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The adenylate cyclase UvAc1 and phosphodiesterase UvPdeH control the intracellular cAMP level, development, and pathogenicity of the rice false smut fungus Ustilaginoidea virens
Fungal Genetics and Biology 129 (2019) 65–73
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The adenylate cyclase UvAc1 and phosphodiesterase UvPdeH control the intracellular cAMP level, development, and pathogenicity of the rice false smut fungus Ustilaginoidea virens Weiwen Guo, Yixin Gao, Zhaomeng Yu, Yuhan Xiao, Zhengguang Zhang, Haifeng Zhang
T
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Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Adenylate cyclase Phosphodiesterase cAMP level Fungal development Pathogenicity
The cyclic adenosine monophosphate (cAMP) signaling pathway plays pleiotropic roles in regulating development and pathogenicity in eukaryotes. cAMP is a second messenger that is important for the activation of downstream pathways. The intracellular cAMP level is modulated mainly by its biosynthesis, which is catalyzed by adenylate cyclases (ACs), and hydrolysis by phosphodiesterases (PDEs). Here, we identified the AC UvAc1 and the cAMP high-affinity PDE UvPdeH in the rice false smut fungus Ustilaginoidea virens; these enzymes are homologs of MoMac1 and MoPdeH in Magnaporthe oryzae (rice blast fungus). A heterogenous complementation assay revealed that UvAc1 and UvPdeH partially or completely rescued the defects in ΔMomac1 and ΔMopdeH mutant M. oryzae. UvAc1 and UvPdeH play important roles in the development and virulence of U. virens. ΔUvac1 and ΔUvpdeH mutant fungi showed defects in conidial production, morphology, and germination; reduced toxicity against germinating rice seeds; and reduced virulence on rice panicles. ΔUvac1 exhibited increased sensitivity to Calcofluor White (CFW) and sodium chloride (NaCl), and decreased sensitivity to Congo Red (CR), while ΔUvpdeH showed increased sensitivity to sodium dodecyl sulfate, CR, sorbitol, and hydrogen peroxide, and decreased sensitivity to CFW and NaCl. High-performance liquid chromatography revealed that the intracellular cAMP level was significantly increased in ΔUvpdeH and decreased in ΔUvac1. Taken together, our results demonstrate that UvAc1 and UvPdeH are conservative components of the cAMP pathway that are important for conidiogenesis, stress responses, virulence, and regulation of the intracellular cAMP level in U. virens.
1. Introduction
In fungal pathogens, the core components of the cAMP signaling regulatory pathways are well conserved and play critical roles in development and virulence. In the rice blast fungus Magnaporthe oryzae, the AC Mac1 and PDE PdeH play opposite roles in modulating the intracellular cAMP level, and are important for appressorium formation and virulence (Choi et al., 1997; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). The AC-associated protein Cap1 binds to the actin cytoskeleton and is involved in Mac1 activation during appressorium formation and pathogenicity (Zhou et al., 2012). The PKA catalytic subunit CpkA is activated by cAMP and is involved in appressorium formation and virulence (Mitchell and Dean, 1995; Xu et al., 1997). Additionally, several components of the heterotrimeric G-protein signaling pathway, including Rgs1, Rgs3, Rgs4, and Rgs7 (regulators of Gprotein signaling); Gα subunits MagA and MagB; Gβ subunit Mgb1; and Gγ subunit Mgg1, have been characterized and reported to play important roles in the development and pathogenicity of M. oryzae (Liang
Eukaryotic cells possess well-conserved and complex signal transduction pathways, which mediate the responses to physiological and environmental stimuli. Such pathways include the heterotrimeric guanine-nucleotide binding protein (G-protein) signaling pathway, cyclic adenosine monophosphate (cAMP) signaling pathway, and mitogenactivated protein kinase (MAPK) signaling pathway (Li et al., 2012). The cAMP signaling pathway regulates morphogenesis and pathogenesis in fungal pathogens by modulating the intracellular level of cAMP (Kronstad et al., 2011), a ubiquitous second messenger that plays a critical role in activating downstream signaling components, such as by phosphorylating protein kinase A (PKA) (Daniel et al., 1998). The level of cAMP is dependent on the actions of diverse proteins that affect its synthesis and degradation, including adenylate cyclases (ACs) and phosphodiesterases (PDEs) (Colicelli et al., 1990). ⁎
