magnaporthe grisea trehalose-6-phosphate (T6P) synthase (Tps1) is responsible for regulating the pentose phosphate pathway, intracellular levels of NADPH and fungal virulence. Tps1 integrates glucose-6-phosphate (G6P) metabolism with nitrogen source utilisation, and thereby regulates the activity of nitrate reductase. Activity of Tps1 requires an associated regulator protein Tps3, which is also necessary for pathogenicity. Tps1 controls expression of the nitrogen metabolite repressor gene, NMR1, and is required for expression of virulence-associated genes. Functional analysis of Tps1 indicates that its regulatory functions are associated with binding of G6P, but independent of Tps1 catalytic activity. Taken together, these results demonstrate that Tps1 is a central regulator for integration of carbon and nitrogen metabolism, and plays a pivotal role in the establishment of plant disease. Introduction Trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) is a non-reducing sugar that is among the most widespread disaccharides in living cells, occurring in bacteria, plants, insects, some invertebrates and fungi. Trehalose has been implicated in the cellular response to numerous environmental stresses, such as heat-shock, starvation, hyperosmotic shock and desiccation, but it is becoming increasingly clear that trehalose and its immediate precursor, trehalose-6-phosphate (T6P), also regulate growth and development. The absence of T6P synthase (Tps1) in Arabidopsis thaliana, for example, causes embryo lethality (Eastmond et al, 2002; Gómez et al, 2006), while T6P phosphatase is required for inflorescence development in maize (Satoh-Nagasawa et al, 2006). The sites of action of T6P and trehalose in the regulation of growth and development in multicellular eukaryotes, however, are largely unresolved. Recently, we discovered that Tps1 is essential for establishment of rice blast disease by the filamentous fungus Magnaporthe grisea (Foster et al, 2003). Rice blast is a devastating problem in cultivated rice (for a review see Talbot, 2003) and the fungus infects host plants using specialised infection structures called appressoria, which develop on the rice leaf surface (Veneault-Fourrey et al, 2006). T6P synthase was found to be essential for development of functional appressoria and M. grisea Δtps1 mutants are non-pathogenic (Foster et al, 2003). In addition, Δtps1 mutants sporulated very poorly, suggesting that Tps1 may play a role in two distinct cellular differentiation events in M. grisea—conidiogenesis and appressorium formation (Foster et al, 2003). Loss of Tps1 in yeasts has previously been shown to have severe effects on sugar metabolism. Deletion of the homologous TPS1 gene in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, for instance, prevents their growth in the presence of glucose. This is associated with overactive hexokinase activity, an increased flux of glucose into glycolysis, irreversible and catastrophic depletion of ATP and free inorganic phosphate (Pi), and accumulation of hexose phosphate intermediates (Luyten et al, 1993; Winderickx et al, 1996; Ernandes et al, 1998; Arguelles, 2000; Gancedo and Flores, 2004). In these yeasts, T6P inhibits hexokinase activity and therefore acts as a means of regulating the entry of glucose into glycolysis. Consequently, glucose-containing media are toxic for tps1Δ mutants. In S. cerevisiae tps1Δ mutants, deletion of HXK2, which encodes hexokinase II, remediates the growth defect on glucose, presumably by slowing entry of glucose into glycolysis (Hohmann et al, 1993). Furthermore Tps1, which is part of a multi-enzyme complex with three additional proteins, Tps2, Tps3 and Tsl1 (Reinders et al, 1997; Voit, 2003; Elbein et al, 2003), may act directly on hexokinase, modulating its activity (Bonini et al, 2003). We