PKA activity is essential for relieving the suppression of hyphal growth and appressorium formation by MoSfl1 in Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and suppression of genes important for hyphal growth.


Introduction
Magnaporthe oryzae is the causal agent of rice blast, which is one of the most important rice diseases worldwide. In the past two decades, M. oryzae has been developed as a model organism to study fungal-plant interactions because of its economic importance and the experimental tractability [1][2][3]. For plant infection, the fungus forms a highly specialized infection cell called an appressorium to penetrate plant cuticle and cell wall [4]. After penetration, the narrow penetration peg differentiates into bulbous invasive hyphae [5] that grow biotrophically inside penetrated plant cells [6]. Various apoplastic and cytoplasmic effectors are known to play critical roles in suppressing plant defense responses during different stages of invasive growth [7]. At late infection stages, lesions are formed and the pathogen produces conidiophores and conidia on diseased plant tissues under favorable conditions. Appressorium formation is initiated when conidia land and germinate on plant surfaces. On artificial hydrophobic surfaces that mimic the rice leaf surface, M. oryzae also forms melanized appressoria. On hydrophilic surfaces, appressorium formation can be induced by cAMP, IBMX, or cutin monomers [8]. Although late stages of appressorium formation is regulated by the Pmk1 MAP kinase, the cAMP-PKA (protein kinase A) pathway is involved in recognizing surface hydrophobicity to initiate appressorium formation, appressorium turgor generation, and invasive growth [9][10][11]. Deletion of the MAC1 adenylate cyclase (AC) gene results in mutants that are defective in appressorium formation [12]. In addition to Cap1 AC-interacting protein [13], heterotrimeric G-proteins and Rgs1 have been shown to function upstream from the cAMP-PKA pathway [2,14]. The PdeH high-affinity cAMP phosphodiesterase is also important for successful establishment and spread of the blast disease [15].
The PKA holoenzyme consists of two regulatory subunits and two catalytic subunits. Binding of cAMP with the regulatory subunit results in the detachment and activation of the catalytic subunits [16]. In M. oryzae, the CPKA gene encoding a catalytic subunit of PKA is dispensable for hyphal growth but the cpkA mutant was delayed in appressorium formation and defective in appressorium turgor generation and plant penetration. In addition, the cpkA mutant still responds to exogenous cAMP for appressorium formation on hydrophilic surfaces [10,11], suggesting that another PKA catalytic subunit gene must exist and play a role in surface recognition and infection-related morphogenesis in M. oryzae. In the budding yeast Saccharomyces cerevisiae, three genes, TPK1, TPK2, and TPK3, encode PKA catalytic subunits and the triple mutant is inviable [17]. The fission yeast Schizosaccharomyces pombe has only one PKA catalytic subunit gene, PKA1, that is important but not essential for normal growth [18]. In the human pathogen Aspergillus fumigatus, the pkaC1 pkaC2 double mutant is delayed in conidium germination in response to environmental nutrients and is significantly reduced in virulence [19]. In the wheat scab fungus Fusarium graminearum, deletion of both CPK1 and CPK2 caused severe defects in growth and conidiation. The cpk1 cpk2 double mutant was sterile in sexual reproduction and nonpathogenic [20]. In the basidiomycete Ustilago maydis, the phenotype of the adr1 uka1 double mutant has similar phenotype with the adr1 mutant and is defective in yeast growth, mating, and plant infection [21].
In S. cerevisiae, Sfl1 is one of the downstream transcription factors of the cAMP-PKA pathway. When functioning as a repressor, it is involved in the repression of flocculation-related genes, including FLO11 and SUC2 [22,23]. As an activator, SFL1 is involved in the activation of stress-responsive genes such as HSP30 [24]. The major PKA catalytic subunit Tpk2 negatively regulates its repressor function [25]. In M. oryzae, deletion of MoSFL1 has no obvious effect on vegetative growth but results in reduced virulence and heat tolerance [26]. Several Sfl1-interacting proteins have been identified in the budding yeast, including Cyc8, Tup1, and various mediator components [23,27]. Although it lacks intrinsic DNA-binding activities, the Cyc8-Tup1 (also known as Ssn6-Tup1) co-repressor complex interacts with various transcription factors with sequence-specific DNA binding motifs, including Sfl1, Mig1, Crt1, and α2, to negatively regulate different subsets of genes [27,28]. In S. cerevisiae, Cyc8 functions as an adaptor protein required for the interaction between Tup1 tetramers and DNA-binding transcription factors [29].
To further characterize the roles of PKA in growth and infection, in this study we generated and characterized the cpk2 and cpkA cpk2 double mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions. The cpkA cpk2 double mutant had severe defects in growth and conidiation and failed to form appressoria or infect plant through wounds. Spontaneous mutations or deletion and truncation mutations in MoSFL1 suppressed the defects of cpkA cpk2 in hyphal growth and appressorium formation but not invasive growth and lesion development. In affinity purification and co-IP assays, MoCyc8 interacted with the full-length but not truncated MoSfl1. Treatment with exogenous cAMP also reduced the interaction of MoSfl1 with MoCyc8 and MoTup1. Furthermore, the S211D mutation in MoSFL1 suppressed the growth defect of cpkA cpk2. In F. graminearum, 29 of 30 suppressor strains of cpk1 cpk2 mutant had mutations in FgSFL1, with 15 of them truncated of its C-terminal region. Taken together, our results indicate that PKA activity is essential for appressorium formation in M. oryzae, and phosphorylation of MoSfl1 by PKA likely relieves its interaction with the Cyc8-Tup1 corepressor and suppression of genes important for hyphal growth and appressorium development. The inhibitory function of Sfl1 orthologs on hyphal growth is likely conserved in filamentous fungi because similar suppressor mutations in FgSFL1 were identified in spontaneous suppressor strains of the cpk1 cpk2 mutant in F. graminearum.

