Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Two Phosphodiesterase Genes, PDEL and PDEH, Regulate Development and Pathogenicity by Modulating Intracellular Cyclic AMP Levels in Magnaporthe oryzae

  • Haifeng Zhang,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Kaiyue Liu,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Xing Zhang,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Wei Tang,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Jiansheng Wang,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Min Guo,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Qian Zhao,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Xiaobo Zheng,

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

  • Ping Wang,

    Affiliation Department of Pediatrics and the Research Institute for Children, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America

  • Zhengguang Zhang

    zhgzhang@njau.edu.cn

    Affiliation Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China

Two Phosphodiesterase Genes, PDEL and PDEH, Regulate Development and Pathogenicity by Modulating Intracellular Cyclic AMP Levels in Magnaporthe oryzae

  • Haifeng Zhang, 
  • Kaiyue Liu, 
  • Xing Zhang, 
  • Wei Tang, 
  • Jiansheng Wang, 
  • Min Guo, 
  • Qian Zhao, 
  • Xiaobo Zheng, 
  • Ping Wang, 
  • Zhengguang Zhang
PLOS
x

Abstract

Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies.

Introduction

Heterotrimeric G protein signaling is one of the most important mechanisms by which eukaryotic cells sense extracellular signals and integrate them into intrinsic signal transduction pathways, such as cAMP-dependent signaling pathway. cAMP is a ubiquitous second messenger produced in cells in response to hormones and nutrients [1]. The level of cAMP is dependent on the actions of many different proteins that affect its synthesis and degradation, such as PDEases. As a second messenger, cAMP plays an important role in activating downstream signaling components, such as phosphorylating enzyme protein kinase A (PKA). Both cAMP and PKA play key roles in the phosphorylation and regulation of enzyme substrates involved in intermediary metabolism [1].

In fungi, the highly conserved components of the cAMP-signaling cascade have been co-opted for a variety of purposes. In Saccharomyces cerevisiae, intracellular cAMP levels are regulated by the activity of the low- and high-affinity PDEases, Pde1 and Pde2, respectively. Pde2 is a high-affinity PDEase expressed in many organisms ranging from fungi to mammals [2]. In contrast, the low-affinity PDEase Pde1 is less well characterized but has been found in a wide range of organisms, including S. cerevisiae, Schizosaccharomyces pombe, Candida albicans, Dictyostelium discoideum, Vibrio fischeri, Leishmania mexicana, Trypanosoma brucei, and Trypanosoma cruzi [3], [4], [5], [6], [7], [8], [9], [10].

In S. cerevisiae, Pde2 modulates the basal level of cAMP, which is important in regulating nutrient sensing, pseudohyphal differentiation, cell cycle progression, and stress signaling [11], [12], [13], [14], [15], [16]. In contrast, Pde1 does not significantly affect basal levels of cAMP and does not have any obvious mutant phenotype. In S. pombe, mating, sporulation, and gluconeogenesis are regulated by cAMP/low-affinity Pde1 [17], [18], [4], [19]. In both S. cerevisiae and S. pombe, Pde1 regulates cAMP levels in responding to the presence of glucose [20], [21]. In S. cerevisiae, the cAMP degradation activity of Pde1 is positively regulated by the PKA catalytic subunits [22], whereas in S. pombe the regulation of Pde1 activity is also seen to be dependent on the PKA catalytic subunits or allosteric activation by cAMP [20], [23]. Pde2 was shown to control intracellular cAMP levels of human pathogenic fungus C. albicans and deletion of PDE2 encoding Pde2 leads to defects in filamentation, nutrient sensing, entry into stationary phase, and cell wall and membrane integrity [24], [25]. Unlike S. cerevisiae and C. albicans, deletion of PDE2 in the human fungal pathogen Cryptococcus neoformans resulted in subtle mutant phenotypes, in contrast to deletion of PDE1, which led to elevation of the basal intracellular cAMP levels. The Δpde1 mutant displayed certain defects in sexual differentiation and several important characteristics of virulence, and, moreover, the Pde1 activity is regulated through PKA-derived phosphorylation [26]. Additionally, cAMP signaling is known to modulate dimorphic transition and virulence of the plant pathogenic fungus Ustilago maydis [27], [28], [29], [30], [31] and morphogenesis, cell polarity, and asexual development of Neurospora crassa and Aspergillus nidulans [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42].

Magnaporthe oryzae, the causal agent of rice blast disease, is the most destructive pathogen of cultivated rice worldwide [43]. Several different stages of the disease cycle are essential for successful disease development. Upon contact, asexual conidia become firmly attached to the host leaves, with the aid of the mucilage stored in their tips. Subsequently, the conidia germinate and form appressoria towards the end of germ tubes. Enormous turgor pressure is generated to penetrate the plant cuticle by the appressoria [44], [45]. Initiation of appressorium formation in M. oryzae was shown to require cAMP signaling, because deletion of MAC1 encoding adenylyl cyclase resulted in a defect in appressorium formation [46]. This defect could be restored by adding exogenous cAMP or by a second-site mutation in SUM1 encoding the regulatory subunit of PKA, resulting in constitutive activation of the PKA catalytic subunit [47]. Consistent with these observations, cPKA (the catalytic subunit of PKA) replacement mutants showed delayed appressorium formation and formation of small, misshapen, and nonfunctional appressoria [48], [49]. Moreover, disruption of the MAGB gene encoding a Galpha subunit resulted in significant reductions in vegetative growth, conidiation, appressorium formation, and pathogenicity [50]. Conversely, expression of a dominant active MagB allele caused appressoria to form on non-inductive surfaces and addition of cAMP can restore appressorium formation in the ΔmagB mutant [50], [51], [52]. A regulator of G protein signaling Rgs1 was recently shown to negatively regulate G protein signaling and the cAMP pathway of M. oryzae [53]. Deletion of RGS1 resulted in a significant increase in intracellular cAMP levels and formation of appressoria on hydrophylic surfaces, indicating that Rgs1 is an important negative regulator of appressorium development [53]. The low- and high-affinity PDEases, PdeL (Pde1) and PdeH (Pde2), were recently described for M. oryzae in an elegant study by Ramanujam and Naqvi [54]. The study demonstratied that PdeH is a key regulator of asexual and pathogenic development [54]. Here, we provide further evidence indicating that PdeL also plays a role in regulating the intracellular cAMP level, asexual development, and conidial morphology. Additionally, PdeH has a role in regulating intracellular cAMP levels during pathogenic and invasive growth, as deletion of PDEH resulted in defects in conidial morphology, cell wall integrity, surface hydrophobicity, and attenuated pathogenicity. Moreover, disruption of PDEH partially rescued mutant phenotypes of ΔmagB and Δpka1, and additional studies suggested that PdeH may function through a feedback mechanism to regulate expression of the pathogenicity factor Mpg1, which is involved in the regulation of the surface hydrophobicity and pathogenicity of M. oryzae.

Materials and Methods

Strains and culture conditions

Guy11 was used as a wild type strain in this study. All strains were cultured on complete medium (CM) agar plates [55]. Liquid CM medium was used to prepare the vegetative mycelia to extract DNA and RNA. For conidiation, strain blocks were maintained on straw decoction and corn agar media (SDC: 100 g of straw, 40 g of corn powder, 15 g of agar in 1 L of distilled water) at 28°C for 7 days in the dark followed by 3 days of continuous illumination under fluorescent light.

Targeted gene deletion and complementation analysis

The PDEL and PDEH gene deletion mutants were generated using the standard one-step gene replacement strategy. First, two 1.0 kb of sequences flanking of targeted gene were PCR amplified with primer pairs FL656 & FL657, FL658 & FL659 (for PDEL) and FL660 & FL661, FL662 & FL663 (for PDEH) (Table S1), then a ∼2 kb fragment containing the two flanking sequences was amplified with primers FL656/FL659 (for PDEL) and FL660/FL663 (for PDEH) by overlap PCR. All amplified sequences and fragments were sequenced and ligated to flank the hygromycin resistance cassette in pMD19-T vector (Takara Co. Dalian, China). The ∼3.4-kb fragments amplified by primers FL656/FL659 (for PDEL) and FL660/FL663 (for PDEH) were transformed into protoplasts of wild type Guy11. The 3.3-kb and 4.9-kb fragments which contained the entire PDEL and PDEH genes were amplified by PCR with primers FL1033/FL1034 and FL1035/FL10346, respectively, and inserted into pCB1532 (sulphonylurea resistance) to complement the ΔpdeL and ΔpdeH strains. For double gene deletion in the ΔpdeH mutant, the same strategy was used and the hygromycin resistance cassette was replaced by sulphonylurea (SUR) resistance cassette to screen the transformants.

Hyphal autolysis and surface hydrophobicity assays

For hyphal autolysis assay, small agar blocks were cut from the edge of 4-day-old cultures and placed onto CM medium with 1 M sorbitol and cultured in the dark at 28°C for two weeks. The size and morphology of the colonies were examined every day and photographed at the 14th day. The experiment was performed in triplicate. For surface hydrophobicity assay, the strains were plated onto CM agar plates and incubated at 28°C for 14-day. Sterile distilled water (10 ml) was placed on the surface of cultures. In addition, wettability of the aerial hyphae to solution containing both 0.02% SDS and 5 mM EDTA was also assessed as previously described [56].

Conidiation and appressorium formation assays

For conidiation, 10-day-old conidia were collected with 3 ml of distilled water, filtered through three layers of lens paper and counted with a haemacytometer under a microscope. The conidial size was measured by a built-in microscope ruler. More than 200 conidia were measured for each strain. For appressorium formation, conidia were resuspended to a concentration of 5×104 spores per milliliter in sterile water. Droplets (30 µl) of conidial suspension were placed on plastic cover slips (hydrophobic) and Gelbond films (hydrophylic) and incubated under humid conditions at room temperature as described previously [57]. Appressorium formation rate was counted at 24 hours post-inoculation (hpi) under the microscope, more than 200 appressoria were counted for each strain. Photographs were taken at 24 days post inoculation (hpi).