previously noted that in addition to the developmental phenotypes associated with loss of Tps1 in M. grisea, Δtps1 mutants were also unable to grow on glucose, but unlike the yeast species their growth could be restored by addition of free amino acids to the medium, suggesting that cross-talk between sugar signalling and nitrogen metabolism occurs in the fungus (Foster et al, 2003). In this study, we set out to determine the role of trehalose biosynthesis in plant infection by M. grisea. We decided to explore, in particular, the relationship between the role of Tps1 in sugar metabolism, and its requirement for fungal pathogenicity. Here, we present evidence to show that the apparent inability of M. grisea Δtps1 to grow on glucose as a sole carbon source is not due to glycolytic misregulation, as predicted by studies in yeast, but is instead due to a role for Tps1 in the control of NADPH levels, via regulation of the oxidative pentose phosphate pathway. Furthermore, Tps1 regulates nitrogen utilisation by controlling NMR1 gene expression and nitrate reductase activity. We also establish that the role of Tps1 in fungal pathogenicity results from regulatory functions of the protein and its control of virulence gene expression, rather than its catalytic function. Taken together, our results demonstrate that the Tps1 protein is a central regulator of infection-related processes in a plant pathogenic fungus. Results M. grisea TPS1 restores glycolytic regulation to a S. cerevisiae tps1Δ mutant In order to investigate the biological function of Tps1 in M. grisea, we first investigated the functional relationship between M. grisea Tps1 and S. cerevisiae Tps1, where a role in the regulation of glycolysis has been clearly established (Hohmann et al, 1993). The M. grisea TPS1 gene was expressed in S. cerevisiae tps1Δ∷ura3 mutant MB062 under control of the galactose-inducible GAL1 promoter. Transformants were grown on growth medium containing galactose and then replica plated onto medium with glucose as sole carbon source (Figure 1A). All transformed strains, either with or without a functional TPS1 gene, were able to grow on galactose, but only the wild-type W303 strain and the tps1Δ∷ura3:GAL1(p):MgTPS1:URA3 strain expressing M. grisea Tps1 could grow on glucose, when transferred by replica plating. We conclude that expression of M. grisea TPS1 is sufficient to restore glycolytic regulation to a yeast tps1Δ mutant. Figure 1.Expression of M. grisea TPS1 in S. cerevisiae and analysis of sugar phosphate metabolism. (A) Complementation of S. cerevisiae tps1Δ strain MB062 with MgTPS1. Yeast strains were plated onto minimal media (MM) with galactose as sole carbon, and compared to growth on glucose yeast medium (GMM). (i, ii) tps1Δ∷ura3:GAL1(p):MgTPS1:URA3 (yeast tps1Δ deletion strain transformed with MgTPS1); (iii) tps1Δ∷ura3:GAL1(p): URA3 (yeast tps1Δ deletion strain transformed with empty vector); (iv) W303 (isogenic strain of S. cerevisiae with functional TPS1 gene); (v) tps1Δ∷ura3 (tps1Δ deletion strain MB062). (B) Sugar phosphate accumulation in mycelium of M. grisea Guy-11 and Δtps1. Mycelium was grown in CM for 48 h before transferring to CM (closed bars) or GMM (open bars) for 24 h. G6P (G-6-P) and fructose-6-phosphate (F-6-P) levels were determined by HPAE-PAD using a PA100 column (Dionex). Error bars represent s.d. of three replications of the experiment. Download figure Download PowerPoint Characterisation of the relationship between Tps1 and the regulation of glycolysis in M. grisea S. cerevisiae tps1Δ mutants accumulate early glycolytic sugar phosphate intermediates when grown with glucose as carbon source, which results from stalled glycolysis (Gancedo and Flores, 2004). We therefore carried out high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) to measure sugar