Results
Both CpkA and Cpk2 interact with the PKA regulatory subunit Sum1 In M. oryzae, the PKA regulatory subunit is encoded by SUM1, a suppressor of the mac1 deletion mutant [30]. In an effort to identify proteins interacting with known virulence factors, including Sum1, we generated the SUM1-S-tag fusion and transformed it into the wild-type strain 70-15. Total proteins were isolated from the resulting transformant B22 (Table 1) and subjected to affinity purification and MS analysis after trypsin digestion as described [13,31]. CpkA and Cpk2 (MGG_02832) were among the Sum1-interacting proteins identified in all three independent biological replicates (S1 Table). Cpk2 shares 48% amino acid identity with CpkA but it has a shorter N-terminal region (S1 Fig).
To confirm their interaction by co-immunoprecipitation (co-IP) assays, the SUM1-S, CPKA-3×FLAG, and CPK2-3×FLAG constructs were generated and transformed into the wild-type strain Guy11 in pairs. Western blot analysis with the resulting transformants showed that both PKA catalytic subunits strongly interact with Sum1 (S2 Fig). The cpkA cpk2 double mutant has severe growth and conidiation defects To determine the function of PKA catalytic subunits, we generated the cpk2 and cpkA cpk2 deletion mutants (Table 1; S1 Fig). On complete medium (CM) plates, the cpkA mutant had no obvious growth defects but the cpk2 mutant was slightly reduced in growth rate. The cpkA cpk2 double mutant was viable but it was significantly reduced in growth rate (Table 2; Fig  1A). Unlike cpkA and cpk2, the cpkA cpk2 double mutant rarely produced conidia. In cultures Regulation of appressorium formation and hyphal growth by PKA induced for conidiation, the double mutant produced only a few conidia per plate. Under the same conditions, over 1×10 7 conidia/plate were produced by the wild type ( Table 2).

PKA activities are essential for appressorium formation and plant infection
Unlike cpkA that was delayed in appressorium formation, cpk2 had no obvious defects in appressorium formation (Table 2). However, the cpkA cpk2 mutant failed to form appressoria on hydrophobic plastic coverslips and GelBond membranes although conidium germination was normal ( Fig 1B; Table 2). Even after prolonged incubation up to 72 h, no appressorium formation was observed in the double mutant. The cpkA cpk2 mutant also failed to form appressoria on barley and rice leaves (S3 Fig).
In spray infection assays with two-week-old seedlings of rice cultivar CO-39, numerous blast lesions were observed on leaves sprayed with Guy11 or the cpk2 mutant but no lesions were caused by the cpkA and cpkA cpk2 mutants ( Fig 1C). Because the cpkA cpk2 mutant failed to form appressoria, we conducted injection infection assays. Whereas the cpk2 mutant was as virulent as the wild type, the cpkA mutant failed to cause lesions on intact leaves but caused limited necrosis at the wounding sites. No lesions or necrosis at the wounding sites were observed on leaves inoculated with the cpkA cpk2 mutant (Fig 1C). These results indicate that PKA activities are essential for appressorium formation and invasive growth after penetration in M. oryzae.
The cpkA cpk2 mutant still recognizes surface hydrophobicity Although the cpkA cpk2 mutant was blocked in appressorium formation, we noticed that majority (over 83%) of its germ tubes were curved one direction after incubation on hydrophobic side of GelBond membranes for 24 h (Fig 1B). Interestingly, when assayed for appressorium formation on the hydrophilic side of GelBond membranes, the majority of the cpkA cpk2 conidia failed to germinate ( Fig 1B; Table 3). For the ones (<25%) germinated, germ tubes of cpkA cpk2 mutant failed to form appressoria (Fig 1B). Conidia of the wild-type, cpkA, and cpk2 strains germinated but failed to form appressoria under the same conditions. In the presence of 5 mM cAMP, over 76% of the wild-type germ tubes formed appressoria on hydrophilic surfaces. However, exogenous cAMP had no stimulatory effects on either conidium germination or appressorium formation in the cpkA cpk2 mutant (Fig 1B; Table 3). Over 75% of the double mutant conidia failed to germinate and the ones germinated failed to form appressoria or display germ tube curling defects in the presence of 5 mM cAMP. These results indicate that the cpkA cpk2 mutant still recognizes surface hydrophobicity for germination and germ tube growth but not for appressorium formation.  Elevated intracellular cAMP levels in the cpkA and cpkA cpk2 mutants We then assayed the intracellular cAMP level in vegetative hyphae harvested from liquid CM cultures. In the cpk2 mutant, the intracellular cAMP level was similar to that of the wild type. However, the cpkA and cpkA cpk2 mutants had higher intracellular cAMP levels than the wild type (Fig 2A). In comparison with the wild type, the double mutant was increased approximately 3-fold in intracellular cAMP. These results suggest that reduced or lack of PKA activities results in an increase in intracellular cAMP in M. oryzae.