Pathogenicity assay

Conidia were harvested as described above and resuspended to a concentration of 5×104 spores per milliliter in a 0.2% (w/v) gelatin solution. Two-week-old seedlings of rice (Oryza sativa cv CO39) and 7-day-old seedlings of barley (cv Four-arris) were used for the assay. Three 20 µl droplets were placed onto the upper side of the detached barley leaves maintained on 4% (w/v) water agar plates. Pictures were taken 5 days after incubation at 25°C. For spray inoculation, 5 ml of conidial suspension of each treatment was sprayed onto rice with a sprayer. Inoculated plants were kept in a growth chamber at 25°C with 90% humidity and in the dark for the first 24 hours, followed by a 12/12 hours light/dark cycle. Lesion formation was observed daily. Photographs were taken 7 days after inoculation.

Rice sheath penetration and turgor assay

For microscopic observation of penetration and infectious hyphae expansion on rice tissue, rice cultivar CO-39 was prepared as previously described [58] and inoculated with 100 µl of conidial suspension (1×104 spores per milliliter) on the inner leaf sheath cuticle cells. After 48 hours incubation under humid conditions at room temperature, the leaf sheaths were observed under a microscope. Appressorium turgor was measured by incipient cytorrhysis (cell collapse) assay using a 1–5 M glycerol solution as described previously [44].

Laccase activity assay

Laccase activity was monitored on 0.2 mM 2, 2′-azino-di-3-ethylbenzath- iazoline-6-sulfonate (ABTS) CM agar plate assays using mycelial blocks at 2 dpi in dark at 28°C. The enzyme activity was measured from 2-day-old CM liquid cultures. Mycelia were removed completely by filtration and centrifugation (5,000 g at 4°C) and processed using a colorimetric determination as described previously [59].

Intracellular cAMP measurement

Two-day-old liquid mycelial cultures were harvested, frozen in liquid nitrogen and lyophilized for 16 hours. Intracellular cAMP extraction was followed as previously described [53]. The cAMP levels were quantified according to the cAMP Biotrak Immuno-assay System (Amersham Biosciences, NJ, USA).

Construction of MPG1 over-expression construct

To generate MPG1 over-expression construct, the genomic DNA of Guy11 was amplified by PCR with primers FL3972/FL3973. The resulting PCR product contained MPG1 gene sequence driven by TrpC promoter. It was then digested with HindIII and KpnI and cloned into pCB1532, resulting in the MPG1 expression vector pCB1532::MPG1. After transforming the pCB1532::MPG1 into ΔpdeH mutant, the SUR resistant transformants were screened by qRT-PCR with primers MPG1_QF/MPG1_QR. The ΔpdeH MPG1-3 was one of the transformants with the high MPG1 expression level. The ΔpdeH MPG1-2 with the low MPG1 expression level than ΔpdeH mutant was used as a control.

Nucleic acid manipulation and DNA microarray

The standard Southern blot protocol was utilized. The target gene probe and HPH probe were amplified with primer pairs FL467/FL2180 (for PDEL), FL468/FL2181 (for PDEH) and FL1111/FL1112 (for HPH), respectively. Probe labeling, hybridization and detection were performed with the DIG High Prime DNA Labeling and Detection Starter Kit I. RNA was isolated from frozen fungal mycelia, conidia (10-day old), appressoria (24 hpi) and infected rice leaves (72 hpi) with TRIzol Reagent (Invitrogen, USA). Quantitative Real-time PCR (qRT-PCR) was run on the Applied Biosystems 7300 Real Time PCR System with SYBR Premix Ex Taq™ (Perfect Real Time, Takara, Japan). Normalization and comparison of mean Ct values were performed as described [60]. The experiment was conducted twice with three independent biological replicates.

For microarray, RNA was purified by QIAGEN RNAeasy mini kit (Qiagen Inc., Valencia, CA, USA) and quality analysis and quantification performed using the Agilent Bioanalyzer (Agilent Technologies, Inc., Wilmington, DE, USA) and the Nano Drop (NanoDrop Wilmington, DE, USA). Three biological replicates of each RNA sample were performed. Microarray data collection and correlation analysis was carried out by SBC (Shanghai Biochip Co., Ltd. China).

The above primers used in this paper were listed in Table S1.

Gene Ontology and functional annotation

Orthologs were identified between M. oryzae predicted proteins and proteins in the GO database [61] via searching reciprocal best hits with the following cut-offs; e-value, 1.0e-3, and identity, 20%. Results from local alignment using BLAST, functional domain comparisons from NCBI and prediction of signal peptides from SignalP 3.0 software and a manual literature review were used to make final assignments to GO functional categories.

Results

PDEH is highly expressed during plant colonization but not in mycelial growth

To gain insight into functions of PdeL and PdeH, we first examined the gene expression profiles at different stages of fungal development by qRT-PCR. There was no significant difference in the abundance of PDEL transcripts. However, higher levels of PDEH expression were found in conidia, appressoria, and infection stages than in mycelia. In the infection stage, the expression of PDEH increased by almost 23-fold (Table 1). These observations suggested that PdeH might have an important role in in planta infection.

thumbnail
Table 1. Real-time RT-PCR quantification of PdeL and PdeH expression in M. oryzae.

https://doi.org/10.1371/journal.pone.0017241.t001

Targeted PDEL and PDEH gene replacement

To examine the roles of PdeL and PdeH, PDEL and PDEH gene replacement constructs (Figure S1A) were generated (see Materials and Methods) and transformed into the protoplasts of the wild-type strain Guy11. The resulting hygromycin-resistant transformants were screened by PCR and confirmed by Southern blotting and RT-PCR analysis. No fragment was detected in the ΔpdeL or ΔpdeH mutants when hybridizing with gene-specific probes (probe1). RT-PCR analysis indicated that there was no PDEL or PDEH transcript in the respective mutants (Figure S1B). The ΔpdeLΔpdeH mutant, as well as ΔpdeHΔmagB and ΔpdeHΔpka1 double mutants, was also confirmed by Southern blotting and RT-PCR analysis (Figure S1B).

PdeL but not PdeH is indispensable for conidiogenesis

To examine functions of PdeL and PdeH, conidiation of Guy11, ΔpdeL, ΔpdeH mutants, and complemented mutant strains were examined. Conidiation in 10-day-old cultures of the ΔpdeL mutant was markedly reduced to approximately 8% of the wild-type strain, while conidia in the ΔpdeH mutant were only reduced approximately 80% (Table 2). In contrast, the wild-type strain and complement strains showed normal sporulation under the same conditions. The data suggested that PdeL is required for conidiation in M. oryzae.

PdeL and PdeH are involved in conidial morphogenesis but dispensable for mycelial growth

To further explore the role of PdeL in conidiation, conidia of the ΔpdeL mutant was examined, along with and the ΔpdeH mutant and other control strains. Surprisingly, both ΔpdeL and pdeH mutants produced elongated and thinner conidia, which were uniform and readily detected under the microscope (Figure 1A). The conidia of the mutants were, on average, longer by ∼7 µm and thinner by ∼4 µm than those of the controls (Figure 1B). Frequent branching and curly tips were also observed at the terminal hyphae of the ΔpdeH deletion mutant. However, Calcofluor white (CFW) staining of mycelial cell walls showed that the septa were normal except for shorter intervals (Figure 1C). To determine whether PdeL and PdeH are involved in vegetative growth, the mutant and control strains were cultured on a variety of media including CM, OMA, SDC, or V8 juice agar media. No significant difference in colony morphology or growth rate was observed (Table 2). Combined, these findings suggested that PdeL and PdeH are indispensable for conidium morphology and but dispensable for mycelial growth.

thumbnail
Figure 1. The ΔpdeL and ΔpdeH mutants have defects in conidial morphology and hyphal branching.

(A) Conidia of the wild type and mutants were observed under an epi-fluorescence microscope. Conidia were stained with 1 µg/ml Calcofluor white (CFW) for 5 min in dark. (B) Conidial size of the wild type and mutants. Values are the mean ±SD from 100 conidia for each strain, which were measured using a microscope ruler. Length is the distance from the base to apex of conidia. And width is the size of the longest septum. (C) Branching patterns of mycelia on complete media slides at day 2 after incubation. Frequent branching occurs at the terminal mycelia of ΔpdeH and ΔpdeHΔpdeL double mutants. Calcofluor staining of mycelia is used to show the distance of septa.

https://doi.org/10.1371/journal.pone.0017241.g001

PdeH is essential for maintenance of cell wall integrity

While the growth of the ΔpdeH mutant appeared normal on CM agar plates (Table 2), the ΔpdeH mutant did undergo progressive autolysis of mycelia after prolonged incubation for at least 14 days (Figure 2, top panel). Autolysis began at the center of the colony and radiated outward. The autolysis of the ΔpdeH mutant can be suppressed by addition of 1 M sorbitol to the culture medium (Figure 2, middle panel). The autolysis tended to be more severe in the ΔpdeLΔpdeH mutant than ΔpdeH, similar to the Δmps1 and Δmck1 mutants that exhibited a defect in cell wall integrity [62], [63]. Interestingly, the ΔpdeH mutant did not undergo autolysis on DSC medium under the same conditions (Figure 2, bottom panel), indicating that PdeH may also be involved in sensing nutrients in the maintenance of cell wall integrity.

thumbnail
Figure 2. ΔpdeH mutants have a defect in cell-wall integrity.

