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The putative GEF RicA mediates upstream signaling for growth and 1
development in Aspergillus 2
3
Nak-Jung Kwon1#, Hee-Soo Park1,2, Seunho Jung3, Sun Chang Kim4 and Jae-Hyuk Yu1,2 † 4
5
1 Departments of Bacteriology and Genetics, 2 Molecular and Environmental Toxicology Center, 6
University of Wisconsin, Madison, WI, USA, 3 Department of Bioscience and Biotechnology, 7
and Center for Biotechnology Research in UBITA, Konkuk University, Seoul, Republic of Korea 8
4 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Dae-9
Jon, Republic of Korea 10
11
# Present address: Macrogen Inc, Gasan-dong, Seoul, Republic of Korea 12
13
† Corresponding Author: 14
Dr. Jae-Hyuk Yu 15
Departments of Bacteriology and Genetics 16
University of Wisconsin-Madison 17
1550 Linden Drive, Madison, 53706, USA 18
Tel: 1-608-262-4696 19
Fax: 1-608-262-9865 20
Email: [email protected] 21
22
Running title: Aspergillus GEF RicA 23
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00255-12 EC Accepts, published online ahead of print on 21 September 2012
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ABSTRACT 24
Heterotrimeric G proteins (G proteins) govern growth, development and secondary metabolism in 25
various fungi. Here, we characterize ricA encoding a putative GDP/GTP exchange factor for G 26
proteins in the model fungus Aspergillus nidulans (Ani) and the opportunistic human pathogen 27
Aspergillus fumigatus (Afu). In both species, ricA mRNA accumulates during vegetative growth 28
and early developmental phases, but is not present in spores. The deletion (Δ) of ricA results in 29
severely impaired colony growth and the total (Ani) or near (Afu) absence of asexual sporulation 30
(conidiation). The overexpression (OE) of AfuricA restores growth and conidiation in the 31
ΔAniricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. 32
A series of double mutant analyses reveal that the removal of RgsA (an RGS protein of the GanB 33
Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restores vegetative growth and conidiation in 34
ΔAniricA. We further demonstrate that RicA can physically interact with GanB in yeast and in 35
vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects 36
caused by ΔAniricA, indicating that RicA mediated growth and developmental signaling is 37
primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA 38
mRNAs accumulate at normal levels in the ΔricA mutant. In addition, and OEfluG or OEbrlA fail 39
to restore development in the ΔAniricA mutant. These suggest that the commencement of asexual 40
development requires unknown RicA-mediated signaling input in A. nidulans. 41
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INTRODUCTION 48
Heterotrimeric G proteins (G proteins) are conserved in all eukaryotes and involved in almost all 49
biological processes (22, 43, 58). Basic units of the heterotrimeric G protein signaling system 50
include a G protein-coupled receptor (GPCR), a G protein composed of α, β and γ subunits, and 51
variety of effectors, relaying the signal into the cells to elicit appropriate physiological and 52
biochemical responses (6). 53
54
G proteins are regarded as biological switches that oscillate between ON and OFF states (7). In 55
non-stimulated conditions, the inactive Gα-GDP::Gβγ trimeric complex prevails in the cell 56
membrane and the signaling pathway remains OFF. Typically, G proteins are turned ON by the 57
guanine nucleotide exchange caused by ligand-bound (sensitized) GPCRs, causing the 58
dissociation of the GTP-bound Gα subunit and the Gβγ heterodimer, which then transduce signals 59
by interacting with various effectors including adenylyl cyclase-PKA, phospholipase C, ionic 60
channels, and MAP kinases (18, 45). The signal is turned OFF when Gα-GTP is hydrolyzed back 61
to Gα-GDP by the intrinsic GTPase activity of Gα subunit, forming the inactive trimeric complex. 62
One key element facilitating the inactivation is the regulator of G-protein signaling (RGS) that 63
accelerates GTP hydrolysis catalyzed by the Gα subunit (15). While GPCR-mediated signaling 64
accounts for the majority of G protein regulated cellular control mechanisms, the evolutionary 65
conserved RIC-8 (Resistance to Inhibitors of Cholinesterase-8) protein is a proven critical 66
guanine nucleotide exchange factor (GEF) activating a subset of Gα subunits (24). Ric-8 interacts 67
with a monomeric Gα-GDP, stimulates the release of GDP, forms a stable nucleotide free 68
transition state complex with Gα subunit , and catalyzes the exchange of GDP for GTP (Fig. 1A) 69
(5, 24). 70
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In fungi, G protein signaling governs cell growth, morphogenesis, sexual/asexual development, 72
mating, pathogenicity, secondary metabolism and many more processes (33, 34, 69, 73). The 73
model filamentous fungus Aspergillus nidulans contains three Gα subunits (FadA, GanB and 74
GanA) (10, 69, 74), one Gβ subunit (SfaD) (48) and one Gγ subunit (GpgA) (53). Genetic studies 75
have revealed that both FadA (Gα) and SfaD::GpgA (Gβγ) mediate signaling that promotes 76
vegetative growth while inhibiting development and biosynthesis of the carcinogenic mycotoxin 77
sterigmatocystin (ST) (23, 48, 73, 74). Further studies showed that FadA signaling is in part 78
transduced via the cAMP-dependent protein kinase PkaA (56). This FadAPkaA mediated 79
signaling in turn inhibits asexual development (conidiation), which is activated by the 80
FluGBrlA pathway and completed by VosA (2, 44, 69, 70). FlbA is the cognate RGS protein 81
whose primary role is to negatively control FadA-mediated vegetative growth signaling (31, 74). 82
Both flbA- and constitutively active FadA mutations (G42R, R178C and Q204L; defective 83
intrinsic GTPase) result in the fluffy autolytic phenotype (64, 74). Importantly, this FadA-84
mediated signaling for vegetative growth, development and toxigenesis is conserved in the 85
aflatoxin producing fungi Aspergillus parasiticus and Aspergillus flavus (23, 49) and the 86
opportunistic human pathogen Aspergillus fumigatus (38, 69). 87
88
The GanB Gα subunit negatively regulates conidiation and plays a positive role in the 89
germination of conidia, whereas GanA’s role is not yet understood (10). Additional studies have 90
revealed that GanB and SfaD::GpgA constitute a functional heterotrimer controlling cAMP-PKA 91
signaling and conidial germination in response to glucose, where GanB is the primary signaling 92
element and SfaD::GpgA functions in proper activation of GanB (30). Among the three 93
additional RGS proteins RgsA, RgsB and RgsC (69), RgsA acts as the negative regulator of 94
GanB signaling in A. nidulans (21). The lack of RgsA results in phenotypes similar to those 95
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caused by constitutive activation of GanB (Q208L), i.e., germination of conidia in the absence of 96