Absence of PKA activities interferes with the Pmk1 and Mps1 MAP kinase pathways
Because the Pmk1 MAP kinase is essential for appressorium formation [34], we assayed its activation with an anti-TpEY phosphorylation specific antibody. To our surprise, although its expression was not affected, Pmk1 phosphorylation was increased in the cpkA cpk2 mutant ( Fig 2B). However, the double mutant was reduced in the expression and phosphorylation levels of Mps1 MAP kinase ( Fig 2B) that is required for appressorium penetration and conidiation [33]. These results suggest that over-activation of the Pmk1 MAP kinase pathway is not sufficient to stimulate appressorium formation in the absence of PKA activities, and reduced Mps1 activities may be related to conidiation defects of cpkA cpk2.
Spontaneous suppressors of cpkA cpk2 are partially recovered in growth but not pathogenesis Interestingly, the cpkA cpk2 mutant was unstable when cultured on the oatmeal agar (OTA) plates and fast-growing sectors caused by spontaneous suppressor mutations often became visible in cultures older than 10 days ( Fig 3A). Twenty suppressor strains with faster growth rate than the original mutant were isolated. All of them had similar colony morphology and produced more aerial hyphae than the cpkA cpk2 mutant. On average, the growth rate of suppressor strains recovered to approximately 83% of that of the wild type (S4 Fig). Conidiation also was partially rescued in these suppressor strains although to a much lesser degree than the recovery in growth rate (S4 Fig).
Although they varied slightly in growth rate and conidiation (S4 Fig), all the 20 suppressor strains had similar defects as strain CCS1 that was described and presented in figures below. Besides having similar colony morphology, suppressor strains produced melanized hyphal tips in aerial hyphae of 10-day-old OTA cultures ( Fig 3B). In infection assays with rice seedlings, none of the suppressor strains caused lesions on intact or wounded leaves ( Fig 3C). Therefore, mutations occurred in these suppressor strains only suppressed the defects of the cpkA cpk2 mutant in hyphal growth but not plant infection.

Appressoria are formed by the suppressor strains of cpkA cpk2
On artificial hydrophobic surfaces, over 95% of the conidia from the suppressor strains formed appressoria after incubation for 24 h. Interestingly, they also developed appressoria on the hydrophilic surface of GelBond membranes ( Fig 3D). However, approximately 40% of appressoria formed by suppressor strains were abnormal in morphology on hydrophobic or hydrophilic surfaces ( Fig 3E). Unlike normal dome-shaped appressoria, the majority of appressoria formed by suppressor strains had irregular shapes ( Fig 3E). Although they were still melanized, many appressoria formed by the suppressor strains had projections at one side ( Fig 3E). These results indicate that suppressor mutations in these strains also only partially rescued the defect of cpkA cpk2 in appressorium morphogenesis.
Deletion of MoSFL1 rescues the defects of the cpkA cpk2 mutant In M. oryzae, deletion of MoSFL1 had no effects on hyphal growth although it was reduced in virulence [26]. To confirm whether insertion or truncation mutation in MoSFL1 has suppressive effects, the  Table 4), including recovered growth rate and increased conidiation in comparison with cpkA cpk2. Melanized appressoria were efficiently formed by the triple mutant but it failed to cause lesions on rice leaves, further confirming that loss-of-function mutations in MoSFL1 rescue the growth defect of cpkA cpk2. Therefore, MoSFL1 must function as a negative regulator of vegetative hyphal growth and phosphorylation of MoSfl1 by PKA relieves its suppressive effects.

The C-terminal region is important for MoSFL1 function as a negative regulator
In suppressor strain CCS1, the 10-bp insertion in MoSFL1 causes frameshift and results in the truncation of its C-terminal 414-588 aa. Sequence alignment showed that this C-terminal region of MoSfl1 is well conserved among its orthologs from other filamentous fungi, including  Table 4). These results suggested that the C-terminal region of MoSfl1 is essential for its negative regulator function although it has no known protein motifs. Whereas the N-terminal region of MoSfl1 is involved in DNA binding, the C-terminal region may be responsible for protein-protein interactions to suppress the expression of its target genes important for hyphal growth.