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.fgb.2019.04.017 Received 29 January 2019; Received in revised form 15 April 2019; Accepted 29 April 2019 Available online 04 May 2019 1087-1845/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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study was obtained from Dr. Yongfeng Liu’s lab, Jiangsu Academy of Agricultural Sciences. All the U. virens strains were routinely cultured on potato sucrose agar (PSA, 200 g of peeled potato, 20 g of sucrose, and 15 g of agar in 1 L of distilled water). For vegetative growth, 2 × 2 mm mycelial blocks were cut from the edge of 10–15 day old cultures and placed onto freshly prepared potato dextrose agar (PDA, 200 g of peeled potato, 20 g of dextrose, and 15 g of agar in 1 L of distilled water), and YT (0.1% yeast extract, 0.1% tryptone, and 1% glucose) plates (Tanaka et al., 2011), and cultured in the dark at 25 °C for 14 days. For stress response assay, mycelial blocks were inoculated onto the YT agar plates with 0.4 M NaCl, 0.5 M sorbitol, 0.07% H2O2, 0.03% (w/v) sodium dodecylsulphate (SDS), or 70 μg/ml Congo red (CR), 100 μg/ml calcofluor white (CFW), respectively, and incubated in the dark at 25 °C for 14 days. Conidiation were assayed with 6-day-old liquid YT cultures, and photographed under a microscope. Freshly harvested conidia were resuspended to 1 × 106 conidia/ml in liquid YTS (0.1% yeast extract, 0.1% tryptone, and 1% sucrose) assayed for germination after incubation for 2, 6, 10, 12, 16, 20 and 24 h (Zheng et al., 2016). The M. oryzae Guy11 strains was used as wild type (WT), the ΔMopdeH mutant was acquired in our previous study (Zhang et al., 2011a), and the ΔMomac1 mutant was as a gift from Dr. Jin-Rong Xu’s lab, Purdue University (Zhou et al., 2012). Both mutants were used for protoplast transformation in this study. All M. oryzae strains were cultured on complete medium (CM) at 28 °C. Protoplast preparation and transformation were performed as described previously (Talbot et al., 1993). For vegetative growth, 2 × 2 mm mycelia blocks were cut from the edge of 7-day-old cultures and placed onto freshly prepared CM, straw decoction and corn (SDC, 100 g straw, 40 g corn powder, 15 g agar in 1 L of distilled water) agar plates, followed by incubation in the dark at 28 °C. The radial growth was measured at 7 dai. For conidium production, strains were maintained on SDC agar plates at 28 °C for 7 days in the dark followed by 3 days of continuous illumination under fluorescent light and analyzed as described previously (Zhang et al., 2010).
et al., 2006; Liu and Dean, 1997; Nishimura et al., 2003; Zhang et al., 2011b). In the saprophytic soil fungus Aspergillus flavus, the AC AcyA and PDE PdeH regulate development, aflatoxin biosynthesis, and virulence (Yang et al., 2017, 2016). In the Fusarium head blight fungus Fusarium graminearum, the cyclase-associated protein FgCap1 and PKA catalytic subunits Cpk1 and Cpk2 regulate development, toxin production, and plant infection (Guo et al., 2016; Hu et al., 2014; Yin et al., 2018a); PDE Pde2 negatively regulates toxin biosynthesis and virulence (Jiang et al., 2016). In the maize pathogen Fusarium verticillioides and the anthracnose fungus Colletotrichum lagenarium, both Cpk1 and the AC Ac1 play multiple roles in fungal development and virulence in cooperation with the MAPK pathway (Choi et al., 2010; Yamauchi et al., 2004). In the gray mold fungus Botrytis cinerea and in the human pathogens Candida albicans and Cryptococcus neoformans, the PDEs BcPdeH, CaPde2, CnPde1, and CaPka1 control development and virulence by modulating the intracellular cAMP level (Harren et al., 2013; Hicks et al., 2005; Jung et al., 2005). In the maize pathogen Ustilago maydis, PKA catalytic subunits Adr1 and Uka1 are required for virulence and fungal morphogenesis (Durrenberger et al., 1998). These reports suggest that the cAMP-PKA signaling pathway is vital in a variety of fungal pathogens; moreover, AC and PDE, as conserved and key components of this pathway, are involved in multiple steps of fungal development and infection. Rice false smut, caused