phosphate levels in mycelium of a wild-type M. grisea strain Guy-11 and the isogenic Δtps1 mutant (Foster et al, 2003), as shown in Figure 1B. Significantly higher amounts of G6P (P⩽0.01) and fructose-6-phosphate (P⩽0.01) were observed when M. grisea Δtps1 mutants were incubated in glucose minimal medium (GMM), compared to the wild-type strain Guy-11, consistent with misregulation of glycolysis. However, unlike in yeast, fructose-1,6-bisphosphate levels were not elevated in Δtps1 strains (Supplementary Figure 1A, available online). To investigate whether control of glycolysis is altered in Δtps1 mutants in the same way as in yeast (Hohmann et al, 1993), we used bioinformatics to identify the sole hexokinase gene, HXK1, of M. grisea (Dean et al, 2005) and disrupt its function by targeted gene replacement, both in Guy11 and in a Δtps1 mutant background. To study the effect of deleting HXK1 in M. grisea, we grew Guy-11, Δtps1, Δhxk1 and Δtps1 Δhxk1 double mutants in CM for 48 h, followed by growth in GMM, with nitrate as nitrogen source, for 16 h. Hexokinase activity was assayed from all four strains and was reduced in Δhxk1 and Δhxk1 Δtps1 mutants (Figure 2A). Consistent with this, the levels of G6P were reduced four-fold in the Δhxk1 and Δhxk1 Δtps1 mutants compared to Guy-11, and reduced almost 14-fold compared to Δtps1 strains (Figure 2B). The residual enzyme activity observed in our assay (Figure 2A) in the mutants is likely due to G6P production by glucokinase, although this does not appear to play a major role in G6P synthesis in M. grisea (Figure 2B). Trehalose levels were also reduced in Δhxk1 mutants, which may result from reduction in the Tps1 substrate G6P (Figure 2C). The ability of M. grisea Δtps1 Δhxk1 mutants to grow in the presence of glucose was partially restored compared to Δtps1 mutants (Figure 2E), but there was no restoration of sporulation (Figure 2D), or their ability to cause rice blast disease (Figure 2F). Furthermore in S. cerevisiae T6P is a potent inhibitor of HXK2 activity (Gancedo and Flores, 2004), whereas hexokinase activity in a Guy-11 extract is not significantly affected by the presence of T6P (Figure 2G). Interestingly, S. cerevisiae tps1Δ diploids do show a sporulation defect that is not repressible by deletion of Hxk2 (Neves et al, 1995), suggesting that some effects of this mutation are independent of its role in glycolytic regulation. When considered together, our results suggested that, unlike in yeast, M. grisea Δtps1 growth phenotypes cannot be remediated by deletion of HXK1 and that Tps1 may serve distinct functions in sugar metabolism, asexual development and the control of fungal virulence. Figure 2.Characterisation of M. grisea Δhxk1 mutants. (A) Hexokinase activity was measured and compared in Δtps1, Δhxk1, Δtps1 Δhxk1 and Guy-11 strains. Strains were grown in complete medium (CM) for 48 h followed by growth in glucose minimal medium (GMM) for 16 h. (B) G6P levels in the same strains and the same conditions. Bars represent standard error of three independent analyses. (C) Trehalose levels in the same strains and conditions. Bars represent standard error of three independent analyses. (D) Effect of Δhxk1 mutation on sporulation. Δtps1, Δhxk1, Δtps1 Δhxk1 and Guy-11 strains were grown on GMM plates (n=4) for 10 days, followed by harvesting of the spores for counting. Bars represent standard error. (E) Effect of Δhxk1 mutation on vegetative growth. Δtps1, Δhxk1, Δtps1 Δhxk1 and Guy-11 strains were grown on GMM plates for 10 days. (F) Virulence of Δtps1, Δhxk1, Δtps1 Δhxk1 and Guy-11 strains on rice seedlings. Rice seedlings were inoculated at a concentration of 5 × 104 spores/ml. (G) Hexokinase inhibition by 1 mM T6P. Bars represent standard error of three independent analyses. Download figure Download PowerPoint M. grisea tps1 