Growth of wild type and mutant strains on complete media (CM) without 1 M sorbitol (top); growth of strains on CM with 1 M sorbitol (middle); growth of strains on straw decoction and corn media (SDC) without sorbitol (bottom). The ΔpdeH and ΔpdeH ΔpdeL mutants undergo progressive autolysis on CM in the absence of osmotic stabilization. Radial growth rates are identical to those of the wild-type strains.

https://doi.org/10.1371/journal.pone.0017241.g002

PdeH is required for surface hydrophobicity

In previous studies, disruption of several M. oryzae hydrophobin genes, including MPG1 and MHP1, resulted in a water- or detergent-soaked easily wettable phenotype [64], [65], [66], [55], [67], [68], [69]. To determine whether PdeL and PdeH are involved in surface hydrophobicity, the mutant and wild type strains were tested with water and detergent solutions. Unlike Δmpg1, the ΔpdeH mutant did not show an easily wettable phenotype when incubated with water droplets (10 µl) for 24 hours. However, aerial hyphae of ΔpdeH mutants that were grown on CM agar were more readily wettable by a solution containing both 0.02% SDS and 5 mM EDTA within 5 min (Figure 3A), as seen in the Δmhp1 mutant. The detergent-wettable phenotype shown by ΔpdeH mutant was stably maintained up to four successive generations, suggesting that this phenotype is mitotically stable (data not shown). Based on the results described above, we suggested that the surface hydrophobicity defect of ΔpdeH and ΔpdeLΔpdeH mutants might be related to Mpg1 and Mhp1. To assess this, we examined the levels of MPG1 and MHP1 expression in the mutant and wild-type strains. The MPG1 expression level showed a 50% decrease in the ΔpdeL mutant compared with the wild-type control, while the expression reduced more than 90% in the ΔpdeH and ΔpdeLΔpdeH mutants (Figure 3B). In contrast, there was no apparent difference in MHP1 expression in any of the mutants compared with the wild-type control (data not shown). These results indicated that the surface hydrophobicity defects of ΔpdeH and ΔpdeLΔpdeH mutants were likely due to the lowed MPG1 expression.

thumbnail
Figure 3. Detergent wettable phenotype of ΔpdeH and ΔpdeHΔpdeL mutants.

(A) Ten microliters of water or detergent solution containing 0.02% SDS and 5 mM EDTA were placed on the colony surfaces of the wild type and mutants strains and photographed after 5 min. (B) Expression analysis of MPG1 gene in wild type and mutant strains. The error bars indicate SD of three replicates. Asterisk indicates significant differences at P = 0.01.

https://doi.org/10.1371/journal.pone.0017241.g003

Over-expression of MPG1 partially restores the surface hydrophobicity and pathogenicity to the ΔpdeH mutant

As mentioned above, the expression level of MPG1 was significantly decreased in the ΔpdeH mutant. Additionally, Mpg1 is known as an important pathogenicity factor in M. oryzae [55]. We over-expressed MPG1 in the ΔpdeH mutant to determine whether Mpg1 could restore the defects in surface hydrophobicity and pathogenicity. We screened the ΔpdeH/MPG1 transformants by qRT-PCR to check the MPG1 expression level and obtained four MPG1-over-expressing transformant strains. One of these, ΔpdeH/MPG1-3, in which the MPG1 expression level was increased to 150% of that in the wild-type, was able to restore the defects of surface hydrophobicity and pathogenicity of the ΔpdeH mutant. However, the transformant ΔpdeH/MPG1-2, in which MPG1 expression level was almost the same as that of the ΔpdeH mutant, could not complement the defects (Figure 4A and 4B). We concluded that the hydrophobicity and pathogenicity defects in the ΔpdeH mutant were primarily due to the low level of MPG1 expression, and it is likely that the expression level of MPG1 must remain at 150% of wild-type or more to maintain the surface hydrophobicity.

thumbnail
Figure 4. Over-expression MPG1 in the ΔpdeH mutant partially restores the surface hydrophobicity and pathogenicity defects.

(A) Ten microliters of water or detergent solution containing 0.02% SDS and 5 mM EDTA were placed on the colony surfaces of wild type, mutants and Mpg1 over-expression strains and photographed after 5 min. Expression analysis of MPG1 gene in wild type and mutants and MPG1 over-expression strains. The error bars indicate SD of three replicates. Asterisk indicates significant differences at P = 0.01. (B) Spraying assay. Five milliliters of conidial suspension (5×104 spores/ml) of each strain were sprayed on rice seedlings. Diseased leaves were photographed 7 days after inoculation.

https://doi.org/10.1371/journal.pone.0017241.g004

PdeL and PdeH regulate intracellular cAMP levels

Abundant studies have shown that PDEases can regulate intracellular cAMP levels in various organisms [3], [4], [5], [6], [7], [8], [9], [10]. To determine whether PdeL and PdeH also regulate cAMP levels in M. oryzae, we measured the intracellular cAMP levels in the hyphal stage. The results indicated that the ΔpdeL mutant accumulated only ∼1.5-fold higher levels of cAMP than the wild-type strain, while the ΔpdeH mutant and the ΔpdeL ΔpdeH double mutant accumulated ∼3-fold and ∼4.5-fold higher levels cAMP compared with the wild-type, respectively (Figure 5).

thumbnail
Figure 5. PdeL and PdeH regulate intracellular cAMP levels during pathogenesis.

Loss of PDEL and PDEH leads to increased accumulation of cAMP levels. Bar chart showing quantification of intracellular cAMPs in the mycelia of the indicated strains cultured for 2 days in complete medium. Two biological repetitions with three replicates were assayed. The error bars represent SD of three replicates.

https://doi.org/10.1371/journal.pone.0017241.g005

PdeH is required for appressorium differentiation, full virulence and elicitation of plant defense responses

Physical cues of inductive surfaces, such as hardness and hydrophobicity, are necessary for appressorium formation [53]. The wild-type strain is unable to form appressoria on non-inductive surfaces, except in the presence of exogenous cAMP or inhibitors of PDEases [70]. To verify the effects of increased cAMP levels in the ΔpdeL and ΔpdeH mutants, we examined appressorium formation on inductive and non-inductive surfaces. The conidia and hyphal tips of the ΔpdeL and ΔpdeH mutants formed normal melanized appressoria on inductive surfaces, similar to the wild-type strain (Figure 6A, Table 2). However, the ΔpdeH mutant was able to form normal melanized appressoria on non-inductive surfaces (Figure 6B, Table 2), indicating that PdeH is an important negative regulator of appressorium formation.

thumbnail
Figure 6. Appressorium formation assays.

(A) Conidia of each strain were incubated on hydrophobic surfaces for 24 hours (up panel); hyphal plugs of each strain were incubated on hydrophobic surfaces for 48 hours (bottom panel). (B) Conidia of each strain were incubated on hydrophilic surfaces for 24 hours.

https://doi.org/10.1371/journal.pone.0017241.g006

To determine whether PDEL and PDEH are involved in pathogenesis, conidial suspensions were sprayed onto susceptible rice seedlings and hyphal plugs were placed onto detached rice seedling leaves (CO-39 cv. oryzae) to develop rice blast lesions. In a spray-inoculation test, the ΔpdeH mutant produced tiny and restricted lesions, whereas the ΔpdeL mutant, wild-type, and complement strains showed susceptible-type spreading lesions (Figure 7A). In a detached-inoculation test, lesions caused by ΔpdeH mutant hyphal plugs spread very slowly and the lesion sizes were small, while those caused by ΔpdeL mutant, wild type, and complement strains spread rapidly and the neighboring lesions joined together by 5 days after inoculation (Figure 7B).

thumbnail
Figure 7. The loss of PDEH leads to reduced pathogenicity and induction of strong plant defense responses.

(A) Spraying assay. Five milliliters of conidial suspension (5×104 spores/ml) of each strain were sprayed on rice seedlings. Diseased leaves were photographed 7 days after inoculation. (B) Detached leaf assay. The hyphal plugs of each strain were placed onto the upside of detached rice seedling leaves. Diseased leaves were photographed 5 days after inoculation. (C) Observation of infectious growth. Excised rice sheath from 4-week-old rice seedlings was inoculated with conidial suspension (5×104 spores/ml of each strain). Infectious growth was observed 48 hours after inoculation. (D) The expression of rice pathogenesis-related (PR) genes over time after inoculation. The transcriptional expression of PR1a and PBZ1 in the infected rice was analyzed using quantitative RT-PCR. The graph was generated with three replicates in a representative data set, and similar results were obtained in another independent biological repetition. The error bars indicate SD of three replicates.

https://doi.org/10.1371/journal.pone.0017241.g007

Because no defects in appressorium development by the ΔpdeH mutant were observed on hydrophobic surface, the development of infectious hyphae within the host cells was examined using excised leaf sheaths. In the wild-type, ΔpdeL mutant, and complement strains, infectious hyphae grew actively and extended into 3–4 cells neighboring the primary infected cells by 48 hours after inoculation (Figure 7C). However, infectious hyphae of the ΔpdeH mutant was mostly restricted to the primary infected cells and almost no infectious hyphae extended into neighboring cells, and accumulation of dark brown granules was seen along the infectious hyphae (Figure 7C). These results were consistent with those of rice infection assays. Additionally, the expression levels of the rice defense-related genes PR1a and PBZ1 in response to ΔpdeH infection were much higher than those associated with infection by the wild-type, ΔpdeL mutant, and complement strains when analyzed by quantitative RT-PCR (Figure 7D). These observations indicated that the induction of plant defense responses in rice challenged by ΔpdeH mutant might contribute to the retardation of infectious hyphal development.