external C-source and enhanced stress response (21). Furthermore, the overexpression of rgsA 97
causes elaboration of asexual developmental structures (conidiophores) in liquid submerged 98
culture as observed in the ΔganB or GanBG207R mutants (10, 21). In A. fumigatus, GpaB (GanB 99
homologue)-mediated signaling is associated with the activation of the predominant PKA 100
catalytic subunit PkaC1, which governs hyphal growth and development (36, 37). 101
102
Despite such a pivotal role of G proteins in many aspects of Aspergillus biology, upstream 103
mechanisms of signal activation remain to be understood. While at least 16 putative GPCRs have 104
been identified in the genome of A. nidulans (69), none have been proven to specifically activate 105
FadA or GanB-mediated signaling. In an effort to understand the upstream activation of G protein 106
signaling in Aspergillus, we have identified and characterized the Ric-8 ortholog RicA in A. 107
nidulans and A. fumigatus. Functional studies of the ricA gene reveal that it plays a crucial (or 108
essential) role in vegetative growth and development in both species, with partially conserved 109
function. Genetic and biochemical studies further indicate that RicA primarily activates the 110
GanBPkaA signaling cascade in A. nidulans. Finally, as normal or elevated expression of key 111
developmental activators fails to trigger conidiation in the absence of RicA, it is proposed that an 112
unknown RicA-mediated signal input, independent of the FluGBrlAVosA pathway, is 113
required for asexual development in A. nidulans. 114
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MATERIALS AND METHODS 120
Strains and culture conditions: A. nidulans (Ani) and A. funigatus (Afu) strains used in this 121
study are listed in Table 1. Glucose minimal medium (MMG) and MMG with 0.5% (w/v) yeast 122
extract (YE) with appropriate supplements were used for general culture of A. nidulans strains 123
(26, 47). For A. fumigatus pyrimidine and arginine auxotrophic mutant strains (AF293.1 and 124
AF293.6; (67), MMG + 0.1% YE was supplemented with 5 mM uridine, 10 mM uracil (for 125
pyrG1) and 0.1% arginine (for argB1). Minimal medium with 100 mM threonine as a sole carbon 126
source (MMT) with 0.5% YE was used for alcA(p)-mediated overexpression. To check the 127
phenotype of the overexpression strains under the alcA (40, 63) promoter in A. nidulans and A. 128
fumigatus, wild type (WT) and overexpression strains were inoculated on the MMG, MMT + 0.5% 129
YE solid media, and incubated at 37°C for 5 days. Effects of overexpression of the target genes 130
under the niiA (4) promoter in A. nidulans were examined by growing the strains in both MM 131
with 0.2% (w/v) ammonium tartrate (MM + AT, non-inducing) and MMG (inducing, containing 132
0.6% (w/v) sodium nitrate). For Northern blot to confirm overexpression by the alcA promoter, 133
strains were cultured in liquid MMG at 37°C, 220 rpm for 12 hours, and the mycelial aggregates 134
were collected, rinsed with liquid MMT, transferred into liquid MMT, and further induced at 135
37°C, 220 rpm for 6 h. Overexpression under niiA(p) was done by culturing the strains in liquid 136
MMG 16 h at 37°C, 220 rpm. The Saccharomyces cerevisiae L40 strain (Clontech) was used to 137
check the protein-protein interaction between RicA fused DNA binding domain and Gα subunits, 138
FadA, GanA and GanB with the activation domain in Y2H. L40 strain was grown in the synthetic 139
dropout minimal medium (SD) with necessary supplements (10 g/l leucine, 2 g/l tryptophan, and 140
2 g/l histidine) (55), and incubated at 30°C for 2-3 days. Escherichia coli DH5α and DH10B 141
were grown in the Luria-Bertani (LB) medium with ampicillin (50 µg/ml, Sigma) or zeocin (20 142
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µg/ml; Invitrogen) for plasmid amplification and construction. Oligonucleotides used in this 143
study are listed in Table S1. 144
145
Database analyses, nucleic acid isolation and manipulation: The putative RicA proteins were 146
retrieved from NCBI BlastX (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using A. nidulans ricA. 147
Phylogenetic tree of the putative 21 RicA proteins was created by EMBL-EBI, 148
http://www.ebi.ac.uk/Tools/clustalw2/index.html. The construction and analysis of phylogenetic 149
tree was carried out from EMBL-EBI, http://www.ebi.ac.uk/Tools/clustalw2/help.html#tree. The 150
AniricA and AfuricA genes were PCR-amplified from A. nidulans (FGSC4) and A. fumigatus 151
(AF293) genomic DNA. cDNA of AniricA was isolated from A. nidulans cDNA library (provided 152
by K.Y. Jahng, Chonbuk National University, Korea) with the primer pairs oNK-39 and 394. 153
AfuricA cDNA was isolated from reverse-transcriptase treated total RNA using oNK-391 and 392. 154
Genomic DNA and total RNA isolation for Northern blot analysis was carried out as described 155
(38, 72). About 10 �g of total RNA isolated from each sample was separated by electrophoresis 156
using the 1.1% (w/v) agarose gel containing 3% (w/v) formaldehyde and ethidium bromide, then 157
transferred onto Hybond-N+ membrane (Amersham). The probes of brlA (1) and vosA (44) in A. 158
nidulans and brlA, abaA and wetA in A. fumigatus for Northern blot analyses were prepared by 159
PCR-amplification with primer pairs oNK-556, 557 (AnibrlA), oNK-14, 15 (AnivosA), oNK-594, 160
595 (AfubrlA), oHS-382, 383 (AfuabaA) and oTL-7, 8 (AfuwetA), respectively (Table S1). 161
162
Construction of deletion, complementation and overexpression strains: The ricA deletion 163
(∆ricA) mutants in A. nidulans and A. fumigatus were generated by double-joint PCR (DJ-PCR) 164
as described (72). The flanking regions of each ricA gene were amplified by PCR with primer 165
pair, oNK-352, 353 (Ani5’ with AfupyrG tail), oNK-354, 355 (Ani3’ with AfupyrG tail), oNK-166
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352, 474 (Ani5’ with AnipyroA tail), oNK-475, 355 (Ani3’ with AfupyroA tail), oNK-358, 359 167
(Afu5’ with AnipyrG tail), oNK-360, 361 (Afu3’ with AnipyrG tail), oNK-358, 933 (Afu5’ with 168
AniargB tail) and oNK-934, 361 (Afu3’ with AniargB tail) from the both genomic DNAs, 169
respectively. The AnipyrG, AnipyroA, AniargB and AfupyrG markers were amplified with the 170
primer pairs oBS-08:oBS-09, oNK-395:oNK-396, oNK-104:oNK-105, and oJH-83:oJH-86, 171
respectively. The final deletion constructs were amplified with the nested primer set oNK-172
356:oNK-357 (A. nidulans) and oNK-362:oNK-363 (A. fumigatus), respectively. The final PCR 173
products were introduced into RJMP1.59 (N.P. Keller; veA+) and RNIW3 (M. Ni; veA1) for A. 174
nidulans, AF293.1 or 293.6 for A. fumigatus (67) using the Vinoflow FCE lysing enzyme (Novo 175
Nordisk) (59). For the deletion mutants of flbA, sfgA, rgsA, rgsB and rgsC in A. nidulans, each 176