Identification of proteins differentially interacting with MoSfl1 and MoSfl1 ΔCT
Because deletion of residues 496-588 is suppressive to cpkA cpk2, this C-terminal region of MoSfl1 is likely responsible for interacting with other proteins as a negative regulator. To identify proteins differentially interacting with MoSfl1 and MoSfl1 ΔCT , the 3×FLAG-MoSFL1 and 3×FLAG-MoSFL1 ΔCT constructs were generated and transformed into the cpkA cpk2 mutant. Total proteins were isolated from the resulting 3×FLAG-MoSFL1 and 3×FLAG-MoSFL1 ΔCT transformants and used for affinity purification with anti-FLAG M2 beads. Proteins co-purified with MoSfl1 or MoSfl1 ΔCT were identified by mass spectrometry (MS) analysis after trypsin digestion as described in previous studies [13,31]. Based on MS results from three biological replicates, MGG_03196 was the only protein that co-purified with MoSfl1 but not MoSfl1 ΔCT (Table 5). Its ortholog in yeast, Cyc8 (Ssn6), forms a transcriptional co-repressor complex with Tup1 to regulate genes involved in a wide variety of physiological processes [28,41,42]. Interestingly, the Tup1 ortholog, MGG_08829, was one of the proteins that were commonly co-purified with MoSfl1 and MoSfl1 ΔCT (Table 5). However, the number of MoTup1 peptides identified by MS analysis was significantly lower in the  MoSFL1 ΔCT transformant than in the MoSFL1 transformant (Table 5), suggesting a weaker interaction of MoSfl1 with MoTup1 when its C-terminal region is deleted. Based on the conserved nature of Tup1, Cyc8, and other components, it is likely that the Cyc8 and Tup1 orthologs also form a transcriptional co-repressor complex with MoSfl1 in M. oryzae, which is consistent with the interaction of Sfl1 with the Cyc8-Tup1 complex in yeast [27].

The C-terminal region of MoSfl1 is important for its interaction with Cyc8
To confirm the importance of the C-terminal region of MoSfl1 in its interaction with Cyc8, the CYC8-S-tag construct was generated and co-transformed into the cpkA cpk2 mutant with 3×FLAG-MoSFL1 or -MoSFL1 ΔCT . The resulting transformants CYS15 and CNC19 (Table 1) were confirmed by western blot analyses for the expression of transforming constructs. In co-IP assays, the MoSfl1 band was detected in both total proteins and elution from anti-S-tag agarose beads in the transformant expressing the CYC8-S and 3×FLAG-MoSFL1 constructs ( Fig  5). However, the MoSfl1 ΔCT band was detected only in total proteins isolated from the transformant expressing CYC8-S and 3×FLAG-MoSFL1 ΔCT (Fig 5). These results confirmed that Cyc8 interacts with the full-length MoSfl1 but not MoSfl1 ΔCT in M. oryzae. Interestingly, additional bands smaller than MoSfl1 or MoSfl1 ΔCT were detected by the anti-FLAG antibody in transformants CYS15 and CNC19 but not in Guy11, suggesting that MoSfl1 proteins may be cleaved in vegetative hyphae.

Exogenous cAMP reduces the interaction of MoSfl1 with MoCyc8
Because deletion of the C-terminal region of MoSFL1 suppressed the growth defect of cpkA cpk2, phosphorylation of MoSfl1 by PKA may affect its interaction with the Cyc8-Tup1 complex. To test this hypothesis, the MoCYC8-S and 3×FLAG-MoSFL1 constructs were co-transformed into the wild-type strain Guy11. In the resulting transformant, treatment with 5 mM cAMP significantly reduced the MoCyc8-MoSfl1 interaction compared to treatment with 10 μM PKA inhibitor (PKI) H-89 (Fig 6A). These results indicate that stimulation of PKA activities by exogenous cAMP reduces the interaction of MoSfl1 with MoCyc8. Therefore,

MoTUP1 also interacts with MoSfl1 and is important for hyphal growth and pathogenesis
We also generated transformants of Guy11 expressing the MoTUP1-S and 3×FLAG-MoSFL1 constructs. MoTup1 was found to interact with MoSfl1 in co-IP assays with the resulting transformant ( Fig 6B). Treatment with exogenous cAMP also significantly reduced the interaction of MoSfl1 with MoTup1 ( Fig 6B). These results indicate that stimulation of PKA activities by exogenous cAMP reduces the interaction of MoSfl1 with MoCyc8 and MoTup1.
To determine the function of MoTUP1, we generated the Motup1 deletion mutant in the wild-type strain Guy11. The resulting deletion mutant MTU35 (Table 1) was significantly reduced in growth rate and it rarely produced aerial hyphae and conidia (Fig 6C). Unlike the cpkA cpk2 mutant, the Motup1 mutant was normal in appressorium formation. One distinct phenotype of the Motup1 mutant was the production of swollen bodies in the subapical regions in hyphae grown on CM (Fig 6C), suggesting cell wall integrity defects. The phenotype differences between the Motup1 mutant and Mosfl1 or cpkA cpk2 mutant indicate that the MoCyc8-MoTup1 co-repressor is involved in regulating different sets of genes by interacting with transcription factors other than MoSfl1.