PdeL and PdeH regulate laccase activities

A previous study indicated that laccases are involved in the pathogenicity of certain fungi [71], [97], and the laccase activity can be detected readily using the specific substrate ABTS (2,2'-azino-di-3-ethylbenzthiazoline-6-sulfonate) [72], [73]. To determine whether PDEases are involved in the regulation of laccase activity, we tested ΔpdeL and ΔpdeH mutant strains on CM agar plates and in liquid medium supplemented with 0.2 mM ABTS. In each case, decreases in laccase activity were observed in ΔpdeL, ΔpdeH, and ΔpdeLΔpdeH mutants, with a less-oxidized dark purple stain around the colonies of the mutants and a lower level of the laccase activity in the culture filtrate compared with the wild-type strain (Figure 8A and 8B). Consistent with these observations, the expression of two extracellular peroxidase genes, MGG_08200.6 and MGG_01924.6, was significantly down-regulated in all the mutants (Figure 8C). Meanwhile, the results also indicated that the laccase activity was decreased to a greater extent in the ΔpdeL mutant than in the ΔpdeLΔpdeH and ΔpdeH mutants (Figure 8A and 8B), suggesting that PdeL may have a more prominent role in regulating the enzyme activity.

thumbnail
Figure 8. PdeL and PdeH are related to the activity of extracellular laccases.

(A) Laccase activity was tested on CM agar medium containing 0.2 mM ABTS at final concentration. Discoloration was observed on day 2. (B) Laccase activity measured by ABTS oxidizing test (see Materials and Methods for details). (C) Quantitative RT-PCR analysis of two laccase genes in wild type and mutants. Expression data were normalized using the ACTIN gene. Error bars represent standard deviation.

https://doi.org/10.1371/journal.pone.0017241.g008

Genome-wide identification of genes regulated by PdeL and PdeH

To identify genes regulated by PdeL and PdeH, we compared gene expression profiles of ΔpdeL, ΔpdeH, and ΔpdeLΔpdeH mutants with the wild-type strain during the hyphal stage. In total, 1582 genes were up-regulated and 1724 genes were down-regulated in ΔpdeL, ΔpdeH, and ΔpdeLΔpdeH mutants (Figure 9A). Of the 1582 up-regulated genes, we found 373 that were regulated by PdeL, 459 by PdeH, and 1248 by PdeL and PdeH together. Of the 1724 down-regulated genes, the corresponding numbers were 509, 652, and 1450, respectively. Those genes in which the expression ratio was greater than 2-fold were functionally grouped into GO categories as described in the Materials and Methods (Figure 9B, Tables S2, S3 and S4). Overall, we noted that genes associated with protein and amino acid degradation, lipid degradation, secondary metabolism including melanin biosynthesis, and cellular transportation exhibited marked decreases in expression. Among genes that were up-regulated by PdeL, PdeH, or PdeL and PdeH, we were able to positively assign two genes, MGG_07881.6 and MGG_03508.6, to PdeH, in contrast to many by PdeL or PdeL and PdeH (Table S5).

thumbnail
Figure 9. Functional categorization of the consensus genes.

(A) Expression profiles were combined and showing PdeL- and PdeH-dependent. (B) Up-regulation and down-regulation of more than two-fold change genes were grouped according to their putative function (see Supplemental data for details).

https://doi.org/10.1371/journal.pone.0017241.g009

To confirm gene expression patterns derived from our microarray experiments, we performed real-time RT-PCR with four selected genes that were down-regulated in the ΔpdeH mutant. Two genes, MPG1 and PTH11, which are required for pathogenesis, were significantly down-regulated (∼10-fold; Figure 4B and 10). Two laccase genes, MGG_11608.6 and MGG_13464.6, were down-regulated to a greater extent (∼100-fold) in the ΔpdeH mutant (Figure 8C). Although the magnitudes of fold changes in laccase gene expression were much higher than the microarray results, the real-time RT-PCR data supported those of the microarray analyses.

thumbnail
Figure 10. PTH11 gene expression in ΔpdeH and ΔpdeL mutants.

RNA was extracted from mycelia cultured in liquid CM medium for 2 days. ACTIN was used for normalization, and the values were calculated by 2-ddCT methods with quantitative RT-PCR data. Values represent mean ±SD from two independent experiments with three replicates each. Asterisk indicates significant differences at P = 0.01.

https://doi.org/10.1371/journal.pone.0017241.g010

Characterization of novel genes regulated by PdeH

To further explore the role of PdeH in M. oryzae, we deleted nine down-regulated genes identified from the ΔpdeH mutant microarray profile. MGG_00311.6 encodes an acid protease involved in protein and amino acid degradation, MGG_11608.6 encodes an oxidase enzyme laccase that is found in many plants, fungi, and microorganisms, MGG_10631.6 encodes a glycoside hydrolase involved in carbohydrate metabolism, MGG_07571.6 encodes a putative cell wall degrading protein with a LysM domain, MGG_07218.6 encodes a transcription factor containing a GAL4-like Zn2-Cys6-binuclear cluster DNA-binding domain at the N-terminal, MGG_06326.6 encodes a vacuolar ATP synthase 16-kDa proteolipid subunit, and, finally, MGG_12214.6encodes a polyketide synthase. Two putative ion transporters, P-type ATPase MGG_04852.6 and sugar transporter MGG_10293.6, were also characterized. Overall, mutant conidia produced normal melanized appressoria on hydrophobic surfaces, and none of them formed appressoria on noninductive surfaces (Figure 11 and data not shown). However, the mutant strain for transcription factor MGG_07218.6 showed significantly reduced pathogenicity with tiny and slow-spread blast lesions, a phenotype similar to that of the ΔpdeH mutant (Figure 11).

thumbnail
Figure 11. Assessment for appressorium formation and pathogenicity of PdeH targeted gene deletion mutants.

Gene expression was assessed according to description in text. Appressorium formation and pathogenicity assessment were also performed as described in text.

https://doi.org/10.1371/journal.pone.0017241.g011

Discussion

Cyclic AMP signaling plays an important role in regulating the growth and differentiation of eukaryotic organisms, including rice blast pathogen M. oryzae. A recent study by Ramanujam and Naqvi [54] has demonstrated that phosphodiesterase PdeL and PdeH have both conserved and distinct functions in regulating cAMP levels and pathogenicity of M. oryzae. Here, our presented our independent study that not only corroborates with that of recent publication but also further illustrates roles of PdeL and PdeH in this fungus. We found that deletion of PDEH resulted in defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. We also found that PDEH disruption partially rescued the ΔmagB and Δpka1 mutant phenotypes. Moreover, we propose that PdeH might function through a feedback mechanism to regulate the expression of Mpg1, which is a demonstrated pathogenicity factor involved in surface hydrophobicity and pathogenic development of M. oryzae.

The fungal cell wall plays an important role in maintaining cell shape and mediating exchanges between the cell and its environment [74]. Although it is rigid, its organization and structure must be remodeled constantly for growth and development [63]. Thus, in pathogenic fungi, the ability to maintain cell wall integrity is critical to the establishment of disease within the host. Several cell wall integrity-associated genes have been characterized in S. cerevisiae, including MEKK (Bck1), a pair of redundant MEKs (Mkk1/2), and a MAP kinase (Slt2) [75]. The Bck1 and Slt2 homologs Mck1 and Mps1 were described as also essential for cell wall integrity and pathogenicity of M. oryzae [76], [63]. In both S. cerevisiae and C. albicans, deletion of PDE2 affected cell wall integrity, stress response, hyphal development, and/or virulence [25], [77], [78]. As we failed to reveal any phenotypic changes between the ΔpdeH mutant and the wild-type control in the response to stress agents, such as ionic stress (Na+ and Cu2+), oxidative stress (H2O2), heavy metal (CoCl2) stress, osmotic stress (sorbitol), or cell wall-disturbing agents (CFW, SDS, and lysing enzyme) M. oryzae may have an alternative approach of responding to stress signals or may possess different stress sensors.

We found that ΔpdeLΔpdeH double mutant undergo faster autolysis than the ΔpdeH mutant on CM agar plates, and the ΔpdeLΔpdeH mutant also showed a higher level of cAMP level than the ΔpdeH mutant. This suggests that cAMP plays an important role in the maintenance of cell wall integrity and, while both are involved in regulating intracellular cAMP levels and cell wall integrity, PdeH might have a more prominent role. Moreover, the ΔpdeH single and ΔpdeLΔpdeH double mutants did not undergo autolysis or showed only very slight autolysis when cultured on SDC media (poor nutrition), suggesting that PdeH may act as a nutrition sensor in modulating intracellular cAMP levels, which may affect cell wall integrity. Regardless, further studies are required to determine whether and how PdeH senses nutritional signals.

Most hydrophobins confer surface hydrophobicity on fungi forming a spore rodlet layer. Disruption of several hydrophobin genes, including Mpg1, resulted in a water- or detergent-soaked easily wettable phenotype [64], [65], [66], [55], [67], [68]. The Mpg1 mutant is defective in conidiation and appressorium formation, and less pathogenic than wild-type strain, indicating that Mpg1 plays an important role in multiple infection-related processes. Here, deletion of PDEH resulted in a significantly reduced level of MPG1 expression and defects in surface hydrophobicity and pathogenicity [55]. In Δmpg1 mutants, the defect in appressorium formation can be restored by adding exogenous cAMP, suggesting that Mpg1 functions upstream of cAMP signal for appressorium formation [55]. Here, over-expression of MPG1 in the ΔpdeH mutant partially rescued the surface hydrophobicity and pathogenicity defects, suggesting that Mpg1 could function downstream of PdeH and cAMP signaling. Thus, we propose that PdeH may function through a feedback mechanism to activate Mpg1 and regulates its expression for surface hydrophobicity and pathogenic development.