flanking region was PCR-amplified using primer pairs, oNK-412, 413 (5’ flbA with AnipyroA 177
tail), oNK-414, 415 (3’ flbA with AnipyroA tail), oNK-397, 398 (5’ sfgA with AfupyroA tail), 178
oNK-399, 400 (3’ sfgA with AfupyroA tail), oNK-540, 541 (5’ rgsA with AfupyroA tail), oNK-179
542, 543 (3’ rgsA with AfupyroA tail), oNK-562, 563 (5’ rgsB with AfupyroA tail), oNK-564, 567 180
(3’ rgsB with AfupyroA tail), oNK-568, 569 (5’ rgsC with AfupyroA tail) and oNK-603, 604 (3’ 181
rgsC with AfupyroA tail), respectively. The final deletion constructs were amplified with oNK-182
416, 417 (AniflbA), oNK-401, 402 (AnisfgA), oNK-544, 545 (AnirgsA), oNK-605, 606 (AnirgsB) 183
and oNK-607, 608 (AnirgsC). These deletion mutants were used to generate double deletion 184
mutants with ΔAniricA by subsequent transformation. 185
186
To generate the complemented strains, genomic DNA fragments of AniricA and AfuricA were 187
PCR-amplified using the primer pairs oNK-870, 871 and oNK-868, 869 from each genomic DNA, 188
then digested with BamHI (followed by treatment with Klenow fragment) and NotI, and cloned 189
between PvuII and NotI of pHS3 (28) containing the ¾ AnipyroA (46) marker and pNJ25 (29) 190
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containing the ¾ AfupyrG (13) marker with the alcA promoter (19), FLAG (DYKDDDDK) tag 191
and the trpC terminator (68), respectively. Each construct was introduced into the recipient 192
ΔAniricA and ΔAfuricA strains, where preferentially a single copy is inserted into the AnipyroA or 193
AfupyrG locus, respectively. The complemented strains were confirmed by PCR amplification 194
using the primers of each vector from the genomic DNA of transformants. 195
196
To generate the ricA overexpression mutant, the ricA genes were amplified by primer pairs, oNK-197
393, 394 (AniricA) and oNK-391, 392 (AfuricA) from each genomic DNA. The amplified genes 198
were digested with restriction enzymes, EcoRI and NotI, and ligated between the alcA promoter 199
and the trpC terminator in pHS3 and pNJ25, respectively. The final plasmids were used to 200
transform TNJ36 and AF293.1, and single integration at the pyroA in A. nidulans and pyrG locus 201
in A. fumigatus was confirmed. 202
203
Autolysis and cell death assays: The AlamarBlue (AB) assay to assess the cell viability by the 204
percent reduction of AlamarBlue was used as described previously (57). 106 conidia of the WT 205
and ΔAfuricA strains were cultured in MMG + 0.1% YE liquid at 37ºC for 7 days. Aliquots (0.5 206
ml) of cultures according to time (day) including the mycelial aggregates and liquid medium 207
were transferred into 24-well plates (Nunc), 1 ml fresh liquid medium containing 150 µl of 208
AlamarBlue® (AbD Serotec) was added to each sample, and then further incubated for 6 h at 209
37ºC. The solution was transferred into 96 well plates excluding mycelial aggregates, and 210
absorbance was read at A570 and A600. A percent AlamarBlue reduction was detected by Synergy 211
HT (BIO-TEK) using a KC4TM v3.1 software, and was calculated by a formula, (117216 x A570 212
of sample – 80586 x A600 of sample) / (155677 x A600 of media – 14652 x A570 of media) x 100 213
(39) as described in Shin et al., (57) 214
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Yeast two hybrid assay: ORFs of RicA (oNK521, 394), FadA (oNK-507, 508), GanA (oNK-216
509, 510) and GanB (oNK-511, 512) were PCR-amplified from the cDNA library of A. nidulans. 217
The ricA ORF was cloned into the pTLexA vector (provided by S. K. Chae, Paichai University, 218
Korea) (11) carrying the LexA DNA binding domain (DBD) (8) generated by modifying 219
pHybLex/Zeo (Invitrogen): insertion of the TRP1 marker from pGBT9 (Clontech). The cDNA 220
derived ORF of fadA, ganA and ganB was each fused under the activation domain of pGAD424 221
(Clontech), respectively. Plasmids were sequence verified and co-introduced into S. cerevisiae 222
L40 using the lithium acetate-polyethylene glycol-mediated yeast transformation (25). The yeast 223
transformants were selected on SD medium in the absence of uracil, tryptophan and leucine (-224
UWL). To test the reporters β-galactosidase and histidine (H) by the interaction of RicA with Gα 225
subunits in yeast, the transformants were inoculated on the -UWL medium containing X-Gal (5-226
bromo-4-chloro-3-indolyl-β-D-galactopyranoside; 40 μg/ml; Sigma) and the -UHWL medium, 227
and the growth and color of the colonies were examined. To confirm and quantify the trans-228
activation activity, five transformants per each test set were tested for β-galactosidase activity (32) 229
using a yeast β-galactosidase assay kit (Pierce). 230
231
GST pull down assay: The AnricA ORF was cloned between EcoRI and NotI into pGEX-5X-1 232
(GE Healthcare), then introduced into E. coli BL21 (DE3) to express the GST-AniRiA fusion 233
protein. E. coli strain was grown up to OD600 = 0.5~0.6 at 37oC, 220 rpm, and 1 mM IPTG 234
(Isopropyl β-D-1-thiogalactopyranoside, Sigma) was added to induce the fusion protein 235
expression. The culture was then further incubated at 30oC, 220 rpm for 3 h. Subsequently, cells 236
were lysed by sonication in ice-cold E. coli lysis buffer (0.1% Triton X-100, 0.1 mM EDTA, 500 237
mM NaCl with 1 protease inhibitor cocktail tablet [Roche] per 50 ml added before use). Then the 238
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cell lysates were cleared of cellular debris by centrifugation, and the supernatant was collected 239
and incubated with Glutathione Sepharose 4B beads (GE healthcare) on a mixer at 4oC overnight. 240
The beads were washed by the lysis buffer three times and resuspended in 500 μl lysis buffer. In 241
addition, ORF of A. nidulans fadA, ganA, ganB and ricA was each cloned into pcDNA3 242
(Invitrogen) to translate in vitro by TNT T7 quick coupled transcription/translation system 243
(Promega). One μg of individual plasmids was incubated with 20 μCi of [35S]-methionine 244
(PerkinElmer) in TNT mixture for 90 min at 30oC. Equal amounts of in vitro translated proteins 245
were added to Glutathione bead-GST-RicA or Glutathione bead-GST (control) suspensions. The 246
mixture was incubated on a mixer at 4oC overnight. After washing with E. coli lysis buffer five 247
times, the samples were mixed with Laemmli sample buffer (BioRad) and loaded to SDS-PAGE 248
gel. After electrophoresis, the gel was dried under a vacuum to three layers of Whatman 3MM 249
filter paper. Autoradiography was performed at -80 oC with the Fuji super RX film. 250
251
Microscopy: The colony photographs were taken using a Sony DSC-T30 digital camera. 252
Photomicrographs were taken using a Zeiss M2Bio microscope equipped with AxioCam and 253
AxioVision digital imaging software. 254
255
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RESULTS 257
Identification of RicA in A. nidulans and A. fumigatus 258
Genome search with the Caenorhabditis elegans RIC-8 protein (GenBank# AF288812.1) resulted 259