Phosphorylation of S211 in MoSfl1 rescues the growth defect of cpkA cpk2
In S. cerevisiae, Sfl1 has two predicted consensus PKA phosphorylation sites S207 and S733 [43] that are conserved in MoSfl1 (S211 and S554) and its orthologs from other fungi (Fig 7A). PSITE analysis identified T441 as the only other consensus PKA phosphorylation site in MoSfl1. To determine their functions, the MoSFL1 S211D , MoSFL1 T441D , and MoSFL1 S554D alleles were generated and transformed into the cpkA cpk2 mutant. Whereas MoSFL1 S211D transformants grew faster, the MoSFL1 T441D and MoSFL1 S554D transformants had similar growth defects with the original cpkA cpk2 mutant (Fig 7B). Similar approaches were used to generate transformants of cpkA cpk2 expressing the MoSFL1 S211A , MoSFL1 T441A , and MoSFL1 S554A alleles ( Table 1). None of these S/T to A mutations had suppressive effects on the growth defect of cpkA cpk2 (Fig 7B). These results indicate that phosphorylation of MoSfl1 at S211 may play a critical role to release its inhibitory functions. However, the MoSFL1 S211D transformant failed to form appressoria on hydrophobic surfaces (Fig 7C). Therefore, the S211D mutation in MoSFL1 could suppress the growth but not appressorium formation defect of cpkA cpk2.  Genes down-regulated in the cpkA cpk2 mutant are enriched for the putative Sfl1-binding site in their promoters To identify genes affected by deletion of both CPKA and CPK2, we conducted RNA-seq analysis with RNA isolated from hyphae collected from 2-day-old CM cultures. Considering the significant reduction in growth rate, it was surprising that only 451 genes were down-regulated in the cpkA cpk2 double mutant in comparison with the wild type (S3 Table). However, many of them are functionally important for growth, including several genes encoding ribosomal proteins (MGG_00546, MGG_03372, MGG_09927, MGG_01113, MGG_06571) and enzymes important for cell wall synthesis (MGG_00592, MGG_03208, MGG_07331, MGG_01575, and MGG_03883). When the promoter regions (1000-bp upstream of the start codon) of genes down-regulated in the double mutant were analyzed, 111 of them contain the putative HSFbinding element AGAA-n-TTCT (n 20) [27]. Among them, 29 genes have more than one HSF-binding elements in their promoter regions. These results indicate that the putative MoSfl1-binding element is enriched among the genes significantly down-regulated in the double mutant.

Spontaneous suppressors of cpk1 cpk2 and mutations in FgSFL1 in F. graminearum
The cpk1 cpk2 double mutant of F. graminearum also had severe growth defects [20]. Similar to the cpkA cpk2 mutant of M. oryzae, fast-growing sectors were often observed in V8 cultures of cpk1 cpk2 mutant that were older than 10 days. We isolated 30 suppressor strains that had similar growth rate with the wild type (Fig 8A). In infection assays with corn silks, suppressor strains of cpk1 cpk2 were still defective in plant infection (Fig 8B). When the FgSFL1 gene was amplified and sequenced, 29 of them had mutations in the open reading frame (ORF) (Fig 8C; S4 Table). Suppressor strain HS29 lacked mutations in the ORF of FgSFL1 although its phenotype was similar to that of other suppressor strains with mutations in FgSFL1, suggesting possible mutations at its interacting site on FgCYC8. The most common mutation is the C1717 to T (Q501 to stop codon) mutation that resulted in the truncation of C-terminal 91 amino acids. A total of 15 suppressor strains had this C1717T mutation. Therefore, truncation of the C-terminal region also suppressed the cpk1 cpk2 mutant in F. graminearum. These results indicate that the function of SFL1 orthologs in hyphal growth is well conserved in M. oryzae, F. graminearum, and possibly other filamentous ascomycetes.

