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Title of article: 1
The Ustilago maydis Nit2 homolog regulates nitrogen utilization and is 2
required for efficient induction of filamentous growth 3
4
Running title: Ustilago maydis Ncr1 controls pathogenicity 5
6
Authors: 7
Robin J. Horst1, Christine Zeh, Alexandra Saur, Sophia Sonnewald, Uwe 8
Sonnewald and Lars M. Voll* 9
10
Division of Biochemistry, Friedrich-Alexander-University Erlangen-Nuremberg, 11
Staudtstr. 5, D-91058 Erlangen, Germany 12
13 *Correspondence: 14
Lars M. Voll 15
Friedrich-Alexander University Erlangen-Nuremberg 16
Division of Biochemistry 17
Staudtstraße 5 18
D-91058 Erlangen 19
Germany 20
Tel.: + 49 (0)9131 852 52 38 21
Fax.: +49 (0)9131 852 82 54 22
Email: [email protected] 23
24 1present address: 25
Department of Biology, University of Washington, Seattle, WA 98195, USA 26
27
28
29
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.05191-11 EC Accepts, published online ahead of print on 13 January 2012
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Summary: 30
Nitrogen catabolite repression (NCR) is a regulatory strategy found in 31
microorganisms that restricts the utilization of complex and unfavored nitrogen 32
sources in the presence of favored nitrogen sources. In fungi, this concept has 33
been best studied in yeasts and filamentous ascomycetes, where the GATA 34
transcription factors Gln3p and Gat1p, and Nit2 / AreA, respectively, constitute 35
the main positive regulators of NCR. It has remained elusive, why functional Nit2 36
homologs of some phytopathogenic fungi are required for full virulence on their 37
hosts. We have identified the Nit2 homolog in the basidiomyceteous 38
phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, 39
positive regulator of nitrogen utilization. By transcriptome analysis of sporidia 40
grown on artificial media devoid of favored nitrogen sources, we show that only a 41
subset of nitrogen responsive genes is regulated by Nit2, including the Gal4-like 42
transcription factor Ton1 (target of Nit2). Ustilagic acid biosynthesis is not under 43
the control of Nit2, while nitrogen starvation-induced filamentous growth is largely 44
dependent on functional Nit2. nit2 deletion mutants show delayed initiation of 45
filamentous growth on maize leaves and exhibit strongly compromised virulence, 46
demonstrating that Nit2 is required to efficiently initiate the pathogenicity program 47
of U. maydis. 48
49
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Introduction 51
52 Heterotrophic microorganisms are capable of utilizing a plethora of different 53
carbon and nitrogen sources. Fungal saprophytes and bacteria strictly rely on the 54
availability of (certain) organic carbon sources, while most have the ability to 55
assimilate inorganic nitrogen in addition to utilizing organic nitrogen. The 56
utilization of available nutrients by microorganisms is not unbiased, since carbon 57
and nitrogen compounds that are easy to metabolize are preferred over more 58
complex compounds that require a higher cost in terms of ATP and reducing 59
equivalents (50). 60
Although the actual mechanisms or regulatory components of the utilization of 61
complex nitrogen sources differ considerably in different classes of bacteria, the 62
output of these signal transduction cascades is the same: metabolization of 63
complex nitrogen sources in the presence of favored nitrogen source is 64
repressed (reviewed in (2, 50)). In eukaryotic organisms, this effect, called 65
nitrogen catabolite repression (NCR) or nitrogen metabolite repression has been 66
best characterized in brewer’s yeast (Saccharomyces cereviseae) and in 67
filamentous fungi of the phylum ascomycota, where extensive work has been 68
performed on Neurospora crassa and Aspergillus sp. (87). 69
In filamentous fungi, the availability of favored nitrogen sources (ammonium and 70
Gln in A. nidulans; ammonium, Gln and Glu in N. crassa) represses the utilization 71
of other nitrogen sources, such as nitrate, peptides or other free amino acids 72
(57). The GATA transcription factors AreA and Nit2 are the central regulators of 73
NCR and control the utilization of unfavored nitrogen sources in A. nidulans and 74
N. crassa, respectively (reviewed in (57, 58)). In the absence of favored nitrogen 75
sources, Nit2 / AreA activates the expression of nitrogen metabolizing genes by 76
binding to the respective promoters of these genes. Functional Nit2 proteins from 77
ascomycetes harbor a highly conserved Zn finger domain in the C-terminal part 78
of the protein as well as a Nit2 / AreA specific sequence motif RMENLTWRMM in 79
the N-terminal part. The outmost C terminus is conserved as well (17, 87). 80
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Deletion of the nit2 / areA gene leads to the inability of filamentous fungi to utilize 81
complex nitrogen sources (17, 47, 57, 66, 82). While Nit2 / AreA are required for 82
the general de-repression of nitrogen metabolizing genes, additional pathway-83
specific activators are needed for the activation of most genes underlying NCR. 84
For nitrate utilization, this activator is called Nit4 / NirA in N. crassa / A. nidulans, 85
respectively, a Gal4-like transcription factor, which can only induce the 86
expression of nitrate and nitrite reductase in the presence of Nit2 / AreA (22). The 87
regulation of AreA activity is best described in A. nidulans. Under N sufficient 88
conditions, AreA is evenly distributed between cytoplasm and nucleus and only 89
strongly localizes to the nucleus under N starvation (80). Furthermore, AreA 90
activity is regulated by mRNA stability (12, 62, 63), a positive autoregulation loop 91
(49) and the negatively acting protein NmrA (3). No DNA-binding activity has 92
been demonstrated for the N. crassa hololog Nmr1 (89), but Nmr1 interacts 93
directly with Nit2 and thus inhibits DNA binding of Nit2 (65, 88). The conserved 94
outmost C terminus of Nit2 is required for the interaction with Nmr1 (65). 95
In yeasts, the utilization of complex nitrogen sources is mediated by two 96
positively acting transcription factors: Gat1p, a closely related homolog of Nit2 / 97
AreA and Gln3p, a GATA-type transcription factor that is not present in 98
filamentous fungi (reviewed in (15, 33, 87)). These two proteins act on a different 99
subset of genes but also share commonly regulated genes. 100
The role of functional Nit2 homologs has been studied in plant pathogenic 101
ascomycetes, where it also regulates nitrogen utilization and is required for full 102
pathogenicity in most cases (9). Nit2 loss of function mutants of Colletotrichum 103
lindemuthianum, Fusarium verticillioides and Fusarium oxysporum showed 104
reduced virulence on their respective hosts (17, 47, 66). The hemibiotrophic C. 105
lindemuthianum clnr1 mutants were specifically affected in the transition from the 106
biotrophic to the necrotrophic phase, while they showed no defects in the initial 107
biotrophic phase itself (66). A depletion of Nit2 homologs in Magnaporthe grisea 108
or in Cladosporium fulvum on the other hand had little or no effect on 109
pathogenicity (23, 67). The reason why Nit2 homologs are required for full 110
pathogenicity in some ascomycetous plant pathogens remains elusive. One 111
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common hypothesis is that plant pathogens are limited in nitrogen supply early in 112
the infection process and that this limitation is an essential signal to start the 113
infection cycle (74). This assumption is based on the observation that expression 114
of the C. fulvum effector Avr9 and the M. grisea pathogenicity factor Mpg1 as well 115
as four other known M. grisea pathogenicity factors are induced in axenic culture 116
under N limitation (19, 77, 83). Expression of Avr9 is thereby under the control of 117
the Nit2 homolog Nrf1 (67). However, only one out of nine known C. fulvum 118
effectors, Avr9, and only five out of 21 known M. grisea pathogenicity factors are 119
induced by N limitation (19, 76, 77, 83). This implies on the one hand that cues 120
other than N limitation play a role in starting the pathogenicity program and on 121
the other hand that the reduced virulence observed in some Nit2 mutants is not 122
solely an effect of reduced effector protein expression. 123
Although the regulation of nitrogen utilization has been well studied in 124
ascomycetes and yeasts, only few reports exist on these regulatory mechanisms 125
in basidiomycete fungi. It is known that nitrate-metabolizing enzymes are 126
transcriptionally repressed by ammonium in Hebeloma cylindrosporum and 127
Ustilago maydis, but the mechanism behind this effect is unknown (4, 38, 39, 51). 128
Gat1, the Nit2 homolog of the basidiomycete human pathogen Cryptococcus 129