in the identification one putative ortholog in each Aspergillus species; Afu4g08820 (AfuRicA; 260
score = 134, e-value = 2.5e-05) and AN1661 (AniRicA; score = 131, e-value = 6.5e-05). To 261
verify its ORF, we isolated and analyzed the ricA cDNA from the A. fumigatus and A. nidulans 262
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cells, and our new AniRicA and AfuRicA sequences and annotations have been deposited in 263
Genbank with the accession numbers JN410838 and JN582330, respectively. Briefly, the 264
AniRicA protein is composed of 466 aa (ORF = 1401 bp with 6 introns) and AfuRicA is 265
composed of 461 aa (ORF=1386 bp with 6 introns) with a predicted mass of 51.7 kDa and 51.2 266
kDa, respectively. Employing these protein sequences, we further identified putative RicA 267
orthologs in other fungi and carried out alignment (Fig. S1) and phylogenetic analyses (Fig. 1B). 268
The RIC8 ortholog is absent in the genomes of S. cerevisiae and plants (65). As presented, A. 269
nidulans RicA is close to that of A. niger, A. oryzae and A. flavus, while A. fumigatus RicA is 270
close to A. clavatus RicA. To characterize the ricA gene, levels of ricA mRNA at different time 271
points in the lifecycle of the two species was examined. As shown in Fig. 1C, in both species, the 272
ricA transcript (each ~2.5 kb) is detectable in a somewhat undulant manner during growth and 273
developmental phases, but not in conidia. It appears that AniricA mRNA is present at low levels 274
in sexual spores (ascospores; AS in Fig. 1C). 275
276
Characterization of ricA in A. fumigatus 277
To understand the function of RicA in A. fumigatus, we generated the AfuricA deletion (ΔAfuricA) 278
mutant by replacing its coding region with AnipyrG+ (for AF293.1) or AniargB+ (for AF293.6 279
for further complementation, see Table 1). Subsequently, using a ΔAfuricA::AniargB+ pyrG1 280
strain as a transformation host, we further generated AfuricA complemented strains by 281
introducing the AfuricA WT allele into the pyrG locus employing pNJ25 (57). We then examined 282
the phenotypes of ΔAfuricA (FNJ12), WT (AF293) and complemented strains (FNJ13, see Table 283
1). Most markedly, the ΔAfuricA mutant exhibited highly restricted colony growth (about 20% of 284
WT) and produced very few and abnormal conidia at the center of colony (Fig. 2A). conidiation 285
at this point was less than 1% compared to that of WT at 5 days after point inoculation on solid 286
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MMG with YE. To test whether the ΔAfuricA abnormal conidia are viable, we tested the rates of 287
conidial germination on solid MMG + 0.1% YE. As shown in Fig. 2B, WT conidia showed 288
germination rates of 90 ± 1.78% at 6 h and 100% at 12 h post inoculation. On the contrary, the 289
ΔAfuricA conidia displayed extremely delayed and defective germination; 0%, 2.9 ± 0.18% and 290
34.5 ± 2.8% of conidia germinated at 6h, 12 h and 18 h, respectively (Fig. 2B), where only ~35% 291
of conidia could germinate eventually (data not shown). These results indicate that AfuRicA is 292
necessary for the functionality (germination) of conidia. 293
294
Aspergillus conidiophore formation requires sequential activities of the central regulatory 295
components brlA abaA wetA (2, 61, 70). As shown in Fig. 2A (middle and bottom panels), 296
the ΔAfuricA mutant produced abnormal conidiophores (marked by black arrow heads) showing 297
improper septation in stalk and incomplete formation of phialides and conidia. As shown with 298
Northern blot analysis, the ΔAfuricA mutant exhibited severely delayed and reduced levels of 299
AfubrlA, AfuabaA and AfuwetA mRNA during the progression of conidiation (Fig. 2C). In WT, 300
AfubrlA mRNA levels increased at 6 h, peaked at 12 h, began to decrease at 24 h, and became 301
almost undetectable at 72 h post developmental induction. Accumulation of the AfuabaA and 302
AfuwetA transcripts followed AfubrlA mRNA accumulation. However, in the ΔAfuricA mutant, 303
AfubrlA mRNA accumulated at low levels at 0 h (= vegetative growth 16 h), gradually increased 304
until 72 h, i.e., reduced, delayed and uncontrolled accumulation of AfubrlA. Moreover, transcripts 305
of AfuabaA and AfuwetA accumulated at low levels even at 48 and 72 h post developmental 306
induction. These results indicate that AfuRicA is necessary for proper expression and regulation 307
of key developmental regulators that coordinate formation, integrity and vitality of spores. 308
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Finally, we tested whether the deletion of AfuricA affects cell death and autolysis in A. fumigatus 310
by AlamarBlue (AB) reduction (57) and dry weight assays, respectively. As shown in Fig. 2D-E, 311
the absence of AfuRicA resulted in delayed cell death and autolysis. Whereas both WT and 312
complemented strains exhibited reduced AB reduction rates at day 4 and only 20% at day 7, the 313
ΔAfuricA mutant exhibited 100% AB reduction at day 5, a decreased rate from day 6 (about 82%), 314
and retained ~58% AB reduction even at day 7 (Fig. 2D). Similarly, while WT and 315
complemented strains exhibited maximum dry weight at day 2 and reduced dry weights from day 316
3, the ΔAfuricA mutant showed peak dry weight at day 5 and gradually reduced weight at day 6 317
and 7 (Fig. 2E). These suggest that AfuRicA is necessary for proper vegetative proliferation and 318
normal progression of cell death and autolysis in A. fumigatus. 319
320
Characterization of AniricA and cross-species complementation 321
The deletion of AniricA resulted in severely impaired growth and the complete lack of asexual 322
and sexual development in A. nidulans. As clearly noticeable from the colonies of WT (TNJ36), 323
ΔAniricA (TNJ21) and complemented (TNJ87) strains grown on solid MMG 37ºC for 5 days, the 324
ΔAniricA mutant formed a very small colony (~30% of WT colony diameter) composed of 325
hyphae without any conidiophores, conidia, cleistothecia (sexual fruiting bodies), or Hülle cells 326
(Fig. 3A). These growth and developmental defects could not be alleviated by changing the 327
growth conditions, e.g., high salt (0.6 M KCl or 0.8 M NaCl), the lack of carbon source, sexual 328
induction, or rich nutrient (0.5% YE) (data not shown). Thus, all further experiments employing 329
the ΔAniricA mutant were done by collecting and inoculating hyphal fragments from a high 330
number of air-exposed mutant colonies. In submerged shake culture following inoculation of the 331
ΔAniricA aerial hyphae, the ΔAniricA mutant exhibited extremely slow hyphal proliferation 332
compared to WT, and the size of ΔAniricA mycelial aggregates was only 15% of that of WT and 333
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complemented strains at 3 days (data not shown). We then asked whether introduction and/or 334