by Ustilaginoidea virens (Cooke) Takah (Teleomorph: Villosiclava virens), is a serious disease that occurs in most rice-growing areas of the world. The major symptom is the conversion of individual grains into smut balls, resulting in floret sterility (Zhou et al., 2008). U. virens is also a producer of toxic secondary metabolites, including ustiloxins, which are toxic to plants and animals, including humans (Tsukui et al., 2015). Although its genome has been sequenced (Zhang et al., 2014), few molecular genetic studies of U. virens have been performed. Only a few genes—including UvHOG1, Uvt3277, UvSUN2, and UvPRO1, which play important roles in the development or virulence of this phytopathogen—have been identified and characterized by deletion or disruption (Lv et al., 2016; Yu et al., 2015; Zheng et al., 2016, 2017). Simultaneously, the targeted gene deletion frequency by conventional gene-replacement approaches or by random insertional mutagenesis was lower than in other plant pathogenic fungi (Liang et al., 2018). Recently, Liang et al. (2018) reported that, compared to Agrobacterium tumefaciens-mediated transformation (ATMT), the targeted gene deletion frequency was greatly increased when using the clustered regularly interspaced short palindromic repeats (CRISPR)associated RNA-guided DNA endonuclease Cas9 system in U. virens. The highest knockout frequency was 90% and 50% for deletion of the USTA ustiloxin and UvSLT2 MAPK genes, respectively. The CRISPR/Cas9 system has been used for gene editing in plants and animals (Cong et al., 2013). It also has been reported to improve the homologous recombination frequency in several ascomycetes, including Trichoderma reesei, M. oryzae, Neurospora crassa, Alternaria alternata, Penicillium chrysogenum, and Aspergillus niger (Arazoe et al., 2015; Kuivanen et al., 2016; Liu et al., 2015; Matsu-Ura et al., 2015; Pohl et al., 2016; Wenderoth et al., 2017). The CRISPR/Cas9 system enables gene deletion in ascomycetes and should greatly accelerate molecular genetic studies of U. virens. Here, we target-deleted two key components (UvPdeH and UvAc1) of the cAMP signaling pathway using the CRISPR/Cas9 system in U. virens and found that UvPdeH and UvAc1 play conserved roles in different plant pathogens. UvPdeH and UvAc1 regulate the intracellular cAMP level in opposite ways, and they control the conidiogenesis, and stress responses, as well as plant infection by U. virens.
2.2. Construction of the replacement vectors and Cas9-gRNA vectors for deletion of UvPDEH and UvAC1 The UvPDEH and UvAC1 gene replacement constructs (pMD19-TUvPDEHKO and pMD19-T-UvAC1KO) were generated according to the homologous recombination principle as described previously (Zhang et al., 2010). For generation of Cas9-gRNA vectors, the spacers were designed with the gRNA designer program for best on-target scores (http://grna.ctegd.uga.edu/) (Table S1). The sense and antisense oligonucleotides of the selected gRNA spacers were synthesized and annealed to generate the corresponding gRNA spacers, and cloned between the two BsmBI sites of pCas9-tRp-gRNA by Golden Gate cloning (NEB) (Arazoe et al., 2015; Ran et al., 2013) to generate the pCas9-tRpgRNA-UvPDEH and pCas9-tRp-gRNA-UvAC1 constructs. Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.fgb.2019.04.017. 2.3. UvPDEH and UvAC1 gene deletion and complementation To obtain the gene deletion mutants, the gene replacement vector and its corresponding pCas9-tRp-gRNA vector were co-transformed into protoplasts of wild type strain P1 according to the Magnaporthe transformation approach (Talbot et al., 1993). Protoplast preparation was performed as described previously with minor modification (Hohn and Desjardins, 1992). Hygromycin-resistant transformants were screened for ΔUvac1 or ΔUvpdeH deletion mutants by PCR, and confirmed by southern blot analysis. For complementation, a fragment containing the entire UvAC1 or UvPDEH gene and its native promoter region (upstream 1.5 kb sequence) was amplified by PCR, and inserted into pYF11 plasmid (bleomycin resistant) using the yeast gap repair approach
2. Materials and methods 2.1. Strains and culture conditions The U. virens wild-type strain P1 (Zheng et al., 2017) used in this 66
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are listed in Table S1.