neoformans regulates nitrogen utilization and glucuronoxylomannan secretion, 130
but deletion of Gat1 has no effect on virulence (48). 131
The basidiomycete Ustilago maydis is the causal agent of corn smut disease and 132
its dimorphic life style has been extensively characterized on a molecular level 133
(see (11, 42) for excellent recent reviews on U. maydis). Haploid sporidia multiply 134
by budding in a yeast-like manner and exhibit a saprophytic life style. After 135
mating on hydrophobic surfaces, growth of infectious filamentous dikaryotic 136
hyphae is initiated and U. maydis can infect its host, maize, via appressoria-like 137
structures, and later proliferates by intracellular and intercellular hyphae, inducing 138
the formation of host-derived tumors. Since U. maydis shows a biotrophic lifestyle 139
in planta, it does not kill its host, but has to continuously acquire nutrients from 140
living host tissue. We reported previously that the induction of tumors provides 141
efficient means of rerouting organic nitrogen sources and soluble carbohydrates 142
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to the infection site at late infection stages (18, 36, 37). The nutritional situation in 143
the early infection stages, however, remains unknown. Although there is no 144
report of induced expression of potential effectors under N starvation in U. 145
maydis, it has been shown that N limitation or low ammonium concentrations can 146
induce the switch to filamentous growth of haploid cells on artificial media (46, 147
73), an effect similar to invasive growth of S. cereviseae under nitrogen 148
starvation (27). It was also shown that N starvation-induced formation of 149
conjugation tubes is dependent on compatible a mating loci in U. maydis, 150
whereas formation of true filaments is dependent on active a and b loci (5). 151
Here we report the identification of a functional Nit2 homolog in U. maydis, which 152
controls nitrogen utilization, and we show that it is required for efficient initiation 153
of filamentous growth and for full virulence on maize leaves. 154
155
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Materials and methods 156
157
Strains and culture conditions 158
Fungal strains used in this study are given in Table 1. U. maydis sporidia were 159
propagated at 28°C on potato dextrose plates or in liquid YEPSlight medium (81) if 160
not specified otherwise. For the determination of nitrogen utilization, minimal 161
medium (MM) plates (35) were supplemented with 2% (w/v) glucose and either 162
0.3% (w/v) KNO3 (NMM) or (NH4)2SO4 (AMM), or 10 mM of the respective 163
nitrogen source. Spermine and spermidine were provided at 2 mM, guanine at 5 164
mM concentration, for nitrogen starvation medium (-N), no nitrogen source was 165
added. For liquid medium, agar was omitted. Resistance to chlorate was tested 166
on MM plates supplemented with 10 mM proline and 250 mM KClO3. For 167
transcript and enzyme activity analyses, sporidia were grown in liquid ammonium 168
MM to an OD600 ~1.0, harvested by centrifugation, washed 2 times in H2O and 169
aliquots were transferred to either liquid AMM, NMM or –N medium. Cells were 170
harvested by centrifugation at different time points after transfer to fresh medium 171
as indicated in the text and immediately snap frozen in liquid nitrogen. 172
173
Infection assay and assessment of pathogenicity 174
Plant infection was conducted as previsously described (26). In brief, a 175
suspension of fungal sporidia (OD600=1) or the same volume of water for mock 176
controls was injected with a syringe into the stems of 7 to 10 day old maize 177
seedlings cv. Early Golden Bantam, which resulted in local infections of leaf 3 178
to 5. Infection symptoms were documented 8, 10, 14 and 20 days post infection. 179
Pathogenicity of the indicated U. maydis strains was assessed by generating a 180
disease index at the indicated time points by classifying symptom severity 181
according to predefined categories as described in (21). 182
183
Construction of U. maydis transgenics 184
U. maydis transgenics were generated in the solopathogenic strain SG200 (45), 185
with the exception of AB31-Δnit2 being in the AB31 background (10), using a 186
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PCR-based technique (44),. A 1 kb flanking region up- (LB) and downstream 187
(RB) of the nit2 gene was amplified using primers 5’-188
TTTCAGCCTTGTCTTCTGTGC-3’ and 5’-189
CACGGCCTGAGTGGCCGAAAGAATCGCAAGACGGAG-3’ (LB for both 190
constructs), and 5’-GTGGGCCATCTAGGCCTTCGGGGTCCGCATCTTC-3’ and 191
5’-GCAACAAAGCCGTAATATCACC-3’ (RB ΔN-nit2) or 5’-192
GTGGGCCATCTAGGCCGGTTTTGTTTAGTGTACCGTCTTTCT-3’ and 5’-193
TCCATCCATGCCAGAATCTCG-3’ (RB Δnit2), respectively. PCR products were 194
digested with SfiI and ligated to the hygromycin resistance cassette of pBS-hhn 195
(44). Primers 5’-TTCGTGAATGGAAGACGTGAGG-3’ and 5’-196
CACGGCCTGAGTGGCCGAAGACGGTGGCAAGATGGAC-3’ (LB) and 5’-197
GTGGGCCATCTAGGCCATCGCTTTCGCTGTTGGTCTCG-3’ and 5’-198
TGCCTGCTGGTGCGGATTGTC-3’ (RB) were used to generate the Δton1 199
construct. For the nit2 complementation assay, a genomic fragment including the 200
Nit2 gene and 1 kb upstream of the start codon was amplified by PCR using 201
primers 5’-CACAAGCTTCGCTCGGTCCTCCTACTCGT-3’ and 5’-202
CACGCGGCCGCACAAAACCTCACTTGCGCTGTC-3’, cloned into the HindIII 203
and NotI sites of p123 (75), a vector that integrates into the ip locus of U. maydis 204
and confers resistance to carboxin. Transformation of U. maydis was performed 205
as previously described (72), selection of transformants occurred on hygromycin 206
or carboxin. 207
208
Sequence analysis 209
The N. crassa Nit2 protein sequence (P19212) was used in a BLASTP search (1) 210
to identify Um10417 as a sequence homolog. Domains were identified using the 211
conserved domains database (56). Protein alignments were performed using the 212
MUSCLE algorithm (20) and distance trees were visualized with the Geneious 213
5.0.4 software (Biomatters Ltd, New Zealand). The following accessions were 214
used: A. nidulans XP_681936, G. fujikuroi P78688, F. oxysporum f.sp. lycopersici 215
ABD60578, C. lindemuthianum AAN65464, M. oryzae XP_366679, S. commune 216
BAA96108, C. neoformans XP_566547, N. crassa P19212.2. 217
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218
Transcript analyses 219
Total RNA from sporidia grown on the indicated media for the given times after 220
transfer from ammonium minimal medium (AMM) was extracted using the 221
RNase-all method (14) and RNA quality was assessed after separation on 222
formaldehyde gels or using an Agilent 2100 Bioanalyzer. For RNA blot analysis, 223
standard molecular techniques were used. Ten µg total RNA were loaded, 224
separated and probed with 32P-labeled, gene-specific probes. nar1 and nrt 225
probes were amplified using primers 5’-TTCCTCGATCCCAAGAAATG-3’ and 5’-226
CTGGATGGGGTCTAAAGCAG-3’, and 5’-CGCACTGCCGACAACGAAA-3’ and 227
5’-GCAGGGATACCGTAGTTGGCA-3’, respectively. Tomato 18S rRNA was 228
used as a loading control. 229
Whole genome transcript analyses with 400 ng total RNA extracted from sporidia 230
cultivated in the indicated media for the given times after transfer from 231
ammonium minimal medium (AMM) were performed using custom U. maydis 232
8x15K microarrays (Agilent, Santa Clara). Annotated genes were downloaded 233
from the MUMDB homepage (http://mips.helmholtz-234
muenchen.de/genre/proj/ustilago/) and specific probes were designed using the 235
eArray software (www.chem.agilent.com/en-236
US/products/instruments/dnamicroarrays/pages/gp50660.aspx). Sample labeling, 237
chip hybridization and scanning, and feature extraction was performed with the 238
One-color spike mix (Agilent) according to the manufactures protocol. Data was 239
analyzed using the GeneSpring XI software with standard settings. Statistically 240
significantly deregulated genes (> 2-fold change in comparison to respective 241
control and p < 0.05 in a t-test, with unequal variance after Benjamini-Hochberg 242
correction) were identified with the volcano plot option of GeneSpring XI. For the 243
comparisons of conditions and genotypes as indicated in the respective text 244
captions. The microarray data presented in this study are deposited at GEO 245
under the accession number GSE28916 (http://www.ncbi.nlm.nih.gov/geo; 246
reviewer access information and password will be provided independently). 247
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The expression pattern of selected genes was verified by quantitative RT-PCR 248
using the Brilliant II SYBR® Green QPCR Master Mix and Mx3000P qPCR 249
system (Stratagene). Gene expression from three biological replicates per 250
condition tested was normalized against U. maydis gapDH. The following primers 251