overexpression of the AfuricA WT allele could restore growth and development in the ΔAniricA 335
mutant. We found that the presence of a genomic fragment of AfuricA including its own promoter, 336
coding region and terminator was not sufficient to restore growth and development in the 337
ΔAniricA mutant (data not shown). However, as shown in Fig. 3B, the overexpression of AfuricA 338
under the control of the AnialcA promoter partially enhanced growth, and fully restored 339
conidiation in the ΔAniricA mutant under the inducing condition (MMT + 0.5% YE). 340
Supplementation with 0.6 M KCl enhanced the developmental restoration by AfuricA in the 341
ΔAniricA mutant grown on MMT + 0.5% YE and even on MMG (non-inducing; Fig. 3B bottom 342
+KCl). These results indicate that AniRicA plays a crucial and essential role for growth and 343
development in A. nidulans, and AfuRicA can partially replace AniRicA. 344
345
Suppression of ΔricA by ΔrgsA in A. nidulans 346
If RicA is a ortholog of Ric-8 in animals, it likely functions in activation of heterotrimeric G 347
protein signaling (24). As RicA is clearly needed for growth and development, we carried out a 348
series of double mutant analyses to examine the genetic interactions of RicA with other key 349
regulators in A. nidulans. The flbA gene encodes an RGS protein required for the attenuation of 350
vegetative proliferation signaling mediated by FadA and activation of conidiophore development 351
in Aspergillus (23, 31, 38, 74). SfgA (suppressor of fluG) is a negative regulator of conidiation 352
functioning downstream of FluG but upstream of other key developmental activators including 353
FlbD, FlbC, FlbB and BrlA (52). As shown in Fig. 4A, the ΔricA ΔflbA and ΔricA ΔsfgA double 354
mutants exhibited the phenotypes identical to the ΔricA single mutant. These indicate that the 355
removal of the key negative regulator of growth (FlbA) or conidiation (SfgA) could not alleviate 356
the growth and developmental defects caused by ΔricA, and that RicA functions either upstream 357
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or independently of the FlbA/FadA growth and FluG/SfgA developmental control pathways. We 358
then asked whether RicA is associated with activating other G protein pathways. RgsA is a RGS 359
protein that inhibits GanB-mediated signaling for germination/developmental control and stress 360
response (10, 21). RgsB and RgsC are putative RGS proteins remain to be characterized (21). As 361
shown in Fig. 4B, while the deletion of rgsB or rgsC failed to suppress ΔricA, the absence of 362
rgsA restored growth and development to the ΔrgsA level in the ΔricA mutant. Furthermore, as 363
found in the ΔrgsA mutant (21), the ΔricA ΔrgsA mutant conidia germinated proficiently in liquid 364
medium lacking an external carbon source, whereas WT conidia did not show any sign of 365
germination (Fig. 4C). These results indicate that RicA mediates signaling for growth and 366
development primarily through the RgsA/GanB signaling pathway. It might do so by activating 367
GanB in the absence of the (unidentified) corresponding GPCR(s). 368
369
Physical interaction between RicA and GanB 370
To further test the hypothesis that the primary target of AniRicA is AniGanB, we first tested the 371
physical interaction between AniRicA and individual Gα subunits employing yeast two hybrid 372
(Y2H) assay. The AniricA ORF PCR fragment derived from cDNA was fused with the LexA 373
DNA binding domain (LexA DBD) in the pTLexA vector (11) and each Gα ORF, AniFadA, 374
AniGanA or AniGanB was fused with the Gal4 activation domain (Gal4 AD) in the pGAD424 375
vector (Clontech). Individual pairs of plasmids were introduced into the yeast, and examined for 376
levels of the β-galactosidase reporter. As shown in Fig. 5A, only the AniRicA-AniGanB pair 377
exhibited blue color on the X-gal SD medium lacking uracil, tryptophan and leucine. 378
Quantification of the β-galactosidase activity of each pair in yeast using o-nitrophenyl-379
galactosidase (ONPG) further demonstrated that only the AniRicA-AniGanB pair resulted in high 380
levels of reporter expression in yeast (Fig. 5B), i.e., about 90% of that of the well-known 381
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transcriptional activator AniAflR (positive control) (44, 71). To map the critical interacting 382
domains, we further tested physical interaction between the truncated AniRicA (aa 1-398 or 51-383
466 of the full length 466 aa) and AniGanB (aa 1-325 or 35-356 of the full length 356 aa), and 384
found that the full length AniRicA and AniGanB are necessary for the interaction in yeast (data 385
not shown). The physical interaction of AniRicA and AniGanB was further tested in vitro by 386
employing the GST-pull down assay (Fig. 5C). The AniricA ORF was fused with GST in the 387
pGEX 5X-1 vector (GE Healthcare), and the AniRicA protein was expressed and purified in 388
Escherichia coli. The ORF region of AnifadA, AniganA or AniganB was cloned under the T7 389
promoter of pCDNA3, and each Gα subunit was translated in vitro and labeled by S35. An equal 390
amount of in vitro translated proteins was added to Glutathione bead-GST-AniRicA or 391
Glutathione bead-GST (control) suspensions and subjected to pull-down. As shown in Fig. 5C, 392
S35 labeled AniGanB, but not AniFadA or AniGanA, could be co-purified with GST-AniRicA 393
specifically, indicating that AniRicA directly binds to AniGanB in vitro. Collectively, these data 394
suggest that GanB is a primary target of RicA-mediated signaling in A. nidulans, and the full 395
length AniRicA and AniGanB are necessary for their physical interaction. 396
397
The RicA GanB PkaA signaling cascade 398
In A. nidulans, early events of conidial germination in response to sensing carbon sources are 399
controlled by GanB (Gα) and SfaD::GpgA (Gβγ), and the cAMP-dependent protein kinase PkaA 400
(17, 30, 56). As GanB is proven to be a primary target of RicA, we asked whether the elevated 401
expression of pkaA could restore growth and development in the ricA deletion mutant. While 402
supplementation of exogenous (up to 10 mM) cAMP and dibutyryl cAMP failed to restore (data 403
not shown), two (one native and an ectopic) copies or the overexpression of pkaA was sufficient 404
to restore growth and development in the ΔricA mutant. As shown in Fig. 6, the niiA(p)::pkaA 405
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single and ΔricA niiA(p)::pkaA double mutants exhibited the identical phenotypes on solid 406
medium with 0.2% ammonium tartrate (non-inducing) and 0.6% sodium nitrate (inducing) as a 407
nitrogen source. The overexpression of pkaA regardless of the presence or absence of RicA 408
resulted in enhanced production of aerial hyphae, and reduced density of conidia per unit area on 409
inducing medium as described by Shimizu and Keller (56). These results suggest that RicA likely 410