(Bruno et al., 2004). The resulting constructs pYF11-UvPDEH and pYF11-UvAC1 were sequenced and transformed into the protoplast of the corresponding mutant. Bleomycin resistant transformants were screened by PCR and selected for phenotype analysis. Primers used in this section are listed in Table S1.
2.7. Pathogenicity assay on rice panicles The strains were cultured in liquid PSB medium (PSB, 200 g of peeled potato, 20 g of sucrose in 1 L of distilled water) for 7 days in shaking. The spore and mycelium mixture was then broken into pieces and adjusted to a concentration of 3 × 106 spores/ml. One milliliter of suspensions was inoculated onto each spike and ten spikes for each strain as described previously (Zheng et al., 2017). The diseased spikes were photographed and the number of false smut balls was counted at 21 dai.
2.4. Toxicity assays with culture filtrates Toxicity assays with culture filtrates were performed according to the method described previously (Zheng et al., 2016). Small agar blocks (2 × 2 mm) were cut from the edge of 10–15 day old cultures and placed into fresh liquid YT medium. After 5 days incubation, mycelia were collected by filtration through two layers of Miracloth (EMD Millipore Corporation, Billerica, MA 01821 USA) and grinded into powder, then lyophilized for 24 h for measurement the dry weight. In addition, culture filtrates were then centrifuged at 3500 rpm for 8 min to collect the supernatants. Seeds of rice cultivar (LYP9) were incubated on sterility filter papers soaked with the culture filtrates at 25 °C. The shoot and root growth were measured after incubation for 5 days.
2.8. Pathogenicity assay of the M. oryzae strains on rice seedlings Pathogenicity assays were performed according to the method described previously (Zhang et al., 2011b). Conidia were harvested from 10-day-old SDC agar cultures, filtered through one-layer Miracloth (EMD Millipore Crop, USA) and resuspended by 0.2% (w/v) gelatin solution to a concentration of 5 × 104 spores/ml. For spraying assay, two-week-old rice seedlings (CO-39) were sprayed with 5 ml of conidial suspension of each strain. The inoculated plants were kept in a growth chamber at 28 °C with 90% humidity and in the dark for the first 24 h, followed by a 12 h/12 h light/dark cycle. Diseased leaves were photographed at 7 days after inoculation (dai).
2.5. Intracellular cAMP measurement and HPLC analysis The 7-day-old YT cultures were collected and quickly ground into powder with liquid nitrogen and lyophilized for 24 h. Intracellular cAMP extraction was performed according to the established procedures (Liu et al., 2007). The cAMP levels were quantified by HPLC analysis as described previously (Liu et al., 2016). 2.6. Reverse transcription-polymerase chain reaction (RT-PCR) and southern blot analysis
2.9. Appressorium formation of the M. oryzae strains on hydrophobic and hydrophilic surfaces
For detection of the transcription of UvPDEH and UvAC1 gene in wild-type and gene deletion strains, total RNA samples were extracted from 7-day-old liquid YT cultures. The stable expression ACTIN gene (UV_6104) was used as an internal control. For southern blot analysis, genomic DNA was isolated from the wild type P1 and candidate gene deletion mutants. A total of 10 μg of DNA from each strain was digested with the corresponding restriction enzyme, separated by electrophoresis and transferred onto a positively charged nylon membrane as described (Zhang et al., 2010). The gene probe and HPH probe were amplified with primer pairs, respectively. Probe labeling, hybridization, and detection were performed with a DIG High Prime DNA Labeling and Detection Starter Kit (Roche). The experiments were repeated three times with three replicates each time. The primers used in this section
Conidia of the wild type, ΔMopdeH, ΔMopdeH/UvPDEH, ΔMomac1 and ΔMomac1/UvAC1 strains were harvested from 10-day-old SDC agar cultures, filtered through one-layer of Miracloth and washed with double-distilled water for three times. Droplets (20 μl) of conidial suspension (5 × 104 spores/ml) were placed onto hydrophobic (cover glasses, Fisher-brand, UK) or hydrophilic (Gelbond Film, GE Healthcare) surfaces and incubated at 28 °C. Photographs and appressorium formation rate were taken and analyzed at 12 or 24 h post-inoculation (Cai et al., 2017). The experiments were repeated three times with three replicates each time.