were used: gapDH 5’-CTCAGGTCAACATCGGTATCAACG-3’ and 5’-252
CCGTGGGTGGAGTCGTACTTG-3’; um01756.2 5’-253
CGGTTGCCCTGCTAAGTACG-3’ and 5’-TGCCAATGAGGAAAGCACCT-3’; 254
um03690 5’-CGCTTCCACTTTGATCTGGTG-3’ and 5’-255
AAAGAGAGGGCGATCGAGATG-3’; um10005 5’-256
ACAGCAAGACTGGCAGGATGC-3’ and 5’-CGGAAGTGAGCGAGCTGAGAG-3’; 257
um04577 5’-GACCAGAGACCGAGGACCACT-3’ and 5’-258
TCAAACGCCGTCTTTTCTTCA-3’; um03847 5’-GACCTGAGCCGTGGTTTGTC-259
3’ and 5’-CATCGAGTGGATTGCCATGA-3’; um11104 5’-260
CTCAGATCGCCGTTTGGAAC-3’ and 5’-CGACCCTTGTCGTCTTCACC-3’; 261
um11105 5’-CTTCTGGCTGCTCGTCACCT-3’ and 5’-262
CGAGGAAACCAGTCGTACCG-3’; um05889 5’-263
GCCTACGTCATCTTGGGAACC-3’ and 5’-TCCCAGAACATCCAGGTGAGA-3’; 264
um02625 5’-CTGCATCCTGCTGTGGTTTTC-3’ and 5’-265
GCCCGACTCCAATACCAGTTC-3’; um00477 5’-266
GTCTGACGTCCGGTGGAATC-3’ and 5’-GCGCTGTATACTCGCCATCC-3’; 267
um04060 5’-CCTGAATCTGGCCAAAGACG-3’ and 5’-268
CGAAAGACCTCGGAACAACG-3’; um06253 5’-269
TTTTTGACCGATTGGATGCAC-3’ and 5’-GCTTGACGAGCTCCCAAAGAT-3’. 270
271
Nitrate reductase activity assay 272
The maximal extractable activity of nitrate reductase in sporidia from the 273
logarithmic growth phase was assayed as described in (25). 274
275
Filamentation assay 276
Filamentation on a hydrophobic surface was performed as described (60). 277
Sporidia were either sprayed in water or in a 1 mM Gln solution on parafilm. For 278
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strains in the AB31 background, sporidia were cultivated to an OD600 of 0.3 in 279
AM-Glc, washed once in distilled water and transferred to an equal volume of 280
either NM-Glc (to relieve NCR with no concomitant induction of the b-locus) or 281
NM-Ara (to relieve NCR and to induce the b-locus concomitantly). After 1h of 282
agitation, sporidia were pelleted for 8 min at 2.500 x g, washed once in distilled 283
water and were finally sprayed onto parafilm at an OD600=0.1. Filamentous 284
growth was evaluated after 16 h and 21 h at 28°C and 100% relative humidity 285
using a Leica DMR fluorescence microscope with DIC optics. Filamentation on 286
planta was assessed 18 h after infection. To visualize fungal material, infected 287
leaf segments were stained 1 min with calcofluor (fluorescence brightener 28, 288
Sigma-Aldrich) and rinsed briefly with ddH2O. Stained samples were immediately 289
examined under a fluorescence microscope using a UV lamp and Leica filter 290
cube A. 291
292
293
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Results 295
296 Ustilago maydis Um10417 is homologous to Nit2 / AreA 297
Functional Nit2 / AreA proteins from ascomycete fungi contain a highly conserved 298
GATA Cys2/Cys2-Zn-finger domain which is followed by an adjacent basic region 299
which is also conserved (70). The U. maydis genome harbors 10 annotated 300
proteins with a GATA Zn-finger domain, of which only URBS1, which is a 301
regulator of iron-siderophore biosynthesis, has been characterized (21, 84). A 302
BLAST analysis (1) using N. crassa Nit2 and A. nidulans AreA as a query 303
showed that Um10417 was the U. maydis GATA transcription factor with highest 304
sequence homology to both Nit2 and AreA. Multiple sequence alignment with 305
functionally characterized regulators of NCR (nitrogen catabolite repression) from 306
ascomycete fungi and with Nit2 orthologs from basidiomycetes revealed that 307
besides the highly conserved, C-terminal zinc finger domain, Um10417 contains 308
a DUF1752 domain that is also conserved among functional Nit2 / AreA proteins 309
(Fig. 1). Nit2 / AreA from ascomycete fungi also harbor a conserved sequence 310
motif at the outmost C-terminus that has been shown to participate in the direct 311
interaction between N. crassa Nit2 and its negative regulator Nmr1 (65). This 312
extension is missing in Um10417 and also in the Nit2 orthologs of the 313
basidiomycetes Cryptococcus neoformans and Schizophyllum commune (Fig. 314
1D). Construction of a phylogenetic tree from the full protein alignment 315
(supplementary Fig. S1) shows that basidiomycete nit2 orthologs diverged from 316
their ascomycete homologs (Fig. 1A). This raises the question, whether the U. 317
maydis Nit2 serves a similar function as the ascomyceteous orthologs in vivo. 318
319
Ustilago maydis Nit2 regulates utilization of unfavorable nitrogen sources 320
The utilization of most non-favorable nitrogen sources is repressed in the 321
presence of favorable nitrogen sources in fungi. Nit2 / AreA proteins are 322
necessary to release this repression in the absence of favorable nitrogen 323
sources. To ascertain whether Um10417 regulates the utilization of unfavorable 324
nitrogen sources like the Nit2 homologs in ascomycetes, we constructed two 325
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independent disruptions of Um10417 in the solopathogenic U. maydis strain 326
SG200 (45) using a targeted gene replacement strategy as described (44). As 327
the 3’ end of the um10417 gene is only 450 bp away from the start codon of the 328
next downstream gene (um10416), we constructed two different knockout strains. 329
The resulting strain SG200-Δum10417 was deleted for the whole predicted CDS 330
of um10417, also potentially affecting the promoter region of um10416, while in 331
strain SG200-ΔN-um10417, the N-terminal 1158 aa were deleted, leaving the 332
potential promoter sequence of the downstream gene intact (Fig. 2). Both strains 333
showed a single integration of the hygromycin resistance cassette at the 334
um10417 locus as assessed by insert-specific PCR and DNA blot analysis (data 335
not shown). SG200-Δum10417 completely failed to accumulate um10417 336
transcript, as suggested by RT-PCR targeting different regions of the predicted 337
full-length cDNA. In contrast, the um10417 3’-end, but not the 5’-end could be 338
amplified from SG200-ΔN-um10417 cDNA (Fig. 2), which suggests the 339
accumulation of a severely truncated 3’ portion of the um10417 transcript in the 340
latter strain. 341
Ustilago maydis has the genetic setup to synthesize every proteinogenic amino 342
acid (59) and is capable of utilizing a plethora of nitrogen sources, e.g. inorganic 343
N like nitrate and ammonium as well as organic nitrogen sources such as 344
biogenic amino acids and nucleobases (34). We assessed the role of um10417 in 345
nitrogen utilization by cultivating SG200, SG200-Δum10417 and SG200-ΔN-346
um10417 sporidia in the presence of different inorganic and organic nitrogen 347
sources. Neither mutant strain showed a growth phenotype when cultured on 348
complex media and grew as well as SG200 on minimal medium supplemented 349
with ammonium as the sole nitrogen source (Fig. 3A). When grown on nitrate 350
minimal medium, however, both mutant strains were unable to grow (Fig. 3B). 351
Growth of the SG200-Δum10417 deletion strain was also assessed on minimal 352
medium supplemented with organic nitrogen sources. While SG200 grew on all 353
nitrogen sources tested except spermine, SG200-Δum10417 sporidia exhibited a 354
similar growth as SG200 only when Gln, Glu, Asn or putrescine were provided. 355
Growth on Tyr was slightly retarded compared to SG200 and no growth of the 356
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Δum10417 strain was observed on all other nitrogen sources tested (Table 2). All 357
defects in the utilization of non-favorable nitrogen sources could be 358
complemented in Δum10417 sporidia by transformation with a wild type um10417 359
copy (Table 2, right column). These results suggest that Um10417 is indeed a 360
functional homolog of Nit2 / AreA, and will subsequently be termed Nit2. 361
In general, Nit2 / AreA-deficient ascomycetes were unable to induce nitrate 362
reductase (NR) activity in the absence of favorable nitrogen sources (58). To test, 363
whether U. maydis SG200-Δnit2 strains also lacked inducibility of NR activity, 364
sporidia were grown on chlorate plates supplemented with proline, a non-365
repressing nitrogen source (Table 2). While SG200 showed only slight growth, 366
quickly followed by death due to the NR-catalyzed reduction of chlorate to toxic 367
chlorite, the two nit2 deletion strains were viable on chlorate plates, but showed 368
strongly reduced growth compared to SG200 grown on proline, due to the 369
inability to efficiently use this nitrogen source. 370
Nitrate reductase activity was completely repressed in SG200 sporidia grown in 371
liquid culture with ammonium, but induced by 2 h after transfer from ammonium 372
to nitrate minimal medium or to minimal medium without nitrogen source (Table 373
3). Δnit2 sporidia failed to induce NR activity upon transfer to non-repressive 374
media, which explains their resistance towards chlorate. 375
376
Transcriptional regulation of nitrate assimilation by Nit2 377