mutants are unable to utilise nitrate as a nitrogen source Previously, it was demonstrated that addition of a rich source of amino acids would restore the ability of Δtps1 mutants to grow in the presence of glucose (Foster et al, 2003). We were intrigued by this observation and therefore grew Δtps1 mutants on both GMM supplemented with single amino acids or alternative nitrogen sources (Supplementary Table 1). Δtps1 mutants could grow well on GMM with single amino acids (except cysteine, see below) and also grew normally on GMM with ammonium (NH4+) or nitrite (NO2−), but not nitrate (NO3−), as sole nitrogen source irrespective of carbon source (Figure 3A). Thus, the original suggestion that Δtps1 mutants cannot grow on glucose (Foster et al, 2003) is actually a conditional response mediated by the nature of the available nitrogen source. Because the metabolism of NO2− differs from that of NO3− by a single enzymatic step, nitrate reductase, we reasoned that M. grisea Δtps1 mutants could be nitrate non-utilising due to the absence, or suppression, of nitrate reductase. To test this idea, we assayed nitrate reductase activity in Δtps1 mutants grown in NO3− and ammonium NH4+-containing medium following a switch from CM. Guy-11 showed increased nitrate reductase activity after growth in NO3− compared to growth on NH4+, while under both growth conditions, Δtps1 mutants showed almost undetectable levels of nitrate reductase activity (Figure 4A). Consistent with this, Δtps1 mutants were resistant to toxic concentrations of potassium chlorate (750 mM) when grown under de-repressing conditions for nitrogen source utilisation (Cove, 1976), confirming the absence of functional nitrate reductase activity in Δtps1 (Figure 3B). Therefore, we conclude that the inability of M. grisea Δtps1 mutants to grow on GMM is not due to glycolytic misregulation, but is instead due to NO3− non-utilisation. Supplementation with individual amino acids, ammonium or nitrite thus restores growth by providing an alternative nitrogen source to the fungus. Figure 3.Nitrogen source utilisation and chlorate resistance of Δtps1 mutants of M. grisea. (A) Guy-11 and Δtps1 mutants were grown on MM containing 1% fructose or glucose as sole carbon sources. Sole nitrogen sources were provided as indicated, at 10 mM concentrations. M. grisea cultures were grown for 10 days at 26°C. (B) M. grisea strains were grown on GMM supplemented with 750 mM potassium chlorate and 3.3 mM NH4+ for 10 days at 26°C. Potassium chlorate is toxic to strains carrying active nitrate reductase enzymes under nitrogen de-repressing conditions (Cove, 1976). Strains were also grown on GMM with 0.1 mg/ml hypoxanthine as sole nitrogen source for 10 days at 26°C. Download figure Download PowerPoint Figure 4.Carbon and nitrogen metabolism is linked via G6P metabolism and NADPH production. (A) Enzyme activities associated with glycolysis, the pentose phosphate pathway and NO3− metabolism from Guy-11 and Δtps1 strains grown in CM for 48 h followed by incubation in GMM with NO3− (closed bars) or NH4+ (open bars) for 16 h. Bars represent standard error of three independent replicates. G-3-PDH is glyceraldehyde-3-phosphate dehydrogenase. G-6-PDH is G6P dehydrogenase. 6-PGDH is 6-phosphogluconic dehydrogenase. (B) Schematic representation of the effect of Δtps1 mutation on carbon and nitrogen metabolism in M. grisea. Differences in metabolite accumulation and enzyme activity between Guy-11 and Δtps1 strains are shown. (C) Transcript analysis of NMR1. Strains were grown for 48 h in CM followed by incubation in GMM with NO3− or NH4+ as a sole nitrogen source for an additional 16 h. Total RNA was extracted and gene expression analysed by RT–PCR. Analysis was performed in triplicate and normalised using levels of actin gene expression. Transcript abundance