mediates signaling through GanB PkaA in A. nidulans. 411
412
Unknown role of RicA-mediated signaling in A. nidulans development 413
Finally, we checked whether the developmental defect caused by ΔricA is due to the defective 414
expression of the key regulators brlA (1) and vosA (44). The mycelial mats of WT, ΔAniricA, 415
niiA(p)::ricA (OEricA), ΔflbA, ΔflbA ΔricA, ΔsfgA, and ΔsfgA ΔricA strains grown on the surface 416
of liquid stationary MMG were collected and subjected to Northern blot analyses. It is important 417
to note that the mutant strains containing ΔricA were cultured for 3 days in order to generate 418
enough hyphal mass on the surface of liquid MMG. Despite the complete lack of conidia, all 419
mutants containing ΔricA showed comparable levels of brlA and vosA mRNAs (Fig. 7A), 420
suggesting that expression of brlA is not sufficient to activate conidiation in the absence of RicA, 421
and that RicA-mediated signaling may provide a critical input for conidiophore development. 422
This was further tested by overexpressing (OE) fluG and brlA in the absence of ricA. Neither 423
OEfluG nor OEbrlA could restore conidiation in the ∆ricA mutant on solid medium (Fig. 7B). 424
Moreover, while OEbrlA ricA+ caused the formation of conidia at the hyphal tip in liquid 425
submerged culture, the OEbrlA ΔricA double mutant showed only reduced mycelial growth 426
without forming conidia (data not shown). These results suggest that expression of brlA is not 427
sufficient for conidiation and unknown RicA-mediated developmental signaling, likely 428
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independent of the fluG brlA pathway, is necessary for conidiophore development in A. 429
nidulans (see Fig. 8 and Discussion). 430
431
432
DISCUSSION 433
During last decade novel families of proteins that can modulate the ON-OFF state of G proteins 434
have been identified, raising the complexity of the regulation of signal transduction (50). Among 435
the newly identified components that may function as positive modulators of G proteins is Ric-8 436
(also known as synembryn; (24). Ric-8 is a cytoplasmic protein initially identified by a genetic 437
screening of the C. elegans mutants that are resistant to the cholinesterase inhibitor Aldicarb 438
(41); and by a yeast two-hybrid screen searching for interacting partners of mammalian Gα 439
subunits (27, 60). Later, it was revealed that RIC-8 is involved in the asymmetric division of C. 440
elegans embryos (3, 12, 42) and Drosophila melanogaster neuroblasts (14, 20, 62). The RIC-8 441
orthologs are present in genomes of animals and filamentous fungi, but not in the baker's yeast 442
and plants. Whereas C. elegans and Drosophila genome contains a single Ric-8 gene, mammals 443
possess two Ric-8 orthologs, Ric-8A and Ric-8B (24). Unlike GPCRs, Ric-8 proteins cannot 444
stimulate the guanine nucleotide exchange activity of Gα subunit associated with Gβγ, i.e., the 445
inactive heterotrimeric complex. Thus, Ric-8 proteins can only function on dissociated (free) 446
monomeric Gα-GDP (see Fig. 1A) (24, 60). It has been hypothesized that RIC-8 was acquired by 447
animals and certain fungi after G proteins and GPCRs evolved, and is therefore a fairly recent 448
addition to G-protein regulatory pathways (65). 449
In this study, we present the experimental evidence that the Ric-8 ortholog (RicA) plays a crucial 450
role in governing vegetative growth and development in two Aspergillus species. The lack of 451
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AniRicA function results in the profound defects in hyphal proliferation and asexual/sexual 452
fruiting. Particularly, the AniricA null mutants were severely impaired in hyphal growth and 453
unable to form conidia or ascospores in A. nidulans. Thus, the ΔAniricA cultures were derived 454
from the mutant hyphal fragments and all double mutants were generated by deleting AniricA 455
from individual single mutants through transformation. Likewise, the ∆AfuricA mutant produces 456
defective conidia many of which are unable to germinate, making it extremely difficult to study 457
the RicA function in both species. Whereas the primary structure of the two Aspergillus RicA 458
proteins is highly conserved (75% identity, 89% similarity and 0% gaps; 459
http://blast.ncbi.nlm.nih.gov/Blast.cgi), the overexpression of AfuRicA only partially restored 460
growth and development in A. nidulans. We show here that RicA interacts with G alpha subunit 461
GanB, but not with FadA or GanA in A. nidulans. Furthermore, RicA is incapable of forming a 462
homodimer or a multimer (Fig. 5). Han et al. described that the deletion of ganB, but not fadA 463
or ganA, suppressed the developmental and metabolic defects caused by ΔrgsA, and concluded 464
that RgsA negatively regulates GanB-mediated signaling in A. nidulans (21). We show that RicA 465
physically interacts with GanB, and only ΔrgsA suppresses the defective growth and development 466
phenotypes caused by ΔricA, and the ΔrgsA ΔricA double mutant exhibits the phenotype identical 467
to the ΔrgsA single mutant (Fig. 4B & C). All these suggest that RicA-mediated signaling is 468
transduced primarily via GanB in A. nidulans (Fig. 8). 469
Studies of the Ric-8 ortholog in Neurospora crassa (66) and Magnaporthe oryzae (35) have 470
revealed that RIC-8 plays a highly conserved role in filamentous fungi. As found in Aspergillus, 471
the N. crassa (Nc) ric8 deletion mutant shows severe defects in growth and development. The 472
phenotypes caused by Ncric8 are similar to those observed in the mutant lacking the Gα genes 473
gna-1 and gna-3. Moreover, Δ Ncric8 results in greatly reduced levels of all three Gα and one Gβ 474
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(GNB-1) subunits (66). Further studies have revealed that NcRIC8 positively regulates of GNA-1 475
and GNA-3, and physically interact with and acts as a GEF for GNA-1 (equivalent to AniFadA) 476
and GNA-3 (AniGanB) in vitro, with the strongest effect on GNA-3. The rice blast fungus M. 477
oryzae RIC8 (MoRIC8) is known to be a novel component of G protein signaling during the 478
infection into plants (35). The deletion of MoRIC8 results in non-pathogenicity and impairment 479
in cellular differentiation associated with sporulation, sexual development, and plant infection. 480
MoRic8 is highly expressed in the appressorium, a specialized plant tissue invading structure, and 481
physically interacts with MagB (AniFadA) but not MagA (AniGanB) in the yeast two-hybrid 482
system. Collectively, these indicate that Ric8/RicA plays a crucial role in upstream activation of 483
various signaling pathways in filamentous fungi and that the primary targets of RicA can vary. 484
Studies in these three fungi (Aspergillus, Neurospora and Magnaporthe) indicate that RIC-8-485
mediated signaling likely involves cAMP-dependent protein kinase signaling and might affect G 486