Fig. 1. UvPDEH and UvAC1 rescued the growth and conidiogenesis defects of the ΔMopdeH and ΔMomac1 mutants in M. oryzae. (A) Mycelial autolysis and vegetative growth of the indicated strains cultured in CM agar plates for 14 or 7 days. (B) Conidiation, conidial morphology and size of the indicated strains. For conidiation, ± SD was calculated from three independent experiments. For conidial size, values are the mean ± SD from 100 conidia for each strain, and asterisks indicate statistically significant differences (p < 0.01).
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Fig. 2. UvPDEH and UvAC1 rescued appressorium formation, virulence and intracellular cAMP level defects of the ΔMopdeH and ΔMomac1 mutants. (A) Appressorium formation of the indicated strains on hydrophilic or hydrophobic surfaces. Appressoria were pointed by black triangles. (B) Pathogenicity assay. Conidial suspensions of the indicated strains were sprayed onto 2-week-old rice seedlings, and photographed at 7 days post-inoculation. (C) Measurement of the intracellular cAMP level in the indicated strains. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01). Bar = 10 μm.
3. Results
homologous recombination (Fig. S1A), and the pCas9-tRNA constructs were made as described previously (Arazoe et al., 2015; Liang et al., 2018). The resulting constructs were co-transformed into the protoplasts of the wild-type strain P1 (Zheng et al., 2017). Hygromycin Bresistant transformants were first screened by PCR and confirmed by RT-PCR and Southern blot analyses (Fig. S1B and C). UvAC1 or UvPDEH was successfully replaced in the two mutants, and a single copy of each gene is present in the U. virens genome.
3.1. UvAC1 and UvPDEH completely or partially rescued ΔMomac1 and ΔMopdeH mutant in M. oryzae The AC MoMac1 and PDE MoPdeH play crucial roles in conidiation, appressorium formation, and virulence by modulating the intracellular cAMP level in M. oryzae (Choi et al., 1997; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). Here, we identified their homologs (UvAc1 and UvPdeH, respectively) in U. virens by a BLAST_P search; 50% and 66% amino acid sequence identities to MoPdeH and MoMac1, respectively, were revealed. To determine whether Ac1 and PdeH have conserved functions in different fungi, UvAC1-GFP and UvPDEH-GFP complementation constructs were generated and transformed into ΔMomac1 and ΔMopdeH mutant protoplasts, respectively. The resulting transformants were screened by PCR and analyzed for phenotype. The results show that UvAC1 and UvPDEH completely or greatly rescued the defects in ΔMomac1 and ΔMopdeH in terms of colony morphology, conidiation, appressorium formation, virulence, and intracellular cAMP level, respectively (Figs. 1 and 2). These findings suggest that Ac1 and PdeH play similar and conserved roles in M. oryzae and U. virens.
Because MoMac1 and MoPdeH play crucial roles in maintaining the intracellular cAMP level in the rice blast fungus, we first determined whether UvAc1 and UvPdeH have similar roles in U. virens. The intracellular cAMP level of ΔUvac1 and ΔUvpdeH was assayed by highperformance liquid chromatography. The cAMP level was significantly decreased (17-fold) in ΔUvac1 and increased 2.6-fold in ΔUvpdeH compared to wild type (Fig. 3). These results suggest that UvAc1 and UvPdeH play opposing roles in regulating intracellular cAMP levels, which may be important for the development and virulence of U. virens.