Several induced genes in plants have been reported to affect fungal pathogenicity, directly or indirectly [79], [55], [80], [81], [69]. The high level of PDEH expression at late infection stages in infected rice leaves also suggests its roles in infectious growth and pathogenicity. Targeted disruption of PDEH significantly reduced virulence in infection assays. The ΔpdeH mutant produced smaller and less numerous lesions than the wild-type strain. The results presented above indicated that appressoria formed by ΔpdeH mutants are probably defective in penetration. It is likely that PdeH regulates the processes involved in a late stage of appressorium development, such as turgor generation or appressorium pore formation. The qRT-PCR results, indicating that PDEH was expressed at high levels in the appressoria, also supported this possibility. We assayed appressorium turgor using the cytorrhysis method described previously [44]. Preliminary data do not indicate any difference in cytorrhysis between appressoria formed by ΔpdeH mutant and wild-type strains (data not shown), suggesting that appressoria of ΔpdeH mutants do not have defects in the generation of turgor pressure. Another possibility is that PdeH may regulate the early stages of appressorium penetration, such as development of the penetration peg or differentiation of infectious hyphae. The reduction in pathogenicity may be due to the reduction in development at the pre-penetration stages or a reduction in infectious growth of ΔpdeH mutants in host cells. This may also explain the high level of PDEH expression during the late stages of infection.

Defense responses induced by the recognition of microbe-associated molecules (pathogen-associated molecular patterns, PAMPs) are often associated with cell wall strengthening, rapid production of reactive oxygen species (ROS), and the transcriptional activation of PR genes [82]. In plants, accumulation of ROS at the site of infection is considered one of the earliest responses during plant PAMP-triggered immunity (PTI) [83], [82]. Plant-derived ROS have various functions during plant–microbe interactions [84], [85], [86], [87], [88], [89]. For pathogens to survive in harsh environments and successfully invade host cells, they must develop mechanisms to scavenge ROS and protect against ROS-induced damage [90], [91], [92], [93]. In M. oryzae, several well-characterized genes have been reported to suppress basal host defenses and to be responsible for ROS detoxification at the site of infection [58], [59], [94]. As rice cells infected by the ΔpdeH mutant showed brown granule accumulation and cell death, it is likely that plant defense responses are involved in virulence attenuation of ΔpdeH mutant. Thus, the defense responses against the wild-type and mutant strains were compared with regard to two genes, PR1a and PBZ1. These two genes are important components of the jasmonic acid (JA) and salicylic acid (SA) pathways, which are involved in JA- and SA-induced plant defense, respectively [95], [96]. The stronger activation of both PR1a and PBZ1 in mutant-inoculated plants compared with wild-type controls suggested that ΔpdeH mutant can still elicit plant defenses, which may be involved in attenuation of the virulence of the ΔpdeH mutant.

In M. oryzae, many evidences suggest that MagB may sense surface cues, stimulate cAMP synthesis, and activate the cAMP signal pathway [50], [51], [52]. Because PdeH can degrade intracellular cAMP, we hypothesize that the decrease in the PdeH activity in the ΔmagB mutant may maintain the balance of intracellular cAMP levels and restore its defects. The result of partially restoration in appressorium formation in the ΔpdeHΔmagB double mutant supported this hypothesis (Table 2). Additionally, the appressorium formation rate on hydrophylic surfaces was reduced to 10% compared with ΔpdeH mutants, suggesting that MagB and PdeH can complement each other in some defects. However, no other defect was restored in the ΔpdeHΔmagB double mutant, all of which were similar to those in the ΔpdeH mutant (Figure 1, 2, 3 and 8). These observations may have been due to the high cAMP level in the ΔpdeH ΔmagB double mutant, because the cAMP level was still much higher in ΔpdeH ΔmagB than in the wild-type (Figure 5). Since cAMP-dependent protein kinase PKA plays a pivotal role in cAMP-dependent pathways of M. oryzae, we compared the phenotype of Δpka1 with that of the ΔpdeH Δpka1 double mutant. The ΔpdeHΔpka1 mutant formed small, misshapen appressoria on hydrophobic surfaces similar to the Δpka1 mutant, while very few appressoria were formed on hydropholic surfaces (Figure 6). However, other phenotypes, such as cell wall integrity and surface hydrophobicity, which may also be due to the high cAMP level, were similar to those of the ΔpdeH mutant (Figure 1, 2, 3 and 8). Based on these results, we conclude that, while MagB and Pka1 activate the cAMP signaling pathway to mediate appressorium formation and pathogenicity, PdeH plays a more critical role in regulating intracellular cAMP levels to affect cell wall integrity and surface hydrophobicity, bypassing the PKA signaling pathway.

Genome-wide analysis of gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared with induction by cAMP revealed new insight into appressorium formation and function in M. oryzae [98]. During appressorium formation, the genes that respond to both stimuli are known to be involved in protein and amino acid degradation, lipid metabolism, secondary metabolism, and cellular transportation. In this study, the levels of expression of two pathogenicity-related genes were significantly decreased in the appressoria treated with cAMP. Targeted deletion of several other changed genes, such as polyketide synthase (MGG_07219.6), subtilisin-like protease (MGG_03670.6), and a transcription factor (MGG_07218.6), affected virulence and other characteristics related to pathogenicity. Similar to these results, our microarray data also provided some novel insight to identify pathogenicity factors. Three genes, MPG1 (MGG_10315.6), PTH11 (MGG_05871.6), and COS1 (MGG_03977.6), which have been reported to be involved in pathogenicity [55], [99], [100], were significantly down-regulated in the ΔpdeH single and ΔpdeLΔpdeH double mutants, but not in the ΔpdeL mutant. Furthermore, deletion of a down-regulated transcription factor, from the ΔpdeH microarray data, also showed reduced pathogenicity, similar to that of the ΔpdeH mutant. These data may explain why ΔpdeH and ΔpdeLΔpdeH mutants attenuated virulence.

In summary, we continued the characterization of the low- and high-affinity cAMP PDEases PdeL and PdeH in M. oryzae. We showed that, while PdeL and PdeH share certain functions, PdeH indeedplays a more prominent role than PdeL in regulating cell wall integrity, surface hydrophobicity and pathogenicity through modulation of intracellular cAMP levels. Our findings also reveal that PdeH may function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1, which is involved in surface hydrophobicity and pathogenic development in M. oryzae.

Supporting Information

Figure S1.

Generation of ΔpdeL and ΔpdeH deletion mutants. (A) Restriction map of the PDEL and PDEH genomic region and knockout construct. Thick arrows indicate orientations of the PDEL, PDEH and hygromycin phosphotransferase (hph) genes. Thin lines below the arrows indicate the probe sequences of each gene. The restriction enzymes are EcoRV (EV), EcoRI (EI), XbaI (XI) and KpnI (KI). (B) Southern blot and RT-PCR analyses of ΔpdeL, ΔpdeH (top) and double-gene (bottom) knockout mutants. Genomic DNA of the wild-type strain and the knockout mutants was digested with corresponding restriction enzymes. Total RNAs of the wild-type strain and the knockout mutants were isolated and the expression levels of target gene were detected using ACTIN as control. WT: wild type; T: transformant.

https://doi.org/10.1371/journal.pone.0017241.s001

(TIF)

Figure S2.

Confirmation of target gene replacement by PCR.

https://doi.org/10.1371/journal.pone.0017241.s002

(PDF)

Table S2.

Categorization of PDEL regulated genes with known function.

https://doi.org/10.1371/journal.pone.0017241.s004

(DOC)

Table S3.

Categorization of PDEH regulated genes with known function.

https://doi.org/10.1371/journal.pone.0017241.s005

(DOC)

Table S4.

Categorization of PDEL&PDEH regulated genes with known function.

https://doi.org/10.1371/journal.pone.0017241.s006

(DOC)

Table S5.

Categorization of genes only dependent on PDEL , PDEH and PDEL & PDEH , respectively.

https://doi.org/10.1371/journal.pone.0017241.s007

(DOC)

Acknowledgments

We thank Dr. Amy Whittington, Louisiana State University Health Sciences Center, New Orleans, LA USA, for commenting on the manuscript.

Author Contributions

Conceived and designed the experiments: HZ X. Zheng ZZ. Performed the experiments: HZ KL X. Zhang WT JW MG QZ. Analyzed the data: HZ X. Zheng PW ZZ. Contributed reagents/materials/analysis tools: HZ KL X. Zhang WT JW MG QZ. Wrote the manuscript: HZ PW ZZ.