Nitrate reductase expression is induced at the transcriptional level by Nit2 / AreA 378
in filamentous fungi in the absence of favorable nitrogen sources only in the 379
presence of nitrate (24). Furthermore, NR has been shown to be subject to 380
transcriptional regulation in U. maydis (4) and induction of the nitrate reductase 381
promoter by nitrate was demonstrated to be tightly controlled (10). Nevertheless, 382
the underlying regulatory mechanisms have remained elusive. The genes coding 383
for nitrate reductase (nar1), nitrite reductase (nir1) and a putative nitrate 384
transporter (nrt) are located in one gene cluster in U. maydis (59), suggesting 385
their coordinated regulation. Thus, the transcriptional response of nar1 and nrt in 386
SG200 and Nit2-deficient SG200 was assessed in response to different nitrogen 387
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regimes by RNA blot analysis. To this end, sporidia were cultured in medium with 388
ammonium as sole nitrogen source and transferred to medium containing 389
ammonium, ammonium plus nitrate, nitrate only or no nitrogen source at all. 390
Samples were taken at different time points after transfer. 391
With ammonium as the sole nitrogen source or with both ammonium and nitrate 392
in the medium, no nar1 and nrt transcripts were detectable in SG200 or the nit2 393
deletion strain, showing that the presence of a favorable nitrogen source 394
completely inhibits the induction of the nitrate assimilation pathway on the 395
transcriptional level (Fig. 4). Upon transfer to nitrate medium, SG200 sporidia 396
showed a strong induction of nar1 and nrt, reaching peak transcript levels 2 h 397
after transfer. Transcript abundance of both genes decreased up to 6 h after 398
transfer but increased again after 24 to 48 h. The similar transcript accumulation 399
patterns of nar1 and nrt in response to changing nitrogen availability further 400
suggest that these two nitrate assimilatory genes are regulated in concert. 401
Although deletion of nit2 completely inhibited the activity of NR on nitrate 402
containing medium (Table 3), transcripts of nar1 and nrt were still detectable in 403
the Nit2-deficient strain, although to a much lesser extent than in SG200 (Fig. 4). 404
The induction kinetics also differed in the Nit2-deficient strain compared to 405
SG200, with peak transcript levels at 6 h, and no detectable transcript 24 and 48 406
h after transfer. In medium without nitrogen, nar1 and nrt were strongly and 407
immediately induced in SG200, while induction was less pronounced and 408
delayed in Nit2 mutant sporidia. 409
These data suggest that nar1 and nrt are subject to NCR in U. maydis, but that 410
Nit2 is not the sole transcriptional activator of these two NCR target genes. 411
However, no NR activity was detected in the Nit2 deprived mutant, despite nar1 412
and nrt transcript accumulation in the absence of favorable nitrogen sources, 413
indicating that NR is additionally regulated at the translational or post-414
translational level. 415
416
417
418
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Nit2-dependent and Nit2-independent regulation of nitrogen-responsive genes 419
As the transcriptional analysis of the nitrate assimilation pathway revealed that 420
Nit2 is not the sole master regulator of nitrogen utilization in U. maydis, 421
microarray analyses were performed to identify nitrogen-responsive genes that 422
are regulated in an Nit2-dependent and -independent fashion. 423
Transcriptome analysis of Nit2-deficient sporidia cultivated either on nitrate or 424
without nitrogen source revealed that the transcript pattern of the deletion strain 425
was very similar in these two N deficient conditions, with only 5 genes 426
significantly regulated more than 3-fold in sporidia cultivated on nitrate and 427
without N source (data not shown). Based on this similarity, further transcriptome 428
analyses with SG200, ΔN-Nit2 and Δnit2 were performed with sporidia that had 429
been transferred from ammonium minimal medium to fresh minimal medium 430
containing ammonium (control) or to fresh medium without any nitrogen source in 431
two completely independent experiments. Samples were taken 2 h after transfer, 432
which was the time point of strongest Nar1 and Nrt induction in the RNA blot 433
experiments (Fig. 4). 434
Forty-six genes were up-regulated under N starvation conditions in both ΔN-nit2 435
and Δnit2 when compared to SG200, while 72 were commonly down-regulated 436
(supplementary Table S1). Classification into functional categories (FunCats) 437
according to the online tool available on the MUMDB homepage 438
(http://mips.helmholtz-muenchen.de/genre/proj/ustilago/) showed that regulated 439
genes were significantly enriched in the categories “metabolism”, “energy” and 440
“transport” (supplementary Table S2). 441
To verify the microarray data and to compare individual transcript accumulation 442
after transfer of sporidia from ammonium to either nitrogen-starvation medium or 443
nitrate medium, transcript abundance of selected genes was quantified in 444
sporidia of SG200 and Δnit2 using qRT-PCR (Table 4). As already shown by 445
RNA blot analysis (Fig. 4), the transcriptional induction of the nitrate assimilatory 446
gene cluster consisting of nar, nir and nrt was only partially Nit2-dependent in 447
response to nitrate or N starvation. However, induction of a putative purine 448
transporter (um01756.2) and a putative purine permease (um03690) was entirely 449
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Nit2-dependent, since no induction was observed when Nit2-deficient sporidia 450
were transferred to nitrate or N starvation medium. A high affinity ammonium 451
transporter (ump2, um05889), which was previously discovered as a nitrogen 452
starvation-induced gene (32) was also strongly induced under nitrogen starvation 453
in our assay. Induction of ump2 was partially Nit2-dependent (Table 4). 454
455
Expression of three genes with strong homology to urea permeases showed 456
distinct expression patterns: While um04577 induction was predominantly Nit2-457
dependent in both nitrate and N starvation medium, um02625 was only induced 458
in an Nit2-dependent fashion when sporidia were transferred to nitrate-containing 459
medium. Under nitrogen starvation this gene was repressed. The third urea 460
permease um06253 was not transcriptionally regulated either in SG200 or in 461
Δnit2 under our experimental conditions. Another level of complexity in the 462
response of U. maydis to changing nitrogen regimes is added by the observation 463
that a putative monocarboxylate transporter (um00477) is repressed on nitrate 464
and N starvation medium, and a putative dicarboxylate transporter (um04060) is 465
Nit2-dependently repressed on nitrate, but not under N starvation, both in an 466
Nit2-dependent fashion. 467
468
The Gal4-like transcription factor Ton1 is a target of Nit2, but not involved in 469
regulating nitrogen metabolism 470
The only transcription factor –besides the deleted Nit2 itself- that was commonly 471
down-regulated in both Nit2 deletion strains was the Gal4-type transcription 472
factor um10005 (supplementary Table S1). The predicted protein sequence 473
harbors an N-terminal Zn2Cys6-type DNA binding domain and does not show any 474
sequence homology to transcription factors from ascomycete fungi outside this 475
DNA-binding domain. Its closest homologs can be found in the basidiomycete C. 476
neoformans (Accession XP_774034.1, e=2e-16), so this transcription factor 477
might represent a novel class of transcription factors specific to basidiomycetes. 478
We termed it Ton1 (target of Nit2) and confirmed the Nit2-dependent induction on 479
nitrate and N starvation medium by qRT-PCR (Table 4). We constructed a 480
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deletion strain termed Δton1 in the SG200 background and tested the ability of 481
this strain to utilize complex nitrogen sources as described for the Δnit2 strains. 482
Deletion of ton1 did not affect growth on any nitrogen source tested, when 483
compared to SG200 sporidia (data not shown), so that a major role of Ton1 in 484
nitrogen utilization is unlikely. 485
To more precisely unravel the function of ton1, a transcriptome analysis was 486
performed with the Δton1 strain. To this end, sporidia were grown on ammonium 487
minimal medium and transferred to ammonium or N starvation medium, and the 488
global expression pattern was compared to the expression pattern of the Nit2 489
deletion strains. Sixty-five genes were commonly up-regulated in the Δnit2 strains 490
as well as in the Δton1 strain under nitrogen limiting conditions, while 21 genes 491
were synonymously down-regulated in these three genotypes (supplementary 492