3.2. Targeted deletion of UvAC1 and UvPDEH using CRISPR/Cas9 in U. virens
3.4. Both UvAc1 and UvPdeH are important for conidiogenesis and conidial germination in U. virens
In the latest study of U. virens, it was reported that the homologous recombination frequency of targeted gene replacement was significantly increased by the CRISPR/Cas9 system (Liang et al., 2018). Here, we target-deleted UvAC1 and UvPDEH in U. virens using the CRISPR/Cas9 system. The replacement constructs were generated by
To investigate whether the alteration of the intracellular cAMP level in ΔUvac1 and ΔUvpdeH related to their phenotypes, we tested the vegetative growth of the indicated strains on yeast extract tryptone (YT) and potato dextrose agar (PDA) plates. After 2 weeks of incubation at 25 °C, the ΔUvpdeH and ΔUvac1 mutant showed similar colony
3.3. UvAc1 and UvPdeH play opposing roles in regulating intracellular cAMP levels
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was evaluated in liquid YTS medium. The ΔUvpdeH mutant formed shorter and thicker germ tubes, while the ΔUvac1mutant formed fewer germ tubes from each conidium compared to wild type and the complemented transformants (Fig. 6). These results suggest that both UvAc1 and UvPdeH regulate conidial production, conidial morphology, and germination in U. virens. 3.5. Culture filtrates of ΔUvac1 and ΔUvpdeH are less toxic to rice seed germination To investigate whether UvAc1 and UvPdeH regulate the production of phytotoxic compounds, we collected YT culture filtrates from 5-dayold wild-type P1, ΔUvac1, ΔUvpdeH mutant cells, and from the complemented strains, and subjected them to rice seed germination assays. The culture filtrates of the wild-type and complemented strains almost blocked rice root growth; very short roots were observed after 5 days. Rice shoot growth was also greatly inhibited by the wild-type and complemented strains. In contrast, rice seeds treated with the ΔUvac1 and ΔUvpdeH mutants showed roots and shoots that were two-fold longer than those treated with the wild-type and complemented strains under the same conditions (Fig. 7A and B).
Fig. 3. UvAc1 and UvPdeH play crucial role in regulating intracellular cAMP levels. Mycelia of the indicated strains were harvested from 2-day old cultures in liquid YT medium, and intracellular cAMP level was measured by HPLC analysis. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01).
3.6. Deletion of UvAC1 and UvPDEH results in defects in plant infection To determine whether UvAc1 and UvPdeH play a role in virulence, we evaluated the infection of rice panicles by inoculating with conidial suspensions of wild-type P1, ΔUvac1, ΔUvpdeH, and the complemented strains. After 3 weeks of incubation, the ΔUvac1 and ΔUvpdeH mutants caused no disease on grains, and no false smut balls were visible on rice panicles. In contrast, approximately 12–20 diseased grains with small false smut balls were found on each spike inoculated with wild-type P1 and the complemented strains (Fig. 8). These results suggest that UvAc1 and UvPdeH play a crucial role in infection by U. virens.
Fig. 4. UvAc1 is involved in pigment biosynthesis. Colony morphology of the wild type P1, ΔUvpdeH and ΔUvac1 mutants, and the complemented transformants ΔUvpdeH/UvPDEH and ΔUvac1/UvAC1 cultured on YT and PDA agar plates. Photographs were taken 14 dai at 25 °C in the dark.