References

  1. 1. Daniel PB, Walker WH, Habener JF (1998) Cyclic AMP signaling and gene regulation. Annu Rev Nutr 18: 353–83.PB DanielWH WalkerJF Habener1998Cyclic AMP signaling and gene regulation.Annu Rev Nutr1835383
  2. 2. Charbonneau H, Beier N, Walsh KA, Beavo JA (1986) Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species. Proc Natl Acad Sci USA 83: 9308–9312.H. CharbonneauN. BeierKA WalshJA Beavo1986Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species.Proc Natl Acad Sci USA8393089312
  3. 3. D’Angelo MA, Sanguineti S, Reece JM, Birnbaumer L, Torres HN, et al. (2004) Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi. Biochem J 378: 63–72.MA D’AngeloS. SanguinetiJM ReeceL. BirnbaumerHN Torres2004Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.Biochem J3786372
  4. 4. De Voti JSG, Beach D, McLeod M (1991) Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO J 10: 3759–3768.JSG De VotiD. BeachM. McLeod1991Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis.EMBO J1037593768
  5. 5. Dunlap PV, Callahan SM (1993) Characterization of a periplasmic 3_:5_-cyclic nucleotide phosphodiesterase gene, cdpP, from the marine symbiotic bacterium Vibrio fischeri. J Bacteriol 175: 4615–4624.PV DunlapSM Callahan1993Characterization of a periplasmic 3_:5_-cyclic nucleotide phosphodiesterase gene, cdpP, from the marine symbiotic bacterium Vibrio fischeri.J Bacteriol17546154624
  6. 6. Hoyer LL, Cieslinski LB, McLaughlin MM, Torphy TJ, Shatzman AR, et al. (1994) A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme. Microbiology 140: 1533–1542.LL HoyerLB CieslinskiMM McLaughlinTJ TorphyAR Shatzman1994A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme.Microbiology14015331542
  7. 7. Kunz S, Kloeckner T, Essen LO, Seebeck T, Boshart M (2004) TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei. Eur J Biochem 271: 637–647.S. KunzT. KloecknerLO EssenT. SeebeckM. Boshart2004TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei.Eur J Biochem271637647
  8. 8. Lacombe ML, Podgorski GJ, Franke J, Kessin RH (1986) Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum. J Biol Chem 261: 16811–16817.ML LacombeGJ PodgorskiJ. FrankeRH Kessin1986Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum.J Biol Chem2611681116817
  9. 9. Nikawa J, Sass P, Wigler M (1987) Cloning and characterization of the low affinity cAMP phosphodiesterase gene of S. cerevisiae. Mol Cell Biol 7: 3629–3636.J. NikawaP. SassM. Wigler1987Cloning and characterization of the low affinity cAMP phosphodiesterase gene of S. cerevisiae.Mol Cell Biol736293636
  10. 10. Rascon A, Viloria ME, De-Chiara L, Dubra ME (2000) Characterization of cyclic AMP phosphodiesterases in Leishmania mexicana and purification of soluble form. Mol Biochem Parasitol 106: 283–292.A. RasconME ViloriaL. De-ChiaraME Dubra2000Characterization of cyclic AMP phosphodiesterases in Leishmania mexicana and purification of soluble form.Mol Biochem Parasitol106283292
  11. 11. Lemaire K, Van de Velde S, Van Dijck P, Thevelein JM (2004) Glucose and sucrose act as agonist and mannose as antagonist ligands of the G proteincoupled receptor Gpr1 in the yeast Saccharomyces cerevisiae. Mol Cell 16: 293–299.K. LemaireS. Van de VeldeP. Van DijckJM Thevelein2004Glucose and sucrose act as agonist and mannose as antagonist ligands of the G proteincoupled receptor Gpr1 in the yeast Saccharomyces cerevisiae.Mol Cell16293299
  12. 12. Jones DL, Petty J, Hoyle DC, Hayes A, Oliver SG, et al. (2004) Genome-Wide Analysis of the Effects of Heat Shock on a Saccharomyces cerevisiae Mutant With a Constitutively Activated cAMP-Dependent Pathway. Comp Funct Genomics 5: 419–431.DL JonesJ. PettyDC HoyleA. HayesSG Oliver2004Genome-Wide Analysis of the Effects of Heat Shock on a Saccharomyces cerevisiae Mutant With a Constitutively Activated cAMP-Dependent Pathway.Comp Funct Genomics5419431
  13. 13. Jones DL, Petty J, Hoyle DC, Hayes A, Ragni E, et al. (2003) Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/ cAMP pathway. Physiol Genomics 16: 107–118.DL JonesJ. PettyDC HoyleA. HayesE. Ragni2003Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/ cAMP pathway.Physiol Genomics16107118
  14. 14. Pan X, Heitman J (1999) Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19: 4874–4887.X. PanJ. Heitman1999Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae.Mol Cell Biol1948744887
  15. 15. Park JI, Grant CM, Dawes IW (2005) The highaffinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses. Biochem Biophys Res Commun 327: 311–319.JI ParkCM GrantIW Dawes2005The highaffinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses.Biochem Biophys Res Commun327311319
  16. 16. Schneper L, Krauss A, Miyamoto R, Fang S, Broach JR (2004) The Ras/protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell cycle progression and proper bud site selection. Eukaryot Cell 3: 108–120.L. SchneperA. KraussR. MiyamotoS. FangJR Broach2004The Ras/protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell cycle progression and proper bud site selection.Eukaryot Cell3108120
  17. 17. Davey J (1998) Fusion of a fission Yeast. Yeast 1529–1566.J. Davey1998Fusion of a fission Yeast.Yeast15291566
  18. 18. Yamamoto M (1996) The molecular control mechanisms of meiosis in fission yeast. Trends Biochemical Sciences. pp. 18–22.M. Yamamoto1996The molecular control mechanisms of meiosis in fission yeast.Trends Biochemical Sciences1822
  19. 19. Higuchi T, Watanabe Y, Yamamoto M (2002) Protein kinase A regulates sexual development and gluconeogenesis through phosphorylation of the Zn finger transcriptional activator Rst2p in fission yeast. Mol Cell Biol 22: 1–11.T. HiguchiY. WatanabeM. Yamamoto2002Protein kinase A regulates sexual development and gluconeogenesis through phosphorylation of the Zn finger transcriptional activator Rst2p in fission yeast.Mol Cell Biol22111
  20. 20. Hoffman CS (2005) Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe. Biochem Soc Trans 33: 261–264.CS Hoffman2005Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe.Biochem Soc Trans33261264
  21. 21. Ma P, Wera S, Dijck PV, Thevelein JM (1999) The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell 10: 91–104.P. MaS. WeraPV DijckJM Thevelein1999The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling.Mol Biol Cell1091104
  22. 22. Nikawa JI, Cameron S, Toda T, Ferguson KM, Wigler M (1987) Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. Genes Dev 1: 931–937.JI NikawaS. CameronT. TodaKM FergusonM. Wigler1987Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae.Genes Dev1931937
  23. 23. Meima ME, Weening KE, Schaap P (2003) Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum. J Biol Chem 278: 14356–14362.ME MeimaKE WeeningP. Schaap2003Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum.J Biol Chem2781435614362
  24. 24. Bahn YS, Staab J, Sundstrom P (2003) Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans. Mol Microbiol 50: 391–409.YS BahnJ. StaabP. Sundstrom2003Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans.Mol Microbiol50391409
  25. 25. Jung WH, Warn P, Ragni E, Popolo L, Nunn CD, et al. (2005) Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans. Yeast 22: 285–294.WH JungP. WarnE. RagniL. PopoloCD Nunn2005Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans.Yeast22285294
  26. 26. Hicks JK, Bahn YS, Heitman J (2005) Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans. Eukaryot Cell 4: 1971–1981.JK HicksYS BahnJ. Heitman2005Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans.Eukaryot Cell419711981
  27. 27. Larraya LM, Boyce KJ, So A, Steen BR, Jones S, et al. (2005) Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis. Eukaryot Cell 4: 2029–2043.LM LarrayaKJ BoyceA. SoBR SteenS. Jones2005Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis.Eukaryot Cell420292043
  28. 28. Regenfelder E, Spellig T, Hartmann A, Lauenstein S, Bolker M, et al. (1997) G proteins in Ustilago maydis: transmission of multiple signals? EMBO J 16: 1934–1942.E. RegenfelderT. SpelligA. HartmannS. LauensteinM. Bolker1997G proteins in Ustilago maydis: transmission of multiple signals?EMBO J1619341942
  29. 29. Durrenberger F, Wong K, Kronstad JW (1998) Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis. Proc Natl Acad Sci USA 95: 5684–5689.F. DurrenbergerK. WongJW Kronstad1998Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis.Proc Natl Acad Sci USA9556845689
  30. 30. Gold SE, Brogdon SM, Mayorga ME, Kronstad JW (1997) The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize. Plant Cell 9: 1585–1594.SE GoldSM BrogdonME MayorgaJW Kronstad1997The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize.Plant Cell915851594
  31. 31. Lee N, Kronstad JW (2002) ras2 Controls morphogenesis, pheromone response, and pathogenicity in the fungal pathogen Ustilago maydis. Eukaryot Cell 1: 954–966.N. LeeJW Kronstad2002ras2 Controls morphogenesis, pheromone response, and pathogenicity in the fungal pathogen Ustilago maydis.Eukaryot Cell1954966
  32. 32. Banno S, Ochiai N, Noguchi R, Kimura M, Yamaguchi I, et al. (2005) A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa. Genes Genet Syst 80: 25–34.S. BannoN. OchiaiR. NoguchiM. KimuraI. Yamaguchi2005A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa.Genes Genet Syst802534
  33. 33. Bencina M, Panneman H, Ruijter GJ, Legisa M, Visser J (1997) Characterization and overexpression of the Aspergillus niger gene encoding the cAMPdependent protein kinase catalytic subunit. Microbiology 143 (Pt 4): 1211–1220.M. BencinaH. PannemanGJ RuijterM. LegisaJ. Visser1997Characterization and overexpression of the Aspergillus niger gene encoding the cAMPdependent protein kinase catalytic subunit.Microbiology143 Pt 412111220