Table S3), indicating that this gene set might be regulated in the Nit2-deficient 493
strains due to ton1 down-regulation. Classification of these genes into FunCats 494
showed that the majority of regulated genes are involved in metabolism and in 495
DNA, RNA and protein metabolism, but no category was significantly enriched 496
(supplementary Table S4). Interestingly, rrm4, an RNA-binding protein involved in 497
cell polarity and also required for full pathogenicity (6, 7) is strongly repressed 498
(>1000-fold) in Δton1 and slightly repressed in Δnit2 (2.3-fold). Deletion of ton1 in 499
the solopathogenic SG200 strain, however, did not affect filamentous growth on 500
charcoal plates, as was shown for rrm4 mutants (supplementary Fig. S2). 501
502
Ustilagic acid biosynthesis is not controlled by Nit2 503
One facet of the nitrogen starvation response of U. maydis is the production and 504
secretion of glycolipids, mainly ustilagic acid (cellobiose lipid) and ustilipid 505
(mannosylerythritol lipid), which act as biosurfactants (31). The biosynthetic 506
genes are organized in gene clusters that have already been characterized (78). 507
Recently, the transcriptional activator Rua1 has been shown to control the up-508
regulation of the ustilagic acid gene cluster under nitrogen limiting conditions 509
(79). We also observed the formation of needle-shaped crystals that are 510
reminiscent of ustilagic acid production after keeping both SG200 and Nit2-511
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deficient sporidia for 4 days on nitrogen starvation medium (Fig. 5A, white arrow 512
heads). Furthermore, analysis of secreted glycolipids isolated from these cultures 513
revealed that the composition was the same for SG200 or Nit2-deficient cultures 514
(supplementary Fig. S3). 515
516
Nit2 is required for full pathogenicity 517
Homologs of Nit2 / AreA have been shown to be required for full pathogenicity in 518
some -but not all- plant pathogenic ascomycete fungi, as their corresponding null 519
mutants showed reduced virulence on planta (summarized in Bolton and 520
Thomma, 2008). To test if deletion of nit2 affects pathogenicity in U. maydis, 521
maize seedlings were infected with Δnit2 strains and the infection phenotype was 522
scored 10 days post infection (Fig. 6C). While SG200 induced the formation of 523
large tumors, leaves infected with Nit2 deletion strains displayed strongly 524
reduced tumor formation and tumor size (Fig. 6A,C). Complementation of the 525
Nit2 deletion strains with the endogenous nit2 restored virulence on maize 526
seedlings nearly to SG200 level, but not completely (Fig. 6A,C). However, 527
transcript amounts of Nit2 in several independent complementation strains were 528
higher than in SG200 (not shown), suggesting post-transcriptional regulation of 529
Nit2 activity or suboptimal gene dosage effects. The disease index scored at 14 530
days post infection (dpi) and at 20 dpi was quite identical to the score obtained at 531
10 dpi (results not shown), indicating that host colonization is impeded in Δnit2, 532
and not just delayed compared to wild type. 533
Since plants infected with Δton1 showed wild type-like infection symptoms (Fig. 534
6B), the reduced virulence of Δnit2 cannot be explained by a secondary effect of 535
ton1 down-regulation in Δnit2 mutants. 536
537
Initiation of U. maydis filamentous growth is disturbed in Δnit2 538
Haploid U. maydis sporidia proliferate by budding under favorable conditions, but 539
filamentous growth has been observed under nitrogen limitation (46). Filaments 540
also formed when sporidia were kept on low ammonium medium and this 541
filamentation has been shown to be dependent on the high affinity ammonium 542
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permease Ump2 (73). The induction of ump2 during nitrogen starvation was less 543
pronounced in the Nit2 deletion strains than in SG200 (Table 4) and furthermore, 544
we also observed that some SG200 sporidia initiated filamentous growth when 545
grown overnight in liquid medium containing nitrate or no nitrogen source, while 546
Δnit2 sporidia did not (Fig. 5A). Thus, we hypothesized that Nit2 might be 547
required to execute the dimorphic switch from budding to filamentous growth. 548
To further investigate if Nit2 is involved in the initiation of filamentous growth, we 549
quantified filament formation on planta and on an artificial hydrophobic surface 550
capable of inducing this morphological differentiation (60). After 16 hours 551
incubation in water on parafilm, 21% of the SG200 sporidia had started to 552
produce filaments, while only 15% of Δnit2 had done so (Table 5). On planta, 553
89% of the SG200 sporidia had produced filaments 16 h post infection (hpi), the 554
majority of which had already exceeded sporidia length. In contrast, only 68% of 555
Δnit2 sporidia had produced filaments at 16 hpi, most of which were shorter than 556
those formed by SG200 (Fig. 5B). 557
Similar to the situation in S. cerevisiae, where invasive filamentous-like growth is 558
initiated under nitrogen limitation (27), filaments were not formed when high 559
concentrations of the favorable nitrogen source ammonium were provided to U. 560
maydis sporidia (data not shown). Furthermore, addition of Gln, another favored 561
nitrogen source for U. maydis, to SG200 sporidia resulted in reduced 562
filamentation in the filamentation assay on parafilm compared to SG200 sporidia 563
kept in water only (Table 5). 564
To test, whether Nit2 acts downstream of the bE/bW heterodimer, we introduced 565
the nit2 knockout into the haploid strain AB31, which carries the bE and bW gene 566
under the control of an arabinose-inducible promoter (10). Without arabinose 567
induction, occurrence of filaments is restricted in both AB31 and AB31-Δnit2 (Fig. 568
5C). After arabinose-triggered induction of b-dependent genes, only 40% of AB31 569
sporidia had not formed filaments at 18 hpi, while 70% of AB31-Δnit2 sporidia 570
failed to form filaments at this time point (Fig. 5C). Likewise, the fraction of 571
sporidia that had formed long filaments had doubled upon the induction of b in 572
the AB31-Δnit2 strain, while it had increased 7-fold in the AB31 strain (Fig. 5C). 573
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This suggests that Nit2 does not act independently of bE/bW during filament 574
induction and that the b-locus is the major player in the induction of filamentous 575
growth. 576
Taken together, these data indicate that Nit2 is a major positive regulator of 577
nitrogen utilization and a molecular player that mediates the dimorphic switch in 578
U. maydis growth downstream of bE/bW. 579
580
581
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Discussion 582
583 Fungi are capable of utilizing a plethora of nitrogen sources, but show preference 584
for nitrogen compounds that are easy to metabolize from the energetic point of 585
view. The regulatory circuit behind this is called nitrogen catabolite repression 586
(NCR) and has been extensively studied in S. cerevisiae and in the filamentous 587
ascomycete fungi N. crassa and A. nidulans, where GATA-type transcription 588
factors act as positive master regulators of nitrogen utilization (reviewed in (15, 589
58). GAT1 is required for full virulence in the opportunistic human pathogen 590
Candida albicans (53) and Nit2 homologs in some, but not all, plant pathogenic 591
ascomycete fungi (summarized in 9). Little is known about the regulatory 592
mechanisms of NCR in basidiomycetes. It has been demonstrated that 593
basidiomycetes possess genes coding for GATA factors homologous to Nit2 / 594
AreA, but lack orthologs of the negative acting factors known from yeast and 595
filamentous fungi (32, 59, 87). 596
This study demonstrates that the U. maydis GATA factor Um10417 is a functional 597
homolog of Nit2 / AreA and that U. maydis Nit2 is a positive regulator of 598
unfavorable nitrogen source catabolism. We further show that Nit2 is required for 599
the initiation of filamentous growth under unfavorable nitrogen conditions and for 600
full virulence on maize leaves. 601
602
Nit2 is a master regulator of nitrogen utilization 603
Ustilago maydis Nit2 showed higher homology to Nit2 / AreA from filamentous 604
fungi than to Gat1p from S. cereviseae and had the same conserved Zn-finger 605
domain and RMENLTWRMM motif in the N-terminal part, a motif that is not 606
conserved in S. cereviseae Gat1p. However, Nit2 and Nit2-homologs from other 607
basidiomycetes exhibit a long N-terminal extension with no homology to any 608
known proteins, and basidiomycete Nit2 homologs are missing the conserved 609
Nmr1 interaction site at the outmost C-terminus that is found in ascomycete Nit2 / 610