was calculated relative to NMR1 expression in Guy-11 on GMM with nitrate. Values with a different letter above are significantly different (Student's t-test, P<0.05). (D) Transcript analysis of NIA1, NII1 and β-tubulin encoding gene. Strains were grown for 48 h in CM followed by incubation in GMM with NO3− as a sole nitrogen source for an additional 16 h. Total RNA was extracted and gene expression analysed by RT–PCR. Analysis was performed in triplicate and normalised using levels of actin gene expression. Transcript abundance was calculated relative to gene expression in Guy-11 on GMM with NO3−. Download figure Download PowerPoint The role of NADPH in nitrate utilisation The role of Tps1 in NO3− utilisation was unexpected, and we therefore set out to determine why a trehalose biosynthetic enzyme should be necessary for nitrate reductase activity. In filamentous fungi, NO3− is metabolised to NO2− by the action of nitrate reductase, followed by reduction of NO2− to NH4+ by nitrite reductase (Cove, 1966). Ammonium is assimilated into amino acids through the action of glutamine synthetase and the GOGAT pathway (Kusnan et al, 1987). Nitrate reductase requires NADPH as a cofactor for function, and NADPH is generated from G6P by the action of G6P dehydrogenase (G-6-PDH) in the oxidative pentose phosphate pathway. G6P is, as described previously, also a substrate of Tps1 (Bell et al, 1998). In Aspergillus nidulans, transfer from growth on NH4+ to growth on NO3− leads to increased nitrate reductase and nitrite reductase activities, and also stimulates hexokinase and the enzymes of the pentose phosphate pathway, thereby increasing NADPH production from G6P (Hankinson and Cove, 1974). We therefore assayed the activities of enzymes involved in synthesis of NADPH and catabolism of NO3− in both Guy-11 and Δtps1 mutants (Figure 4A). Incubation in NO3− led to an increase in hexokinase activity in both strains, suggesting that elevated levels of G6P are required by M. grisea under conditions of NO3− utilisation (Dunn-Coleman and Pateman, 1979) and, secondly, that the increase in hexokinase activity does not require Tps1. In addition, other enzyme activities of the glycolytic pathway, such as glyceraldehyde-3-phosphate dehydrogenase (Figure 4A), phosphofructokinase and phosphoglucose isomerase (Supplementary Figure 1B) were not affected by the Δtps1 mutation, while levels of ATP were similar in both Δtps1 and Guy-11 (Figure 4A). These results underline the fact that glycolytic misregulation and ATP depletion does not occur in M. grisea Δtps1 mutants during growth on glucose-containing media. In the wild-type M. grisea strain Guy-11, a large increase in NADPH levels was observed during growth on NO3− (Figure 4A). In contrast, NADPH levels did not increase in Δtps1 during growth on NO3− and there was also a relative decrease in G-6-PDH activity (Figure 4A) despite the fact that high levels of G6P accumulated in the Δtps1 mutant during growth on both nitrogen sources (Supplementary Figure 1C). We did not detect any inhibitory effect of G6P on G-6-PDH activity in vitro (Supplementary Figure 1D). Glutamine synthetase levels were similar in Guy-11 and Δtps1 strains, suggesting that the GS-GOGAT pathway operates normally in the absence of Tps1. The activity of 6-phosphogluconic dehydrogenase, the next enzyme of the pentose phosphate pathway, is also not reduced in Δtps1. From these data we can conclude that regulation of G6PDH and NADPH production, in response to NO3−, is a Tps1-dependent process in M. grisea (Figure 4B). It is worth noting that two NADPH-dependent proteins are required for the metabolism of L-cysteine in eukaryotes. Cysteine oxygenase (EC 1.13.11.20) converts L-cysteine to 3-sulfinoalanine (Lombardini and Singer, 1969) and precedes conversion of 3-sulfinoalanine to alanine, while