  34. 34. Brakhage AA, Liebmann B (2005) Aspergillus fumigatus conidial pigment and cAMP signal transduction: significance for virulence. Med Mycol 43: Suppl 1S75–82.AA BrakhageB. Liebmann2005Aspergillus fumigatus conidial pigment and cAMP signal transduction: significance for virulence.Med Mycol43Suppl 1S7582
  35. 35. Grosse C, Heinekamp T, Kniemeyer O, Gehrke A, Brakhage AA (2008) Protein kinase A regulates growth, sporulation, and pigment formation in Aspergillus fumigatus. Appl Environ Microbiol 74: 4923–4933.C. GrosseT. HeinekampO. KniemeyerA. GehrkeAA Brakhage2008Protein kinase A regulates growth, sporulation, and pigment formation in Aspergillus fumigatus.Appl Environ Microbiol7449234933
  36. 36. Ivey FD, Kays AM, Borkovich KA (2002) Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa. Eukaryot Cell 1: 634–642.FD IveyAM KaysKA Borkovich2002Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa.Eukaryot Cell1634642
  37. 37. Kays AM, Rowley PS, Baasiri RA, Borkovich KA (2000) Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa. Mol Cell Biol 20: 7693–7705.AM KaysPS RowleyRA BaasiriKA Borkovich2000Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa.Mol Cell Biol2076937705
  38. 38. Kays AM, Borkovich KA (2004) Severe impairment of growth and differentiation in a Neurospora crassa mutant lacking all heterotrimeric G alpha proteins. Genetics 166: 1229–1240.AM KaysKA Borkovich2004Severe impairment of growth and differentiation in a Neurospora crassa mutant lacking all heterotrimeric G alpha proteins.Genetics16612291240
  39. 39. Liebmann B, Gattung S, Jahn B, Brakhage AA (2003) cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages. Mol Genet Genomics 269: 420–435.B. LiebmannS. GattungB. JahnAA Brakhage2003cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages.Mol Genet Genomics269420435
  40. 40. Saudohar M, Bencina M, van de Vondervoort PJ, Panneman H, Legisa M, et al. (2002) Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger. Microbiology 148: 2635–2645.M. SaudoharM. BencinaPJ van de VondervoortH. PannemanM. Legisa2002Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger.Microbiology14826352645
  41. 41. Liebmann B, Muller M, Braun A, Brakhage AA (2004) The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus. Infect Immun 72: 5193–5203.B. LiebmannM. MullerA. BraunAA Brakhage2004The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus.Infect Immun7251935203
  42. 42. Lafon A, Han KH, Seo JA, Yu JH, d’Enfert C (2006) G-protein and cAMP mediated signaling in Aspergilli: a genomic perspective. Fungal Genet Biol 43: 490–502.A. LafonKH HanJA SeoJH YuC. d’Enfert2006G-protein and cAMP mediated signaling in Aspergilli: a genomic perspective.Fungal Genet Biol43490502
  43. 43. Talbot NJ (2003) On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. Annu Rev Microbiol 57: 177–202.NJ Talbot2003On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea.Annu Rev Microbiol57177202
  44. 44. Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci USA 88: 11281–11284.RJ HowardMA FerrariDH RoachNP Money1991Penetration of hard substrates by a fungus employing enormous turgor pressures.Proc Natl Acad Sci USA881128111284
  45. 45. Howard RJ, Valent B (1996) Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annu Rev Microbiol 50: 491–512.RJ HowardB. Valent1996Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea.Annu Rev Microbiol50491512
  46. 46. Choi WB, Dean RA (1997) The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9: 1973–1983.WB ChoiRA Dean1997The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.Plant Cell919731983
  47. 47. Adachi K, Hamer JE (1998) Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. Plant Cell 10: 1361–1373.K. AdachiJE Hamer1998Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea.Plant Cell1013611373
  48. 48. Mitchell TK, Dean RA (1995) The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant Cell 7: 1869–1878.TK MitchellRA Dean1995The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.Plant Cell718691878
  49. 49. Xu JR, Urban M, Sweigard JA, Hamer JE (1997) The CPKA gene of Magnaporthe grisea is essential for appressorial penetration. Mol Plant-Microbe Interact 10: 187–194.JR XuM. UrbanJA SweigardJE Hamer1997The CPKA gene of Magnaporthe grisea is essential for appressorial penetration.Mol Plant-Microbe Interact10187194
  50. 50. Liu SH, Dean RA (1997) G protein alpha subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. Mol Plant-Microbe Interact 10: 1075–1086.SH LiuRA Dean1997G protein alpha subunit genes control growth, development, and pathogenicity of Magnaporthe grisea.Mol Plant-Microbe Interact1010751086
  51. 51. Fang EG, Dean RA (2000) Site-directed mutagenesis of the magB gene affects growth and development in Magnaporthe grisea. Mol Plant-Microbe Interact 13: 1214–1227.EG FangRA Dean2000Site-directed mutagenesis of the magB gene affects growth and development in Magnaporthe grisea.Mol Plant-Microbe Interact1312141227
  52. 52. Ebbole DJ (2007) Magnaporthe as a model for understanding hostpathogen interactions. Annu Rev Phytopathol 45: 437–56.DJ Ebbole2007Magnaporthe as a model for understanding hostpathogen interactions.Annu Rev Phytopathol4543756
  53. 53. Liu H, Suresh A, Willard FS, Siderovski DP, Lu S, et al. (2007) Rgs1 regulates multiple Galpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. EMBO J 26: 690–700.H. LiuA. SureshFS WillardDP SiderovskiS. Lu2007Rgs1 regulates multiple Galpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism.EMBO J26690700
  54. 54. Ramanujam R, Naqvi NI (2010) PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae. PLoS Pathogen 6: e1000897.R. RamanujamNI Naqvi2010PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.PLoS Pathogen6e1000897
  55. 55. Talbot NJ, Ebbole DJ, Hamer JE (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5: 1575–1590.NJ TalbotDJ EbboleJE Hamer1993Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea.Plant Cell515751590
  56. 56. Stringer MA, Timberlake WE (1995) dewA encodes a fungal hydrophobin component of the Aspergillus spore wall. Mol Microbiol 16: 33–44.MA StringerWE Timberlake1995dewA encodes a fungal hydrophobin component of the Aspergillus spore wall.Mol Microbiol163344
  57. 57. Zhang HF, Zhao Q, Liu KY, Zhang ZG, Wang YC, et al. (2009) MgCRZ1, a transcription factor of Magnaporthe grisea, controls growth, development and is involved in full virulence. FEMS Microbiol Lett 2: 160–169.HF ZhangQ. ZhaoKY LiuZG ZhangYC Wang2009MgCRZ1, a transcription factor of Magnaporthe grisea, controls growth, development and is involved in full virulence.FEMS Microbiol Lett2160169
  58. 58. Guo M, Guo W, Chen Y, Dong SM, Zhang X, et al. (2010) The bZIP transcription factor Moatf1 mediates oxidative stress responses and is necessary for full virulence of the rice blast fungus Magnaporthe oryzae. Mol Plant-Microbe Interactions 23: 1053–1068.M. GuoW. GuoY. ChenSM DongX. Zhang2010The bZIP transcription factor Moatf1 mediates oxidative stress responses and is necessary for full virulence of the rice blast fungus Magnaporthe oryzae.Mol Plant-Microbe Interactions2310531068
  59. 59. Chi MH, Park SY, Kim S, Lee YH (2009) A novel pathogenicity gene is required in the rice bast fungus to suppress the basal defenses of the host. PLoS Pathogen 5: e1000401.MH ChiSY ParkS. KimYH Lee2009A novel pathogenicity gene is required in the rice bast fungus to suppress the basal defenses of the host.PLoS Pathogen5e1000401
  60. 60. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods 25: 402–408.KJ LivakTD Schmittgen2001Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method.Methods25402408
  61. 61. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene Ontology: tool for the unification of biology. Nat Genet 25: 25–29.M. AshburnerCA BallJA BlakeD. BotsteinH. Butler2000Gene Ontology: tool for the unification of biology.Nat Genet252529
  62. 62. Xu JR, Staiger CJ, Hamer JE (1998) Inactivation of the mitogen-activated protein kinase MPS1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defence responses. Proc Natl Acad Sci USA 95: 12713–12718.JR XuCJ StaigerJE Hamer1998Inactivation of the mitogen-activated protein kinase MPS1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defence responses.Proc Natl Acad Sci USA951271312718
  63. 63. Jeon J, Goh J, Yoo S, Chi MH, Choi J, et al. (2008) A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Mol Plant-Microbe Interact 21: 525–534.J. JeonJ. GohS. YooMH ChiJ. Choi2008A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae.Mol Plant-Microbe Interact21525534
  64. 64. Stringer MA, Dean RA, Sewell TC, Timberlake WE (1991) Rodletless, a new Aspergillus developmental mutant induced by direct gene inactivation. Gene Dev 5: 1161–1171.MA StringerRA DeanTC SewellWE Timberlake1991Rodletless, a new Aspergillus developmental mutant induced by direct gene inactivation.Gene Dev511611171
  65. 65. Bell-Pedersen D, Dunlap JC, Loros JJ (1992) The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes Dev 6: 2382–2394.D. Bell-PedersenJC DunlapJJ Loros1992The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer.Genes Dev623822394
  66. 66. Lauter FR, Russo VE, Yanofsky C (1992) Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora. Genes Dev 6: 2373–2381.FR LauterVE RussoC. Yanofsky1992Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora.Genes Dev623732381
  67. 67. van Wetter MA, Schuren FHJ, Schuurs TA, Wessels JGH (1996) Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae. FEMS Microbiol Lett 140: 265–269.MA van WetterFHJ SchurenTA SchuursJGH Wessels1996Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae.FEMS Microbiol Lett140265269