AreA proteins (65). Although it was previously reported that the U. maydis 611
genome does not harbor an Nmr1 homolog (87), we could identify a gene coding 612
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for a protein with an NmrA multidomain. The gene um11107 showed 35% 613
sequence identity with Tar1, a functional Nmr1 homolog of C. neoformans (40). 614
Interestingly, an um11107 knockout mutant did not exhibit expression of nar1 in 615
the presence of favorable nitrogen sources (unpublished results), indicating that 616
Um11107 is not directly inhibiting Nit2 activity. In contrast to A. nidulans areA, U. 617
maydis nit2 also did not show changes in transcript accumulation under different 618
nitrogen regimes (unpublished observations), suggesting that it may be subject to 619
completely different regulatory mechanisms than the previously described master 620
regulators of nitrogen metabolism. 621
We generated two independent nit2 deletion strains and tested their ability to 622
utilize different nitrogen sources. Sporidia of the deletion strains were unable to 623
grow on most nitrogen compounds tested, as only ammonium, Gln, Asn, Glu, 624
putrescine and Tyr supported proliferation of Δnit2. While only ammonium and 625
Gln or ammonium, Gln and Glu can be utilized in a Nit2 / AreA-independent 626
manner by A. nidulans or N. crassa, respectively (28), several complex nitrogen 627
sources could still be used by nit2 knock-out mutants of the ascomycete 628
phytopathogens C. lindemuthianum, M. grisea and C. fulvum (23, 66, 67). We 629
therefore hypothesize that the large set of favored nitrogen sources of U. maydis 630
is not basidiomycete-specific, but may reflect an adaptation to the pathogenic 631
lifestyle. 632
Taken together, these observations showed that U. maydis harbors a functional 633
Nit2 homolog, and that it acts as a derepressor of nitrogen utilization in U. 634
maydis. 635
636
Transcript analysis reveals Nit2-dependent and –independent gene expression in 637
response to the nitrogen source 638
The inability of Δnit2 sporidia to utilize nitrate was due to a lack of nitrate 639
reductase activity. Nitrate reductase (NR) activity is regulated by the available 640
nitrogen source (51), which is known to be mediated on the transcriptional level 641
(4, 10). As it was shown that induction of the nitrate assimilatory genes in N. 642
crassa and A. nidulans strictly depend on the global derepressor Nit2 / AreA and 643
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pathway-specific induction (13, 24, 41), we analyzed the induction kinetics of 644
nar1 and the nitrate uptake transporter nrt under different nitrogen regimes in 645
SG200 and Δnit2. Ammonium completely repressed transcript accumulation of 646
nar1 and nrt, even in the presence of nitrate, while removal of the favored 647
nitrogen source ammonium led to a swift induction of these genes. For A. 648
nidulans nitrate reductase (NiaD) it was shown that niaD transcript accumulation 649
is highly dependent on transcript stability, which is mediated by the nitrogen 650
source (12). Based on our data, we cannot exclude that U. maydis nar1 is 651
regulated on the post-transcriptional level as well. 652
In contrast to the regulation during N starvation in N. crassa and A. nidulans, we 653
could detect fast and strong induction of nar1, nrt and other Nit2-regulated genes 654
involved in nitrogen metabolism also in the absence of any nitrogen source. 655
Pathway-independent induction of nitrogen metabolizing enzymes was also 656
observed in the pythopathogenic ascomycetes F. fujikoroi, F. oxysporum and M. 657
griseae (17, 19, 71) and the mycorrhizal basidiomycete Hebeloma 658
cylindrosporum (38, 39) and may, therefore, represent a general adaptation to 659
the pathogenic lifestyle of these organisms. However, a putative urea permease 660
(um02625) showed induction only in the presence of nitrate, but not under N 661
starvation. This indicates that pathway-specific induction may be necessary for 662
some, but not all genes. 663
664
In G. fujikuroi, many of the nitrogen starvation-induced genes were only partially 665
dependent on AreA (71). We also observed this in U. maydis: While a subset of 666
genes that are controlled by the nitrogen source are strictly dependent on Nit2, a 667
subset of genes was identified, whose induction under N starvation was only 668
partially dependent on Nit2. The nitrate assimilation cluster is induced in Δnit2, 669
although at a lower level compared to SG200. Since it was shown that the U. 670
maydis NR protein is subjected to high turnover (51) the low nar1 transcript levels 671
in Δnit2 do not seem to be sufficient to drive NR activity to detectable levels. In 672
turn, this could indicate that there are additional positive regulators that are 673
involved in the transcriptional activation of nar1 and other Nit2 target genes 674
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identified in this study. Alternatively, nar1 expression might also be regulated at 675
the post-transcriptional level that prevented the generation of NR protein from the 676
detectable nar1 transcripts. 677
Ustilago maydis produces and secretes glycolipids under nitrogen limiting 678
conditions (31). Activation of the biosynthetic gene cluster has been shown to be 679
mediated by the transcription factor Rua1 and it has been postulated that Rua1 680
may be under the control of Nit2 (79). Our data suggests that glycolipid 681
production is not under the control of Nit2 and may, therefore, be controlled by 682
additional transcriptional inducers. Recently, it has been shown that the bikaverin 683
biosynthetic cluster in G. fujikuroi is mainly regulated in an AreA-independent 684
manner by the bZIP factor MeaB (86), while gibberellic acid biosynthesis is under 685
strict control of AreA (61), suggesting that biosynthetic genes of different 686
secondary metabolites are regulated independently, albeit being concertedly 687
induced under N starvation. 688
Taken together, these data suggest on the one hand that regulatory circuits differ 689
in U. maydis from those known to control nitrogen utilization in filamentous fungi, 690
and that at least a second positive acting regulator is present that controls 691
expression of nitrogen metabolizing genes. As there is no Gln3p homolog in U. 692
maydis, these additional regulators remain elusive. 693
Amongst the genes that are regulated in a Nit2-dependent manner are also 694
genes involved in carbon metabolism, specifically the putative carboxylate 695
transporters Mct1 and Dic1. These are up-regulated under nitrogen starvation, 696
but seem to be repressed by Nit2. While the closest yeast homolog of Mct1 is the 697
riboflavin transporter Mch5p (68), yeast Dic1p represents a mitochondrial 698
carboxylate transporter (43) that is thought to be required for providing carbon 699
backbones to the mitochondrial matrix (64). The fact that Asp and Glu 700
supplementation could restore growth on ethanol or acetate in yeast dic1 deletion 701
strains indicates potential crosstalk between carbon and nitrogen metabolism via 702
UmDic1 (64). 703
704
705
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Nit2 is required for filamentous growth and full virulence 706
Deletion of nit2 led to a strongly decreased virulence on maize leaves and 707
strongly reduced tumor size and number. Similar effects on pathogenicity have 708
been described for Nit2 / AreA deletion strains of several plant pathogenic 709
ascomycetes (17, 47, 66). Reduced growth of F. verticillioides AreA mutants on 710
mature maize kernels was attributed to the inability to adapt to the low nitrogen 711
conditions, since growth of the mutants was not impaired on kernel blisters that 712
contain high concentrations of free amino acids (47). A similar explanation was 713
found for the reduced virulence of C. lindemuthianium clnr1 mutants on the 714
leaves of common bean (Phaseolus vulgaris), where the initial penetration of the 715
leaf epidermis and the biotrophic phase were not affected, but where the mutant 716
was arrested in the early necrotrophic stage (66). The authors of this study 717
hypothesized that in the biotrophic stage, amino acids are readily available to the 718
pathogen, but are limiting growth in the early necrotrophic stage. 719
Fusarium oxysporum invades plants via growth within the xylem vessels, which is 720
an environment generally low in favored nitrogen sources. Divon et al. (2006) 721
therefore concluded that loss of the AreA ortholog Fnr1 decreases the fitness of 722
the pathogen in this environment. More recently, F. oxysporum MeaB was shown 723
to be an inhibitor of virulence functions under favorable nitrogen conditions and it 724
was demonstrated that MeaB acts independently of Fnr1 (54). While invasive 725