Discussion
Like many other filamentous ascomycetes, the rice blast fungus has two genes encoding catalytic subunits of PKA. Whereas the cpkA mutant is defective in appressorium formation and pathogenesis, deletion of CPK2 had no effects on plant infection and appressorium morphogenesis. Interestingly, unlike cpkA, the cpk2 mutant was slightly reduced in growth rate. Therefore, although CpkA plays a more critical role than Cpk2 for pathogenesis, CPK2 may be more important during vegetative growth. In F. graminearum and A. fumigatus, deletion of the CPK2 ortholog had no detectable phenotype [19,20], which differs slightly from cpk2 in M. oryzae. However, the cpkA cpk2 mutant had more severe defects than the single mutants in growth and conidiation, which is similar to F. graminearum and A. fumigatus [19,20]. Therefore, the overlapping functions of CpkA and Cpk2 orthologs during vegetative growth and asexual reproduction may be evolutionally conserved in filamentous ascomycetes.
Although cAMP signaling is known to be important for appressorium formation in a number of fungal pathogens, including Colletotrichum species [44,45], no mutants deleted of both catalytic subunits have been reported in plant pathogenic fungi except in U. maydis. In U. maydis, the mutant deleted of both catalytic subunits was defective in plant infection but its defects in appressorium formation was not examined [21]. Our results showed that PKA activities are essential for appressorium formation in M. oryzae, which has not been previous reported in plant pathogens.
The cAMP-PKA pathway is responsible for surface recognition in M. oryzae. To our surprise, although no tip swelling or appressorium formation was observed, the majority of cpkA cpk2 germ tubes curled or rotated on hydrophobic surfaces. Therefore, germ tubes of the double mutant still responded to surface hydrophobicity although they were blocked in appressorium formation. Sensing of surface hardness and hydrophobicity likely involves mechanosensor proteins, which may trigger polarity disturbance and germ tube curling independent of cAMP signaling. Several putative mechanosensor genes have been shown to be up- regulated during appressorium formation [46]. It is puzzling that most cpkA cpk2 conidia failed to germinate on hydrophilic surfaces, and curling germ tubes were not observed in a few of them that germinated. In filamentous ascomycetes such as A. nidulans and C. trifolii, cAMP signaling is known to regulate conidium germination [44,47]. However, the cpkA cpk2 mutant was normal in conidium germination on hydrophobic surfaces. Because surface attachment is a cue for stimulating conidium germination in M. oryzae, one likely explanation is that deletion of both CPKA and CPK2 may affect the attachment of conidia to hydrophilic surfaces.
The Pmk1 MAP kinase pathway is essential for appressorium formation in M. oryzae and other plant pathogens [9,34]. The cpkA cpk2 mutant had an increased phosphorylation level of Pmk1 but was defective in appressorium formation. It is possible that PKA activity is required to release the suppressive effect of MoSfl1 on genes important for germ tube tip swelling and appressorium formation. Spontaneous suppressors of cpkA cpk2 produced melanized tips in aerial hyphae and appressoria on hydrophilic surfaces, which is similar to transformants expressing the dominant active RAS2 [48]. Therefore, releasing the repressor role of MoSfl1 or MoCyc8-MoTup1 in the cpkA cpk2 mutant in which Pmk1 is over-activated is sufficient to activate appressorium formation under non-conducive conditions. One likely explanation is that some genes required for tip deformation or appressorium formation are only expressed when MoSfl1 is phosphorylated by PKA although the essential role of Pmk1 in appressorium formation involve other downstream targets. Because deletion of MoSFL1 had no effect on appressorium formation [26], it will be interesting to determine the effects of expressing the dominant active MST7 allele in the Mosfl1 deletion mutant.
In M. oryzae, the mac1 mutant is known to produce spontaneous suppressors and some of them had suppressor mutations in the SUM1 gene [30]. In fact, instability of adenylate cyclase mutant and suppressor mutations in regulatory subunit genes are well characterized in S. cerevisiae, U. maydis, and other fungi [30,49,50]. However, to our knowledge, spontaneous suppressors of PKA mutants have not been reported in other fungi. In S. cerevisiae, the tpk1 tpk2 tpk3 triple mutant is not viable. It will be important to assay whether the mutants deleted of both catalytic subunits also produce spontaneous suppressors in U. maydis and A. fumigatus [19,21]. Because the cpk1 cpk2 mutant of F. graminearum [20] was also found to be unstable and had mutations in FgSFL1 in 29 of the 30 suppressor strains sequenced, it is likely that SFL1 orthologs have a conserved role in the repression of genes important for growth and conidiation, at least in filamentous ascomycetes. For the one F. graminearum and two M. oryzae suppressor strains without mutations in the SFL1 ortholog, identification of the suppressor mutations by whole genome sequencing [51] and characterization of the corresponding genes in these mutants in the future will be helpful to better understand the cAMP-PKA pathway in filamentous fungi.
In yeast, SFL1 can function as either a transcriptional activator or repressor [23,24]. In M. oryzae, deletion of MoSFL1 by itself did not affect vegetative growth but resulted in a reduction in virulence [26]. Phenotype characterization of the Mosfl1 mutant is suitable for characterizing its activator but not repressor functions. In this study, we showed that deletion or truncation of MoSFL1 could suppress the defects of cpkA cpk2 mutant in vegetative growth and appressorium formation but not plant infection and conidiation. Interestingly, the suppressor mutants with mutations in FgSFL1 were also recovered in vegetative growth but not pathogenicity in F. graminearum. Whereas M. oryzae forms melanized appressoria for plant penetration, F. graminearum produces infection cushions and hyphopodia [52,53]. However, after plant penetration, both of them form invasive hyphae inside plant cells that are different from vegetative hyphae in hyphal morphology and possibly cell cycle regulation [52,54,55]. The cAMP-PKA pathway is important for plant infection in M. oryzae [34,56] and F. graminearum [20,53], possibly by regulating the growth of invasive hyphae after penetration in these two plant pathogenic fungi with different infection mechanisms. It is possible that MoSFL1 plays an activator role in regulating genes important for invasive growth but negatively regulates genes important for vegetative growth in M. oryzae. However, it is more likely that the cAMP-PKA pathway regulates genes important for plant penetration and invasive growth via other transcription factors.
In yeast, Sfl1 inhibits the transcription of its target genes by interacting with the Cyc8-Tup1 co-repressor [27]. However, it is not clear which region of Sfl1 interacts with Cyc8 or Tup1. Our data clearly showed that the C-terminal 93 amino acids of MoSfl1 is essential for its interaction with Cyc8 (Fig 9). This C-terminal region of MoSfl1 is well conserved in its orthologs from filamentous ascomycetes. In F. graminearum, 15 of the 29 suppressor strains of cpk1 cpk2 mutant had the nonsense mutation at Q501 (S4 Table) in FgSFL1 resulting in the truncation of its C-terminal region. Other 12 suppressor strains had either nonsense or frameshift mutations upstream from Q501. These results indicate that the C-terminal region of Sfl1 orthologs likely plays a conserved role in regulating the expression of genes important for hyphal growth via its association with the Cyc8-Tup1 co-repressor complex. The difference between MoSfl1 and yeast Sfl1 in the C-terminal region may be directly related to the importance of PKA activities in hyphal growth in M. oryzae, F. graminearum, and possibly other filamentous fungi.
Besides the Cyc1-Tup1 co-repressor, Sfl1 also interacts with the mediator proteins Ssn2, Ssn8, Sin4, and Rox3 in S. cerevisiae [23]. Although their orthologs are conserved in M. oryzae, none of them was identified by affinity purification. One possibility is that phosphorylation by PKA is necessary for MoSfl1 to interact with these mediator components but the cpkA cpk2 mutant was used to identify proteins that differentially interacted with MoSfl1 and MoSfl1 ΔCT and responsible the suppression of PKA deficiency. Nevertheless, it is also possible that their interactions with MoSfl1 is mediated by the Cyc8-Tup1 complex, which may be too dynamic or transient in M. oryzae. Among the MoSfl1-interacting proteins identified by affinity purification, MGG_01588 and MGG_06958 are orthologous to BMH1 and SSA1, respectively that interact with Sfl1 in S. cerevisiae. However, several MoSfl1-interacting proteins in M. oryzae (Table 5) such as the putative pathway-specific nitrogen regulator MGG_03286 are unique to filamentous fungi. It will be important to determine their functions in hyphal growth and pathogenesis in M. oryzae.
In S. cerevisiae, Sfl1 is a substrate of Tpk2 [25,57]. However, it is not clear whether S207D mutation (= S211 of MoSfl1) is sufficient to suppress the growth defect of inviable tpk1 tpk2 tpk3 PKA-deficient mutant [58]. In M. oryzae, expression of the MoSFL1 S211D allele in the cpkA cpk2 mutant rescued its growth but not appressorium formation defects. It is possible that phosphorylation of MoSfl1 by PKA disrupts the interaction of MoSfl1 with the Cyc8-Tup1 co-repressor, which in turn activates the expression of genes important for hyphal growth (Fig 9). Residue S211 of MoSfl1 is well conserved in its orthologs from other filamentous ascomycetes, suggesting that its phosphorylation by PKA likely has a conserved role in the regulation of hyphal growth by the cAMP-PKA pathway. Considering the fact that CpkA and Cpk2 are highly similar in the kinase domain and they have overlapping functions in hyphal growth, it is possible that both of them can phosphorylate MoSfl1 at S211. Nevertheless, MoSfl1 may be phosphorylated by CpkA and Cpk2 at different amino acid residues. Therefore, it will be important to characterize the phosphorylation sites of MoSfl1 in the cpkA and/or cpk2 deletion mutants.