  68. 68. Spanu PD (1998) Deletion of HCf-1, a hydrophobin gene of Cladosporium fulvum, does not affect pathogenicity on tomato. Physiol Mol Plant–Pathol 52: 323–334.PD Spanu1998Deletion of HCf-1, a hydrophobin gene of Cladosporium fulvum, does not affect pathogenicity on tomato.Physiol Mol Plant–Pathol52323334
  69. 69. Kim S, Ahn IlP, Rho HS, Lee YH (2005) MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Molecular Microbiology 57: 1224–1237.S. KimIlP AhnHS RhoYH Lee2005MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization.Molecular Microbiology5712241237
  70. 70. Lee YH, Dean RA (1993) cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea. Plant Cell 5: 693–700.YH LeeRA Dean1993cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea.Plant Cell5693700
  71. 71. Bar-Nunn N, Tal-Lev A, Harel E, Mayer AM (1988) Repression of laccase formation in Botrytis cinerea and its possible relation to phytopathogenicity. Phytochemistry 27: 2505–2509.N. Bar-NunnA. Tal-LevE. HarelAM Mayer1988Repression of laccase formation in Botrytis cinerea and its possible relation to phytopathogenicity.Phytochemistry2725052509
  72. 72. Song WW, Dou XY, Qi ZQ, Wang Q, Zhang X, et al. (2010) R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PLoS ONE 5: e13193.WW SongXY DouZQ QiQ. WangX. Zhang2010R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae.PLoS ONE5e13193
  73. 73. Dou XY, Wang Q, Qi ZQ, Song WW, Wang W, et al. (2011) MoVam7, a conserved SNARE component involved in vacuole assembly, is required for growth, endocytosis, chitin distribution, ROS accumulation, and pathogenesis of Magnaporthe oryzae. PLoS ONE 6: e16439.XY DouQ. WangZQ QiWW SongW. Wang2011MoVam7, a conserved SNARE component involved in vacuole assembly, is required for growth, endocytosis, chitin distribution, ROS accumulation, and pathogenesis of Magnaporthe oryzae.PLoS ONE6e16439
  74. 74. Cabib E, Roh DH, Schmidt M, Crotti LB, Varma A (2001) The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J Biol Chem 276: 19679–19682.E. CabibDH RohM. SchmidtLB CrottiA. Varma2001The yeast cell wall and septum as paradigms of cell growth and morphogenesis.J Biol Chem2761967919682
  75. 75. Levin DE (2005) Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69: 262–291.DE Levin2005Cell wall integrity signaling in Saccharomyces cerevisiae.Microbiol Mol Biol Rev69262291
  76. 76. Xu JR (2000) MAP kinases in fungal pathogens. Fungal Genet Biol 31: 137–152.JR Xu2000MAP kinases in fungal pathogens.Fungal Genet Biol31137152
  77. 77. Park JI, Grant CM, Dawes IW (2005) The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses. Biochem Biophys Res Commun 327: 311–319.JI ParkCM GrantIW Dawes2005The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses.Biochem Biophys Res Commun327311319
  78. 78. Wilson D, Tutulan-Cunita A, Jung WH, Hauser , NC , Hernandez R, et al. (2007) Deletion of the high-affinity cAMP phosphodiesterase encoded by PDE2 affects stress responses and virulence in Candida albicans. Molecular Microbiology 65: 841–856.D. WilsonA. Tutulan-CunitaWH JungHauserNCR. Hernandez2007Deletion of the high-affinity cAMP phosphodiesterase encoded by PDE2 affects stress responses and virulence in Candida albicans.Molecular Microbiology65841856
  79. 79. Pieterse CMJ, Riach MBR, Bleker T, van den Berg-Verthuis GCM, Govers F (1993) Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential hybridization of a genomic library. Physiol Mol Plant Pathol 43: 69–79.CMJ PieterseMBR RiachT. BlekerGCM van den Berg-VerthuisF. Govers1993Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential hybridization of a genomic library.Physiol Mol Plant Pathol436979
  80. 80. Lauge R, Joosten MHA, van den Ackerveken GFJM, van den Broek HWJ, De Wit PJGM (1997) The in planta-produced extracellular proteins ECP1 and ECP2 of Cladosporium fulvum are virulence factors. Mol Plant–Microbe Interact 10: 725–734.R. LaugeMHA JoostenGFJM van den AckervekenHWJ van den BroekPJGM De Wit1997The in planta-produced extracellular proteins ECP1 and ECP2 of Cladosporium fulvum are virulence factors.Mol Plant–Microbe Interact10725734
  81. 81. Stephenson SA, Hatfield J, Rusu AG, Maclean DJ, Manners JM (2000) CgDN3: an essential pathogen city gene of Colletotrichum gloeosporioides necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis. Mol Plant-Microbe Interact 13: 929–941.SA StephensonJ. HatfieldAG RusuDJ MacleanJM Manners2000CgDN3: an essential pathogen city gene of Colletotrichum gloeosporioides necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis.Mol Plant-Microbe Interact13929941
  82. 82. Nürnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 198: 249–266.T. NürnbergerF. BrunnerB. KemmerlingL. Piater2004Innate immunity in plants and animals: striking similarities and obvious differences.Immunol Rev198249266
  83. 83. Apostol I, Heinstein PF, Low PS (1989) Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: Role in defense and signal transduction. Plant Physiol 90: 109–116.I. ApostolPF HeinsteinPS Low1989Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: Role in defense and signal transduction.Plant Physiol90109116
  84. 84. Abramovitch RB, Kim YJ, Chen S, Dickman MB, Martin GB (2003) Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death. EMBO J 22: 60–69.RB AbramovitchYJ KimS. ChenMB DickmanGB Martin2003Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death.EMBO J226069
  85. 85. Bradley DJ, Kjellbom P, Lamb CJ (1992) Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: A novel, rapid defense response. Cell 70: 21–30.DJ BradleyP. KjellbomCJ Lamb1992Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: A novel, rapid defense response.Cell702130
  86. 86. Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79: 583–593.A. LevineR. TenhakenR. DixonC. Lamb1994H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response.Cell79583593
  87. 87. Lin CH, Yang SL, Chung KR (2009) The YAP1 homolog-mediated oxidative stress tolerance is crucial for pathogenicity of the necrotrophic fungus Alternaria alternata in citrus. Mol Plant-Microbe Interact 22: 942–952.CH LinSL YangKR Chung2009The YAP1 homolog-mediated oxidative stress tolerance is crucial for pathogenicity of the necrotrophic fungus Alternaria alternata in citrus.Mol Plant-Microbe Interact22942952
  88. 88. Tanaka A, Christensen MJ, Takemoto D, Park P, Scott B (2006) Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. Plant Cell 18: 1052–1066.A. TanakaMJ ChristensenD. TakemotoP. ParkB. Scott2006Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction.Plant Cell1810521066
  89. 89. Torres MA, Dangl JL (2005) Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr Opin Plant Biol 8: 397–403.MA TorresJL Dangl2005Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development.Curr Opin Plant Biol8397403
  90. 90. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55: 373–399.K. ApelH. Hirt2004Reactive oxygen species: metabolism, oxidative stress, and signal transduction.Annu Rev Plant Biol55373399
  91. 91. Miller RA, Britigan BE (1997) Role of oxidants in microbial pathophysiology. Clin. Microbiol Rev 10: 1–18.RA MillerBE Britigan1997Role of oxidants in microbial pathophysiology. Clin.Microbiol Rev10118
  92. 92. Moye-Rowley WS (2003) Regulation of the transcriptional response to oxidative stress in fungi: Similarities and differences. Eukaryotic Cell 2: 381–389.WS Moye-Rowley2003Regulation of the transcriptional response to oxidative stress in fungi: Similarities and differences.Eukaryotic Cell2381389
  93. 93. Toone WM, Jones N (1999) AP-1 transcription factors in yeast. Curr Opin Genet Dev 9: 55–61.WM TooneN. Jones1999AP-1 transcription factors in yeast.Curr Opin Genet Dev95561
  94. 94. Yi M, Chi MH, Khang CH, Park SY, Kang S, et al. (2009) The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae. Plant Cell 21: 681–695.M. YiMH ChiCH KhangSY ParkS. Kang2009The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae.Plant Cell21681695
  95. 95. Mei C, Qi M, Sheng G, Yang Y (2006) Inducible overexpression of a rice allene oxide synthase gene increases the endogenous jasmonic acid level, PR gene expression, and host resistance to fungal infection. Mol Plant-Microbe Interact 19: 1127–1137.C. MeiM. QiG. ShengY. Yang2006Inducible overexpression of a rice allene oxide synthase gene increases the endogenous jasmonic acid level, PR gene expression, and host resistance to fungal infection.Mol Plant-Microbe Interact1911271137
  96. 96. Qiu D, Xiao J, Ding X, Xiong M, Cai M, et al. (2007) OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. Mol Plant-Microbe Interact 20: 492–499.D. QiuJ. XiaoX. DingM. XiongM. Cai2007OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling.Mol Plant-Microbe Interact20492499
  97. 97. Zhang HF, Zhang X, Liu KY, Song WW, Zhao Q, et al. (2010) A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae . Current genetics 56: 517–528.HF ZhangX. ZhangKY LiuWW SongQ. Zhao2010A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae .Current genetics56517528
  98. 98. Oh YY, Donofrio N, Pan HQ, Coughlan S, Brown DE, et al. (2008) Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae. Genome Biology 9: R85.YY OhN. DonofrioHQ PanS. CoughlanDE Brown2008Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae.Genome Biology9R85
  99. 99. DeZwaan TM, Carroll AM, Valent B, Sweigard JA (1999) Magnaporthe grisea Pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues. Plant Cell 11: 2013–2030.TM DeZwaanAM CarrollB. ValentJA Sweigard1999Magnaporthe grisea Pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues.Plant Cell1120132030
  100. 100. Zhou Z, Li G, Lin C, He C (2009) Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae. Mol Plant Microbe Interact 22: 402–410.Z. ZhouG. LiC. LinC. He2009Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae.Mol Plant Microbe Interact22402410