NADPH-dependant coenzyme A disulfide-glutathione reductase (EC 1.8.1.10; Ondarza et al, 1969) is necessary for the reduction of L-cysteine. Interestingly, the only amino acid that Δtps1 mutants are unable to utilise as a sole nitrogen source is cysteine (Figure 3A), consistent with the observed deficiency in NADPH. Genetic regulation of nitrate utilisation genes via Tps1 The absence of nitrate reductase activity in Δtps1 mutants during growth on NO3− could be due either to an inactive enzyme or to repression of the NIA1 gene, which encodes nitrate reductase. Adding exogenous NADPH to the nitrate reductase activity enzymatic assay did not result in detectable nitrate reductase activity in Δtps1 (Figure 4A), suggesting that there is no nitrate reductase protein present in the cell. We also noted that Δtps1 mutants cannot utilise hypoxanthine as a nitrogen source (Figure 3B). Nitrate reductase and xanthine dehydrogenase (used to metabolise hypoxanthine) both require a molybdenum-containing cofactor, encoded in A. nidulans by the cnx genes (Unkles et al, 1999). Expression of cnx and niaD genes requires a positively acting GATA transcription factor encoded by the areA gene in A. nidulans (Wilson and Arst, 1998) and the NUT1 gene in M. grisea (Froeliger and Carpenter, 1996). The activity of AreA is negatively regulated by the product of nmrA (Andrianopoulos et al, 1998). We performed RT–PCR to analyse the gene expression of NMR1, the equivalent M. grisea gene, in Guy11 and Δtps1 during growth on nitrate and ammonium (Figure 4C). Expression of NMR1 was significantly higher in Δtps1 mutants than in Guy-11 during growth on nitrate, and was comparable to expression levels observed during growth of Guy-11 and Δtps1 on ammonium when it fully represses the action of the AreA GATA factor (Andrianopoulos et al, 1998). Furthermore, high levels of NMR1 transcript in Δtps1 strains corresponded with reduced expression of NIA1 and NII1, encoding nitrate and nitrite reductase respectively, compared to Guy-11 (Figure 4D). In A. nidulans, nitrate and nitrite reductase genes are tightly linked and coordinately regulated from a shared promoter (Cove, 1976). However, M. grisea NIA1 and NII1 genes are not linked and reside on chromosomes III and VI, respectively (Dean et al, 2005). We wondered if, considering Δtps1 strains could grow on NO2− but not NO3− media, there was evidence for differential regulation of nitrate and nitrite reductase activities in M. grisea. Interestingly, unlike in A. nidulans, we were unable to detect nitrate reductase activity in protein extracts of Guy-11 and Δtps1 strains grown in CM followed by a switch to NO2−-containing media (not shown). In addition, NADPH levels of Guy-11 and Δtps1 strains were not elevated during growth on NO2− compared to NH4+ (Supplementary Figure 2). This suggests that nitrate and nitrite reductase activities are not always coordinately regulated, and second that NADPH may not be important for nitrite reductase activity. Studies of Neurospora crassa nitrite reductase (Nason et al, 1954) demonstrated that the enzyme can utilise both NADH and NADPH as cofactor in vitro, and the preferred cofactor for M. grisea NII1 therefore needs to be determined. We conclude that in response to NO3−, Tps1 is required to regulate the activity of G6PDH, which controls levels of G6P, NADPH and nitrate reductase activity, and also to modulate gene expression by control of NMR1. Genetic regulation of Tps1 activity in M. grisea by Tps3 The catalytic subunit of Tps1, encoded by TPS1, is part of a multi-protein complex in yeast, with the Tps2 T6P phosphohydrolase and two other proteins, Tps3 and Tsl1 (Gancedo and Flores, 2004). To investigate the control of M. grisea Tps1 activity, we analysed the genome sequence of M. grisea (Dean et al, 2005), identified a