growth of F. oxysporum is generally inhibited in the presence of ammonium, 726
deletion of MeaB resulted in invasive growth even in the presence of ammonium 727
(54). While U. maydis does not possess a meaB ortholog, it has been described 728
that nitrogen starvation induces filamentous growth of sporidia (46), similar to the 729
pseudohyphal growth of yeast cells observed under nitrogen starvation (27). 730
Furthermore, C. albicans Δgat1 and Δgln3 strains showed impaired filamentous 731
growth under certain nitrogen conditions (16, 52). In U. maydis, the initiation of 732
filamentous growth represents a crucial morphological switch to start the 733
pathogenic program (42), and the activity of the active bE/bW heterodimer is 734
necessary and sufficient to trigger pathogenic development (8). Nitrogen 735
limitation has been shown to trigger the dimorphic switch, but this cue still 736
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requires the activity of bE/bW (5). Using a temperature sensitive bE allele, Wahl 737
and co-workers have shown that not all b-dependent genes in planta are also 738
regulated upon b-induction in axenic cultures (85). The authors suggested that 739
other regulators integrate plant-derived environmental cues into the b-mediated 740
cascade. We observed reduced filamentous growth of Δnit2 sporidia in both in 741
vitro assays and on planta. Furthermore, inhibition of filament formation of SG200 742
sporidia could be achieved by supplementing the favored nitrogen source Gln. 743
The pheromone response factor (prf1) is a central regulator of the dimorphic 744
transition in U. maydis, as prf1 mutants cannot initiate filamentous growth and 745
are sterile and non-pathogenic (29). Prf1 is integrating environmental signals 746
such as availability of carbon sources and relays these signals to the activity of 747
the b locus (30). Neither prf1 itself, nor any of the known proteins regulating Prf1 748
activity (reviewed in (11)) are differentially regulated in Δnit2 sporidia, suggesting 749
that Nit2 acts independently or downstream of Prf1 in the haploid pathogenic 750
strain used in this study. In the absence of Nit2, filament formation could only be 751
partially restored by inducing the expression of bE/bW in haploid sporidia, 752
suggesting that nitrogen-induced filamentation via Nit2 integrates directly into the 753
b-triggered signaling cascade. Vice versa, Nit2 did not influence filament 754
induction by bE/bW, indicating that filamentation is predominantly controlled by 755
the b-locus. Consistently, it has previously been demonstrated that the induction 756
of filamentous growth by nitrogen limitation is dependent on b-triggered gene 757
expression (4). We conclude that Nit2 is one regulator of filamentous growth 758
under low nitrogen conditions that acts downstream of bE/bW. Nevertheless, 759
nitrogen limitation on the plant surface appears to be a cue to induce the 760
dimorphic switch from budding to filamentous growth. 761
The Gal4-like transcription factor Ton1, which is regulated in a Nit2-dependent 762
manner, is involved in inducing expression of rrm4 under nitrogen starvation. 763
While rrm4 mutants were impaired in filamentous growth and pathogenicity (7), 764
deletion on ton1 did not affect either. Most likely, there is residual Rrm4 activity in 765
Δton1 sporidia that is able to promote filamentous growth. 766
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Ho et al., (2007) found the ammonium permease Ump2 to be up-regulated under 767
nitrogen starvation. We observed the same in this study and could show that this 768
up-regulation is dependent of Nit2. Ump2 is a close homolog of S. cereviseae 769
Mep2p, which is involved in sensing low nitrogen conditions that eventually leads 770
to pseudohyphal growth (55). Yeast Δmep2 mutants did not form pseudohyphae 771
on low ammonium, a defect that could be complemented by U. maydis Ump2 772
(73). Deletion of ump2 in U. maydis led to a defect in filament formation of 773
haploid cells, but no effect on pathogenicity of these mutants has been reported 774
(73). Similarly, deletion of the Mep2p homolog Amt2 in C. neoformans also led to 775
impaired invasive growth, but had no effect on virulence in two different 776
cryptococcus assays (69). While reduced induction of ump2 in Δnit2 sporidia 777
under low N conditions may contribute to the impaired filamentation, this 778
reduction alone does not explain the reduced virulence of Δnit2 on planta. 779
The genome of U. maydis harbors clusters of genes coding for small, secreted 780
peptides that are required for full virulence on maize leaves (45). In contrast to 781
the situation in C. fulvum and M. griseae, where expression of a subset of 782
effector genes is induced under N limitation (19, 77, 83), the transcriptome 783
analysis performed in this study did not reveal up-regulation of any putative 784
effector genes under nitrogen starvation and no altered expression of such genes 785
in Δnit2 sporidia. Since these transcriptome analyses were not performed on 786
planta, however, it cannot be excluded that some putative effectors may be 787
dependent on Nit2 at later infection stages. Nevertheless, we hypothesize that 788
the reduced virulence of U. maydis Δnit2 is unlikely to be a consequence of 789
altered effector expression, but rather a consequence of impaired filament 790
formation. 791
At this stage, we cannot rule out that impaired utilization of complex nitrogen 792
sources in later infection stages also contributes to the overall reduced virulence 793
on maize. As U. maydis-induced tumors constitute strong sinks for organic 794
nitrogen and amino acids to accumulate to high concentrations in the vicinity of 795
fungal hyphae (36), it appears unlikely that Nit2 function in utilization of complex 796
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nitrogen sources plays an important role for U. maydis pathogenicity at late 797
infection stages. 798
Our study provides insight into the regulatory mechanisms of nitrogen catabolite 799
repression in a basidiomycete phytopathogen. We showed that the U. maydis 800
Nit2 homolog is a central, but not the only regulator of NCR and that this 801
transcription factor integrates nitrogen metabolism into the induction of 802
filamentous growth, and that Nit2 is therefore required for full pathogenicity. 803
804
805
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Acknowledgements: 806
This work was funded by the Deutsche Forschungsgemeinschaft via the priority 807
program FOR 666, project SO 300 11-2. 808
The authors would like to thank Sabine Karpeles (FAU Erlangen-Nuremberg) for 809
technical assistance, Stephen Reid (FAU Erlangen-Nuremberg) for microarray 810
hybridizations, Marlis Dahl and Christian Koch (FAU Erlangen-Nuremberg) for 811
their advisory support on microbiological, molecular and genetic work with U. 812
maydis, as well as Jörg Kämper (KIT, Karlsruhe, Germany) for the provision of U. 813
maydis strains SG200 and AB31 and Regine Kahmann (Max-Planck Institute for 814
Terrestrial Microbiology, Germany) for the provision of vectors used in this study. 815
Microarray data deposition note: 816
Microarray data generated in this study has been deposited at GEO under the 817
accession number GSE28916 (http://www.ncbi.nlm.nih.gov/geo). 818
819
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Tables 1138
1139
Table 1 1140
Ustilago maydis strains used in this study. P: promoter, ipr: allele of ip conferring 1141
resistance to carboxin. 1142
1143
strain genotype reference SG200 a1mfa2 bW2bE1 (45) SG200-Δnit2 a1mfa2 bW2bE1 Δum10417 this study SG200-ΔN-nit2 a1mfa2 bW2bE1 ΔN-terminus-um10417 this study SG200-Δnit2-nit2 a1mfa2 bW2bE1 Δum10417 ipr[Pum10417-
um10417] this study
SG200-Δton1 a1mfa2 bW2bE1 Δum10005 this study AB31 Pcrg:bw2, Pcrg:bE1 (10) AB31-Δnit2 Pcrg:bw2, Pcrg:bE1 Δum10417 this study 1144 1145
1146
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Table 2 1147
Summary of observed growth of SG200, Δnit2 and Δnit2-Nit2 sporidia on minimal 1148
medium plates supplemented with a single nitrogen source. Sporidia dilution 1149
series of each genotype were plated and growth properties were rated 1 day after 1150
plating as normal growth (+), reduced growth (+-), strongly reduced growth (-) 1151
and no growth (--). nd: not determined. Resistance to chlorate was assessed 7 1152
days after plating in the presence of proline. (*) Growth characteristics in the 1153
chlorate assay are not compared to growth on media without chlorate. 1154
1155
SG200 Δnit2 Δnit2-Nit2