Strains and culture conditions
All the M. oryzae strains used in this study (Table 1) were cultured on oatmeal agar (OTA) or complete medium (CM) plates at 25˚C and stored on desiccated Whatman #1 filter paper at In the wild-type strain, phosphorylation of MoSfl1 by PKA disrupts its interaction with the Cyc8-Tup1 co-repressor. Without its association with MoSfl1, the Cyc8-Tup1 complex fails to block the transcription of MoSfl1 target genes that are important for hyphal growth and other developmental or infection processes. B. In the cpkA cpk2 mutant, the Cyc8-Tup1 co-repressor interacts with un-phosphorylated MoSfl1 to repress subsets of its target genes important for growth. C. Suppressor mutations in MoSFL1 block or reduce the association of MoSfl1 with the Cyc8-Tup1 co-repressor to bypass the requirement for its phosphorylation by PKA. P, phosphorylation; MBS, MoSfl1-binding site.

Generation of the cpk2 and cpkA cpk2 mutants
To generate the CPK2 gene replacement construct by double-joint PCR, its 1.2-kb upstream and downstream flanking sequences of CPK2 were amplified with primer pairs 1F/ 2R and 3F/ 4R (S5 Table), respectively (S1 Fig). The hph cassette was amplified with primers Hyg/F and Hyg/R from pCX63 [62]. The resulting products of double-joint PCR were transformed into protoplasts of the wild-type strain Guy11. Putative cpk2 mutants were screened by PCR with primers 5F and 6R and further confirmed by Southern blot analyses with its downstream flanking sequence as the probe. Vegetative hyphae harvested from two-day-old CM cultures were used for DNA and protein isolation as described [63].
The same strategy was used to generate the CPKA gene replacement construct. The 1.2-kb upstream and downstream flanking sequences of CPKA were amplified with primer pairs A1F/A2R and A3F/A4R, respectively (S1 Fig). The G418 cassette was amplified with primers G418/F and G418/R from pFl7. The products of double-joint PCR were transformed into protoplasts of the cpk2 mutant to generate the cpkA cpk2 mutant.