homologue of TPS3 and generated a Δtps3 mutant. The Δtps3 mutant was impaired in both trehalose (see Figure 6D) and T6P synthesis. HPAE-PAD analysis of mycelial extracts showed concentrations of 99.8±8 pmol per milligram T6P mycelium (n=3) in Guy-11, but no detectable T6P in mycelium of Δtps3 mutants (not shown). The Δtps3 mutant grew better than Δtps1 on NO3−, but was still reduced in sporulation (Figure 5). NADPH was also reduced and showed no increase during growth of Δtps3 mutants on NO3−-containing medium, consistent with the Δtps1 phenotype. Interestingly, however, nitrate reductase activity in Δtps3 mutants was equivalent to that in Guy-11 during growth on NO3− and NH4+ (Figure 5B). Nitrate reductase activity is assayed by addition of exogenous NADPH, so while this result indicates that nitrate reductase is present in Δtps3 mutants, it is unlikely to be active in the cell due to the lack of endogenous cellular NADPH (Figure 5C). Δtps3 mutants are, for example, resistant to potassium chlorate under nitrogen de-repressing conditions (Figure 5A). In addition, Δtps3 does not accumulate G6P when grown on NO3− (Supplementary Figure 1C) or in CM (Supplementary Figure 1E). Taken together, our results indicate that Tps1 and Tps3 are both necessary for T6P synthesis and regulation of the pentose phosphate pathway and NADPH production, while at least Tps1 is also necessary for control of NMR1 expression. Significantly, Δtps3 mutants are also unable to cause rice blast disease (Figure 5D), demonstrating that fungal pathogenicity requires Tps3-dependent Tps1 activity. Figure 5.Genetic control of Tps1 activity by Tps3. (A) Guy-11, Δtps1 and Δtps3 strains were grown on GMM and GMM supplemented with 500 mM potassium chlorate and de-repressing concentrations (3.3 mM) of NH4+. (B) Nitrate reductase activity in Guy-11, Δtps1 and Δtps3 strains following 48 h growth in CM and 16 h growth in GMM with NO3− or NH4+. Activities and concentrations were measured in triplicate. Bars represent standard error. (C) NADPH concentration in Guy-11, Δtps1 and Δtps3 strains following 48 h growth in CM and 16 h growth in GMM. Activities and concentrations were measured in triplicate. Bars represent standard error. (D) Guy-11, Δtps1 and Δtps3 strains were inoculated onto rice seedlings to analyse pathogenicity. Spores were inoculated at a rate of 5 × 104 spores/ml. Download figure Download PowerPoint Figure 6.Site-directed mutagenesis of the G6P-binding pocket of Tps1. (A) Energy-minimised Tps1 homology model structure constructed from OtsA template structures (Gibson et al, 2002) by MODELLER, and showing residues involved in G6P binding. Residues, which were mutated are (clockwise from top) Trp108, Asp153, Tyr99 and Arg22. The locations of G6P (green and red) and UDP-glucose (blue) are based on the template structures to indicate their proximity to the mutated residues. (B) Schematic representation of the relative positions of introduced amino-acid substitutions in the G6P-binding pocket of Tps1. The drawing is not to scale. (C) Spore production was measured for Guy-11, Δtps1∷TPS1, Δtps1, Δtps3, Δtps1∷Tps1R22G, Δtps1∷Tps1Y99V, Δtps1∷Tps1W108S and Δtps1∷Tps1D153G strains. Spores from strains grown for 10 days on CM and GMM were counted from four independent plates. (D) Trehalose concentration in Guy-11, Δtps1∷TPS1, Δtps1, Δtps3, Δtps1∷Tps1R22G, Δtps1∷Tps1Y99V, Δtps1∷Tps1W108S and Δtps1∷Tps1D153G strains (left bar chart) and Guy-11, Δtps1, Δtps1∷otsA and Δtps1∷ScTPS1 (right bar chart). Trehalose was measured in triplicate. Bars represent standard error. (E) The virulence of Δtps1∷TPS1, Δtps1, Δtps1∷Tps1R22G, Δtps1∷Tps1Y99V, Δtps1∷Tps1W108S and Δtps1∷Tps1D153G, Δtps1∷otsA and Δtps1∷ScTPS1 strains was analysed by inoculating spores onto rice seedlings o
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