ammonium + + + nitrate + -- + Ala + - + Arg + - + Asn + + + Asp + - + citrullin + -- + Cys + -- + Gln + + + Glu + + + Gly + -- + His + -- + Ile + -- + Leu + -- + Lys + - + Met + -- + ornithine + - + Phe + -- + Pro + - + Ser + -- + Thr + -- + Trp + - + Tyr + +- + uracil + - + Val + -- + adenine + -- + cytosine + -- + guanine + - + thymine + -- + putrescine + + + spermidine + - + spermine -- -- -- proline-chlorate (*) - + -
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1156 1157 Table 3 1158
Maximum extractable nitrate reductase activity. Sporidia were cultivated on 1159
ammonium minimal medium (AMM) over night and transferred to nitrate minimal 1160
medium (NMM), AMM or to minimal medium without nitrogen source (-N) during 1161
the late exponential growth phase. Nitrate reductase activity was determined 1162
from cells harvested 2 h after transfer to the new medium and is normalized to 1163
OD600=1. NR activity is shown in nmol OD1-1 min-1. The standard error (n=4) is 1164
indicated. ND: not detectable. 1165
1166
AMM NMM -N SG200 ND 6.1 ± 0.8 11.8 ± 1.3 Δnit2 ND ND ND 1167 1168
1169
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Table 4 1170
Verification of transcript accumulation of selected genes by qRT-PCR. SG200 1171
and ∆nit2 sporidia were transferred to nitrate (NMM), nitrogen starvation (-N) or 1172
ammonium minimal medium (AMM) and harvested 2 h after transfer. Shown is 1173
the absolute fold change and standard error of the respective genes in –N or 1174
nitrate minimal medium compared to control determined from three experimental 1175
replicates with three technical replicates each. Values marked with an asterisk 1176
show strong induction due to a basically absent transcript on AMM and have to 1177
be considered qualitative, not quantitative. 1178
SG200 ∆nit2 GeneID Annotation -N vs AMM nitrate vs
AMM -N vs AMM
nitrate vs AMM
Category
um01756.2 purine transporter
12 ± 5 1.7 ± 0.2 0.7 ± 0.1 0.45 ± 0.01
Nit2-dependent induction
um03690 purine permease
671 ± 161 39 ± 7 2.9 ± 0.5 1.9 ± 0.2 Nit2-dependent induction
um10005 Gal4-TF (ton1)
12 ± 2 3.1 ± 0.4 1.6 ± 0.5 1.9 ± 0.2 Nit2-dependent induction
um04577 urea permease
1236 ± 530*
702 ± 114* 7 ± 2 12 ± 5 Nit2-dependent induction
um03847 nar1 131 ± 32 71 ± 8 13 ± 3 23 ± 3 partially Nit2-
dependent induction
um11104 nir1 2484 ± 787*
2013 ± 393* 154 ± 60 294 ± 90 partially Nit2-dependent induction
um11105 nrt 3281 ± 863*
3604 ± 703* 232 ± 70 559 ± 16 partially Nit2-dependent induction
um05889 ump2 996 ± 73* 423 ± 28 177 ± 21 152 ± 24 partially Nit2-dependent induction
um02625 urea
permease 0.0027 ± 0.0004
10 ± 2 0.021 ± 0.007
0.045 ± 0.008
Nit2-dependent induction on NMM only
um00477 mct1 28 ± 8 1.2 ± 0.8 153 ± 42 60 ± 2 Nit2-dependent
repression um04060 dic1 65 ± 10 17 ± 3 52 ± 12 50 ± 9 Nit2 dependent
repression on NMM
um06253 urea
permease NR NR NR NR not regulated
1179 1180
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Table 5 1181 1182 Filament formation on a hydrophobic surface. Sporidia were grown in ammonium 1183 minimal medium, harvested by centrifugation, washed and were finally 1184 resuspended in ddH2O or ddH2O with 1 mM Gln before they were sprayed onto 1185 parafilm, which was incubated 16 h or 21 h (with Gln) at 28°C under 100% 1186 relative humidity. Per condition, at least one hundred sporidia were counted in 4 1187 biological replicates each. The difference between the control (SG200) and the 1188 two other conditions was statistically significant in a student’s t-test with p<0.05. 1189 1190
1191 1192
1193
SG200 Δnit2 SG200 w/ Gln % filament forming sporidia
21 ± 2 15 ± 2 5 ± 1
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Figure legends 1194
1195
Figure 1: Sequence analysis of U. maydis GATA transcription factor Um10417. 1196
A Phylogenetic tree of characterized Nit2 / AreA transcription factors from the 1197
ascomycetes Aspergillus nidulans (AnAreA), Colletotrichum lindemuthianum 1198
(ClClnr1), Fusarium oxysporum (FoFnr1), Gibberella fujikuroi (GfAreA) and 1199
Neurospora crassa (NcNit2), and homologous proteins from the basidiomycetes 1200
Schizophyllum commune (ScGata-6) and Cryptococcus neoformans (CnGat1). B 1201
Protein alignment showing the DUF1752 motif conserved among functional Nit2 / 1202
AreA proteins, C the Zn-finger motif and D the C-terminal motif only conserved in 1203
ascomycete Nit2 / AreA proteins. Multiple sequence alignment was executed 1204
using the MUSCLE algorithm (20). 1205
1206
Figure 2: A Domain structure of Um10417. Deleted regions in the respective 1207
strains and primer binding sites for the verification of transcript abundance via 1208
RT-PCR are indicated. B RT-PCR analysis of Um10417 deletion strains. The 1209
Um10417-specific primer pairs correspond to the ones shown in A. 1210
Glyceraldehyde-3-phosphate dehydrogenase (GapDH) was used as positive 1211
control. Contamination by genomic DNA was excluded beforehand with gDNA 1212
specific primers. 1213
1214
Figure 3: Growth of U. maydis sporidia on minimal medium plates supplemented 1215
with A 0.3% (w/v) ammonium (AMM) or B 0.3% (w/v) nitrate (NMM). Dilution 1216
series (OD600 1 to OD600 10-4 in 10-1 steps from left to right) of SG200, Δnit2, ΔN-1217
Nit2 and Δnit2 complemented with Nit2 (Δnit2-nit2) were plated onto the 1218
respective medium and growth was assessed after incubation at 28°C for 1 day. 1219
1220
Figure 4: Expression analysis of Nar1 and Nrt in U. maydis sporidia. Transcript 1221
abundance was determined using specific probes against Nar1 (top) and Nrt 1222
(middle) at 1 h, 2 h, 3 h, 4 h, 6 h, 24 h and 48 h upon transfer of SG200 and ΔN-1223
Nit2 sporidia from ammonium minimal medium to minimal medium containing i. 1224
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ammonium (left panel), ii. ammonium and nitrate (second panel from the left), iii. 1225
nitrate (second panel to the right) or iv. no nitrogen source (right panel). 1226
Ammonium and nitrate concentrations in the media were 0.3% (w/v), 1227
respectively. As loading control, abundance of 18S rRNA was determined 1228
(bottom). 1229
1230
Figure 5: Filamentation of U. maydis sporidia. A Sporidia were cultivated for 4 1231
days in nitrogen starvation medium (no N source, top panel) or nitrate minimal 1232
medium (NMM, bottom panel). Filamentous growth was assessed using a 1233
microscope at 400x magnification and DIC optics. Black arrows point to sporidia 1234
that had switched to filamentous growth. White arrowheads mark ustilagic acid 1235
crystals. B Filamentous growth on planta. Filamentation of strains SG200 1236
(control), Δnit2 and Δnit2-Nit2 was assessed 18 h after infection by calcofluor 1237
white staining of fungal structures at the injection site. The difference between 1238
SG200 and Δnit2 was statistically significant in a student’s t-test with p<0.05. C 1239
Filamentous growth in vitro. Filamentous growth of strains AB31 and AB31/Δnit2 1240
was assessed 18 h after spraying the sporidia in water onto parafilm. Previously, 1241
the b-locus had been induced with arabinose (Ara) or sporidia were mock treated 1242
with glucose (Glc) for 1 h before spraying. 1243
Per genotype, four replicates were assessed and per replicate, 100 to 200 1244
sporidia were classified into three groups: no visible filaments (black), filaments 1245
shorter then sporidium length (dark grey), filaments longer than sporidium length 1246
(light grey). Error bars represent the standard error (n=4). The difference 1247
between AB31 and AB31/Δnit2 after Ara treatment was statistically significant in 1248
a student’s t-test with p<0.05. 1249
1250
Figure 6: Pathogenicity of U. maydis strains on maize leaves. A Maize leaves 10 1251
days post infection with SG200, Δnit2 or Δnit2 complemented with a genomic 1252
promoter-gene fragment of Nit2. B Maize leaves 8 days post infection with 1253
SG200 or Δton1. Two representative leaves per strain are shown. In total, 1254
between 12 and 20 infected seedlings per strain were analyzed. C Disease index 1255
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for SG200, Δnit2 or Δnit2-Nit2 scored at 10 dpi. Maize seedlings were infected by 1256
injecting sporidia suspension (OD600 = 1.0) of the respective strain into the leaf 1257
canal with a syringe. The incidence of the indicated symptoms was scored as 1258
described in the methods section. (n= 12-20) Results for one representative out 1259
of four experiments are displayed. 1260
1261
1262 1263
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Figures 1264 1265 Figure 1 1266
1267 1268 Figure 2 1269 1270
1271
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Figure 3 1272 1273
1274
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1275 Figure 4 1276 1277
1278 1279 1280
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1281 Figure 5 1282
1283
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1284 Figure 6 1285 1286
1287 1288
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