Appressorium formation, penetration, and plant infection assays
Conidia were harvested from 10-day-old OTA cultures and resuspended to 5×10 4 conidia/ml in sterile water. For appressorium formation assays, 50 μl droplets of conidium suspensions were placed on glass cover slips (Fisher Scientific) or GelBond membranes (Cambrex) and incubated at 25˚C for 24 h as described [13,64]. To assay its stimulatory effects, cAMP was added to the final concentration of 10 μM to conidium suspensions [65]. For infection assays, conidia were resuspended to 5×10 5 conidia/ml in 0.25% gelatin. Two-week-old seedlings of rice cultivar CO-39 were used for spray or injection infection assays as described [66,67]. Lesion formation was examined 7 days post inoculation (dpi).

Assays for the TEY phosphorylation of MAP kinases
Vegetative hyphae were harvested from 2-day-old CM cultures and used for protein extraction as described [68]. Total proteins (approximately 20 mg) were separated on a 12.5% SDS-PAGE gel and transferred to nitrocellulose membranes for western blot analysis [62]. Expression and phosphorylation of Pmk1 and Mps1 were detected with the PhophoPlus p44/42 MAP kinase antibody kits (Cell Signaling Technology) following the manufacturer's instructions.

Assays for the intracellular cAMP levels
Intracellular cAMP was extracted from vegetative hyphae harvested from two-day-old CM cultures as described [69] and detected with the cAMP enzyme immunoassay (EIA) system (Amersham Pharmacia Biotech) following the manufacturer's instructions.

Co-immunoprecipitation (co-IP) assays
The yeast gap repair approach was used to generate the S-tag and 3×FLAG fusion constructs [68]. To generate the SUM1-, TUP1-, and CYC8-S-tag constructs, each gene was amplified and cloned into vector pXY203 [66,70]. MoSFL1 was cloned into vector pFL6 to generate the 3×FLAG-MoSFL1 construct. CPKA and CPK2 were cloned into vector pFL7 [70] to generate the CPKA-3xFLAG and CPK2-3×FLAG fusion constructs. All of the resulting S-tag and 3×FLAG fusion constructs were confirmed by sequencing analysis and transformed into Guy11 or the cpkA cpk2 mutant in pairs. Total proteins were isolated from the resulting transformants and incubated with the anti-S-Tag Antibody Agarose beads (Bethyl Laboratories). Proteins bound to anti-S-tag agarose were eluted and used for western blot analysis [13,31]. The presence of related fusion proteins was detected with the anti-FLAG (Sigma-Aldrich) or anti-S (Abcam) antibody as described [31].

Spontaneous suppressors of the cpkA cpk2 mutant
Fast-growing sectors of the cpkA cpk2 mutant were transferred with sterile toothpicks to fresh oatmeal agar plates. After single-spore isolation, each subculture of spontaneous suppressors was assayed for defects in growth, conidiation, and plant infection [71,72]. To identify suppressor mutations, all the candidate downstream target genes of PKA were amplified with primers listed in S5 Table and sequenced. Mutation sites were identified by sequence alignment with sequences of target genes in the reference genome [1] and their PCR products.
Generation of the cpkA cpk2 Mosfl1 and cpkA cpk2 MoSFL1 ΔCT mutants To generate the cpkA cpk2 Mosfl1 mutant, the upstream and downstream flanking sequences of MoSFL1 were amplified with primer pairs Sfl1ko1F/Sfl1ko2R and Sfl1ko3F/ Sfl1ko4R (S5 Table), respectively, and fused with the ble cassette amplified from pFL6 [73] by double-joint PCR [74]. To generate the cpkA cpk2 MoSFL1 ΔCT mutant, the flanking sequences of MoSFL1 were amplified with primer pairs CCSko1F/CCSko2R (S5 Table) and Sfl1ko3F/ Sfl1ko4R (S5 Table). The resulting MoSFL1 and MoSFL1 CT gene replacement PCR products were transformed into protoplasts of the cpkA cpk2 mutant. Putative Mosfl1 or MoSFL1 CT mutants were screened by PCR analysis and verified for the deletion of MoSFL1 or its C-terminal region (496-588 aa).

Affinity purification and mass spectrometry analysis
The full length MoSFL1 and MoSFL1 ΔCT fragments were amplified with primers listed in S5 Table and cloned into vector pFL6 by yeast gap repair [70]. The 3×FLAG-MoSFL1 and 3×FLAG-MoSFL1 ΔCT fusion constructs were rescued from the resulting yeast transformants and transformed into the cpkA cpk2 mutant. Hyphae of the 3×FLAG-MoSFL1 and 3×FLAG-MoSFL1 ΔCT transformants were homogenized with a glass beater at 4˚C for protein extraction [31,68]. Proteins eluted from anti-FLAG resins (Sigma-Aldrich) were digested with trypsin and the resulting tryptic peptides were analyzed with nanoflow liquid chromatography tandem mass spectrometry (MS) as described [31,[75][76][77]. Proteins were identified by searching MS data against NCBI non-redundant F. graminearum protein database with the SEQUEST™ algorithm [78]. At least three independent biological replicates were analyzed to identify proteins that interact with MoSfl1 WT and MoSfl1 ΔCT .