protein levels. In N. crassa, ΔNcric8 results in low levels of adenylyl cyclase protein. Moreover, 487
ΔNcric8 can be suppressed by a mutation in the PKA regulatory subunit (66). MoRic8 is thought 488
to act upstream of the cyclic AMP response pathway that is necessary for appressorium 489
morphogenesis. In accordance with these, we have shown that two copy and/or overexpression of 490
the primary catalytic subunit of cAMP-dependent protein kinase PkaA suppresses the profound 491
defects caused by the lack of RicA function. Previous studies have also revealed that RIC-8 is 492
also required for normal levels of various G proteins in Neurospora, Drosophila, C. elegans, and 493
mammalian cells. Likewise, the deletion of MoRIC8 causes the down-regulation of Gα subunits, 494
MAGA, MAGB and MAGC as well as Gγ (MGG1) subunit but not Gβ subunit (MGB1) (35). 495
Taken together, these suggest that maintenance of normal levels of G proteins and adenylyl 496
cyclase (and perhaps other yet-unknown regulatory components) is an important conserved 497
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function of the RIC-8 protein (66) and that RicA-mediated signaling is transduced primarily 498
through GanBPkaA in Aspergillus (Fig. 8). 499
While the absence of RicA function essentially abolished conidiation in both species, mRNA of 500
the key developmental activator brlA accumulates to some extent in both A. nidulans and A. 501
fumigatus. Moreover, while abaA and wetA mRNA levels were very low in A. fumigatus ricA 502
null mutant, AnivosA mRNA accumulates in hyphal cells. Furthermore, neither the removal of the 503
key repressor of conidiation SfgA (51, 52) nor the overexpression of the key activators 504
FluG/BrlA rescued the developmental defects caused by the lack of RicA. This suggests that, in 505
addition to the activation of brlA expression, an unknown RicA-mediated signaling input is 506
essential for conidiation in A. nidulans. As the deletion of rgsA and the overexpression of PkaA 507
restored the production of conidia in the ricA null mutant, we speculate that the 508
RicAGanBPkaA signaling input is somehow (indirectly) necessary for the activation of 509
conidiophore development (see Fig. 8). As a possible explanation, we propose that RicA 510
mediated activation of GanB and PkaA singling is necessary for the acquisition of developmental 511
competence (2 and references therein). As previously reported, conidiation does not usually occur 512
in A. nidulans until cells have gone through a defined period of vegetative growth (2 and 513
references therein), supporting the hypothesis that the early aspect of Aspergillus conidiophore 514
development occurs as an integral part of the lifecycle rather than as a response to unfavorable 515
environmental conditions. We speculate that the absence of ricA function abolishes transduction 516
of signals for carbon source sensing, spore germination, vegetative proliferation, and thereby the 517
acquisition of developmental competence: all are thought to be primarily mediated by 518
GanBPkaA (Fig 8). In a previous model, it was proposed that GanB and PkaA signaling inhibit 519
expression of brlA (10, 56). We further speculate that GanB and PkaA play a role in down-520
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regulating brlA expression in order to prevent precocious conidiation and to confer vegetative 521
growth for a certain period of time during the lifecycle of A. nidulans. Further investigation of 522
RicA-mediated signaling and the PkaA downstream components that are associated with the 523
developmental regulation/competence needs to be carried out in Aspergillus species. 524
525
526
ACKNOWLEDGMENTS 527
We thank our lab members for helpful discussions and Ellin Doyle for critically reviewing the 528
manuscript. This work was supported by National Science Foundation (IOS-0950850), USDA 529
CSREES Hatch (WIS01195), and UW Food Research Institute grants to JHY. The work at KU 530
was supported by the National Research Foundation of Korea Grant funded by the Korean 531
Government ( NRF-2011-619-E0002) to SJ. The work at KAIST was supported in part by the 532
Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031955) funded by the 533
Ministry of Education, Science and Technology, Republic of Korea to SCK. 534
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737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
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Table 1. Aspergillus strains used in this study 755
Strain Genotype Source
A. nidulans
FGSC4 veA+ (wild type) FGSCa
RJMP1.59 pyrG89; pyroA4; veA+ (54)
TNJ21 pyrG89; pyroA4; ΔricA::AfupyrG+; veA+ This study
TNJ36 pyrG89 AfupyrG+; pyroA4; veA+ (28)
TNJ42 pyrG89, ΔflbA::AnipyroA+; pyroA4; veA+ This study
TNJ49 pyrG89, ΔflbA::AnipyroA+; pyroA4; ΔricA::AfupyrG+; veA+ This study
TNJ57 pyrG89; ΔsfgA::AfupyrG+; pyroA4; veA+ This study
TNJ58 pyrG89; ΔsfgA::AfupyrG+; pyroA4; ΔricA::AnipyroA+; veA+ This study
TNJ59b pyrG89; pyroA4 niiA(p)::fluG::Flag::3/4pyroA+ (pHS11-fluG); veA+ This study
TNJ60 b pyrG89; pyroA4 niiA(p)::fluG::Flag::3/4pyroA+; ΔricA::AnipyroA+; veA+ This study
TNJ61 pyrG89; pyroA4; ΔrgsA::AnipyroA+; veA+ This study
TNJ62 pyrG89; pyroA4; ΔrgsA::AnipyroA+; ΔricA::AfupyrG+; veA+ This study
TNJ63 pyrG89; ΔrgsB::AfupyrG+; pyroA4; veA+ This study
TNJ64 pyrG89; ΔrgsB::AfupyrG+; pyroA4; ΔricA::AnipyroA+; veA+ This study
TNJ65 pyrG89; pyroA4; ΔrgsC:: AfupyrG+, veA+ This study
TNJ66 pyrG89; pyroA4; ΔricA::AnipyroA+; ΔrgsC:: AfupyrG+, veA+ This study
TNJ68 pyrG89; pyroA4; ganBQ208L::AfupyrG+, veA+ This study
TNJ69 pyrG89; pyroA4; ΔricA::AnipyroA+; ganBQ208L::AfupyrG+, veA+ This study
TNJ85 b pyrG89; pyroA4 niiA(p)::brlA::Flag::3/4pyroA+ (pHS11-brlA); veA+ This study
TNJ86 b pyrG89; pyroA4 niiA(p)::brlA::Flag::3/4pyroA+; ΔricA::AnipyroA+; veA+ This study
TNJ87 b pyrG89; pyroA4, ricA(p)::ricA::ricA(t)::3/4pyroA+; ΔricA::AfupyrG+; veA+ This study
TNJ89 b pyrG89; pyroA4 niiA(p)::pkaA::3/4pyroA+ (pHS11-pkaA); veA+ This study
TNJ90b pyrG89; pyroA4 niiA(p)::pkaA::3/4pyroA+; ΔricA::AfupyrG+; veA+ This study
TNJ94b pyrG89; pyroA4 alcA(p)::AfuricA::3/4pyroA+(pHS3-AfuricA); veA+ This study
TNJ95b pyrG89; pyroA4 alcA(p)::AfuricA::3/4pyroA+; ΔricA::AfupyrG+; veA+ This study
A. fumigatus
AF293 Wild type (9)
AF293.1 pyrG1 (67)
AF293.6 pyrG1; argB1 (67)
FNJ12 pyrG1; ΔricA::AnipyrG+ This study
FNJ13c pyrG1, ricA(p)::ricA::ricA(t)::3/4pyrG+; ΔricA::AniargB+, argB1 This study
aFungal Genetics Stock Center. 756
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bThe ¾ pyroA marker in pHS causes the targeted integration at the pyroA locus. 757 cThe ¾ AfupyrG marker in pNJ25 causes the targeted integration at the pyrG locus. 758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
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FIGURE LEGENDS 782
FIG. 1. Summary of the ricA genes 783
(A) Proposed Ric-8 mediated G protein signaling. (B) A phylogenetic tree of the putative RicA 784
proteins identified in various fungal species (from top to bottom: Magnaporthe grisea, A. terreus, 785
P. chrysogenum, A. nidulans, A. niger, A. oryzae, A. flavus, A. clavatus, A. fumigatus, Penicillium 786
marneffei, Talaromyces stipitatus, Coccidioides immitis, Ajellomyces capsulatus, P. tritici, 787
Phaeosphaeria nodorum, Sclerotinia sclerotiorum, G. zeae, Botryotinia fuckeliana, Podospora 788
anserine and Chaetomium globosum). (C) mRNA levels of ricA during the lifecycles of A. 789
fumigatus and A. nidulans. C and AS indicate conidia (asexual spores) and ascospores (sexual 790
spores in A. nidulans), respectively. Numbers indicate the time (hours) of incubation in liquid 791
MMG or MMG + 0.1% YE (Vegetative) or after transfer onto solid MMG glucose under the 792
conditions favoring asexual development (Asexual) or sexual development (Sexual). Equal 793
loading of total RNA was evaluated by the ethidium bromide (EtBr) staining of rRNA. 794
795
FIG. 2. Phenotypes caused by ΔAfuricA 796
(A) Wild type (WT; AF293), ΔAfuricA (FNJ12) and complemented (C’ AfuricA; FNJ13) strains 797
were point inoculated on solid MMG + 0.1% YE, and incubated at 37°C for 5 d. Photographs of 798
colony size and close-up views of the center of the colony and conidiophores are shown. Black 799
arrows indicate abnormal conidiophores at the center of the ΔAfuricA colony. (B) Germination of 800
WT (AF293) and ΔAfuricA (FNJ12) conidia inoculated on MMG + 0.1% YE plates and 801
incubated for 6 and 12 h. Black arrows in ΔAfuricA indicate germinated conidia at 12 h. (C) 802
Accumulation of brlA, abaA, wetA and γ-actin mRNA post asexual developmental induction of 803
WT (AF293) and ΔAfuricA (FNJ12) strains. Development 0 h indicates vegetative growth in 804
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MMG + 0.1% YE liquid for 18 h. The predicted A. fumigatus γ-actin gene (Afu6g04740) (29) 805
was identified by A. nidulans γ-actin (16). (D) AlamarBlue reduction data indicating relative cell 806
death rates. The mycelial aggregates of WT, ΔAfuricA and complemented strains were mixed 807
with the AlamarBlue (AB) reagent to check the cell viability for 7 days. (E) Dry weights of WT, 808
ΔAfuricA and complemented strain in MMG + 0.1% YE submerged culture were quantified for 7 809
days at 37°C, 220 rpm. 810
811
FIG. 3. Requirement of RicA in A. nidulans growth and development 812
(A) A. nidulans WT (TNJ36), ΔAniricA (TNJ21) and complemented (C’ AniricA; TNJ87) strains 813
were inoculated on MMG and cultured for 5 days at 37°C. Colonies and conidiophores of the 5 814
day old culture solid MMG were observed under a stereo-microscope. (B) Partial 815
complementation by AfuricA overexpression in ΔAniricA. WT (TNJ36), OEAfuricA (TNJ94), 816
ΔAniricA (TNJ21) and OEAfuricA ΔAniricA (TNJ95; OE in Δ) were point inoculated on solid 817
MMG (non-inducing), MMT + 0.5% YE (inducing by the alcA promoter), MMG without KCl 818
and MMT + 0.5% YE with 0.6 M KCl, and incubated for 3 days 37°C. Bar = 200 μm. 819
820
FIG. 4. Double mutant analyses in A. nidulans 821
(A) WT (TNJ36), ΔAniricA (TNJ21), ΔAnisfgA (TNJ57), ΔAnisfgA ΔAniricA (TNJ58) ΔAniflbA 822
(TNJ42) and ΔAniflbA ΔAniricA (TNJ49) strains grown on solid MMG for 5 d at 37°C. (B) WT 823
(TNJ36), ΔricA (TNJ21), ΔrgsA (TNJ61), ΔricA ΔrgsA (TNJ62), ΔrgsB (TNJ63), ΔricA ΔrgsB 824
(TNJ64), ΔrgsC (TNJ65), ΔricA ΔrgsC (TNJ66) were inoculated on MMG, and incubated at 825
37°C for 5 days. (C) Germination of WT (TNJ36), ΔrgsA (TNJ61) and ΔricA ΔrgsA (TNJ62) 826
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conidia in the absence of an external carbon source. Photographs were taken after inoculating 1 X 827
106 conidia into liquid MM without an external C source, and cultured for 16 h at 37°C and 220 828
rpm. Bar = 25 µm. 829
830
FIG. 5. RicA and GanB physically interact in yeast and in vitro. 831
(A) Colony photographs of yeast strains expressing both the LexA DNA binding domain fused 832
AniRicA protein and the Gal4 activation domain fused Gα proteins, AniFadA, AniGanA and 833
AniGanB on the X-Gal medium without uracil, tryptophan and leucine (-UWL/X-Gal). (B) 834
Quantitative analyses of β-galactosidase activities using ONPG in yeast strains including the 835
positive (AflR) (71) and negative (pTLex vector; LexA DNA binding domain alone) controls 836
shown left. (C) GST pull down assay for GST-AniRicA and in vitro translated 35S-Gα proteins. 837
The right lane of each panel shows the in vitro translated FadA, GanA and GanB protein, 838
respectively (10 μl from 50 μl translation reaction volume). The in vitro translated proteins were 839
divided into two parts (each 20 μl) and mixed with the GST-AniRicA protein (left lane) or GST 840
alone (middle lane). The expected protein size of FadA, GanA and GanB is about 39, 40 and 39 841
kDa, respectively. 842
843
FIG. 6. Suppression of ΔAniricA by an ectopic copy or overexpression of pkaA 844
Colony (top) photographs and close-up views (bottom) of A. nidulans WT (TNJ36), ΔAniricA 845
(TNJ21), OEpkaA (TNJ89) and OEpkaA ΔAniricA (TNJ90) strains are shown. These strains were 846
point inoculated on non-inducing (NI; MMG containing 0.2% ammonium tartrate as a nitrogen 847
source) and inducing (I; MMG containing 0.6% sodium nitrate as a nitrogen source) media, and 848
incubated for 5 days at 37°C. 849
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850
FIG. 7. Requirement of AniricA in conidiation independent of fluG and brlA 851
(A) mRNA levels of brlA and vosA in WT (TNJ36), ΔricA (TNJ21), OEricA (TNJ88), ΔflbA; 852
ΔricA (TNJ49) and ΔsfgA ΔricA (TNJ58) cells collected from MMG liquid surface culture. Note 853
the strains including ΔricA were cultured for 3 days on the surface of liquid MMG to obtain equal 854
amount of the hyphal aggregates. (B) WT, ΔricA (TNJ21), OEfluG (TNJ59), OEfluG ΔricA 855
(TNJ60), OEbrlA (TNJ85) and OEbrlA ΔricA (TNJ86) strains were point inoculated on non-856
inducing (NI; MMG with 0.2% ammonium tartrate) and inducing (I; MMG with 0.6% sodium 857
nitrate) solid media, and incubated at 37°C for 5 days. 858
859
FIG. 8. Model for RicA governing A. nidulans growth and development (see text) 860
The putative GEF RicA governs upstream signaling for spore germination, vegetative growth and 861
development in Aspergillus primarily through the GanBPkaA signaling cascade. RicA is also 862
(indirectly) required for conidiophore development, which may involve the acquisition of 863
developmental competence via modulating GanBPkaA signals. In addition, a potential direct 864
role of RicA in activating conidiation, independent of FluG/BrlA, is indicated. It is further 865
speculated that brlA is down-regulated by GanB and PkaA during early phases of vegetative 866
growth in A. nidulans. 867
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