3.7. UvAc1 and UvPdeH are involved in stress responses
morphology and growth rate to the wild-type P1, while the ΔUvac1 mutant showed almost no pigment on both media (Fig. 4), suggesting that UvAc1 plays a role in pigment biosynthesis in U. virens. We then examined the production of conidia and conidial germination in liquid YT medium. After incubation at 25 °C with shaking for 6 days, the ΔUvpdeH and ΔUvac1 mutants exhibited fewer conidia compared to wild type and the complemented transformants (Fig. 5). The number of conidia was decreased by 95.4% in ΔUvpdeH and 93.4% in ΔUvac1 compared to wild type (Fig. 5). Some of the conidia produced by the mutants were abnormal—8.9% of the conidia of ΔUvpdeH and 16.0% of those of ΔUvac1 were larger or longer compared to wild type and the complemented transformants (Fig. 5). Conidial germination
To determine whether UvAc1 and UvPdeH play a role in responding to environmental stresses, wild-type P1, ΔUvac1, ΔUvpdeH, and the complemented strains were inoculated onto YT plates with cell-wall disturbing agents (sodium dodecyl sulfate [SDS], Calcofluor White [CFW], and Congo Red [CR]), osmotic or salt stress (sorbitol and sodium chloride [NaCl]), and oxidative stress (hydrogen peroxide [H2O2]). After incubation for 14 days, the inhibition rate of ΔUvpdeH was significantly increased on the SDS, CR, sorbitol, and H2O2 plates, and decreased on the CFW and NaCl plates in comparison to the wildtype and complemented strains. In comparison, the inhibition rate of ΔUvac1 was significantly increased on CFW, sorbitol, and NaCl plates, and decreased on CR plates (Table 1). Thus, UvAc1 and UvPdeH are involved in the responses of U. virens to environmental stress. Fig. 5. UvAc1 and UvPdeH play an important role in conidial production and morphology. (Upper panel) Conidial morphology of the wild type P1, ΔUvpdeH, ΔUvac1, ΔUvpdeH/UvPDEH and ΔUvac1/ UvAC1 strains. Black triangles indicate abnormal conidia. Bar = 20 μm. (Lower panel) Conidiation and statistical analysis the conidial number of the indicated strains. Photographs were taken from 6-day old YT cultures. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01). Bar = 20 μm.
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Fig. 6. UvAc1 and UvPdeH are involved in conidial germination. Conidia of the indicated strains were cultured in liquid YTS medium. Examination was performed at 2, 6, 10, 12, 16, 20 and 24 h under a microscopy. Bar = 20 μm. Fig. 7. Culture filtrates of the ΔUvac1 and ΔUvpdeH mutants are less toxic to rice seed germination. (A) Seeds of rice cultivar were incubated on filter papers soaked with blank control or filtrates of 5-day-old YT cultures of the indicated strains. Root and shoot growth were examined after incubation at 25 °C for 5 days. CK, liquid YT medium. (B) Statistical analysis the root and shoot growth of the indicated strains. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01).
3.8. Exogenous IBMX caused ΔUvpdeH-like defects in the wild type U. virens The results above showed that the loss of UvPdeH function leads to higher intracellular cAMP level, thus affecting the development and virulence of U. virens. Therefore, we further investigated whether exogenous IBMX (3-isobutyl-1-methylxanthine: a phosphodiesterase inhibitor), which has been extensively used to artificially cause the enhancement of endogenous cAMP levels (Lee et al., 1993; Liu et al., 2007; Mitchell and Dean, 1995; Ramanujam and Naqvi, 2010; Skamnioti et al., 2007) would mimic the ΔUvpdeH mutant. As shown in the Figure, wild type strain treated by IBMX displayed a significant reduction in conidial number and less toxic to rice seed germination, as well as defect in conidial germination, which are similar to the ΔUvpdeH mutant (Fig. 9A–C). These results support our findings that the defects of the ΔUvpdeH mutant are indeed due to increased cAMP levels, caused due to the loss of UvPdeH function in the rice false smut fungus.
Fig. 8. UvAc1 and UvPdeH are important for plant infection. Conidial suspensions collected from the indicated strains were inoculated into rice panicles and photographed at 21 dpi. The diseased grains were separated and displayed.
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Table 1 Sensitivity test of the indicated strains under the treatment of various stressors. Strains
Inhibition rate of colony diameter (%) 100 μg/ml CFW
0.05% SDS Wild type ΔUvpdeH ΔUvac1 ΔUvpdeH/UvPDEH ΔUvac1/UvAC1
30.8 46.4 31.3 32.6 30.8
± ± ± ± ±
B
1.5 1.4A 2.0B 1.9B 2.3B
57.7 53.6 65.5 57.7 57.7
± ± ± ± ±
B
1.2 1.7C 2.1A 1.5B 1.8B
70 μg/ml CR 42.3 57.1 37.5 43.1 42.3
± ± ± ± ±
B
1.5 1.9A 1.9C 2.1B 2.1B
0.5 M Sorbitol 26.9 50.0 37.5 26.9 24.0
± ± ± ± ±
C
1.6 1.8A 1.7B 1.7C 1.6C
0.035% H2O2
0.4 M NaCl 61.5 46.4 81.3 61.5 60.0
± ± ± ± ±
B
1.2 2.0C 1.5A 1.5B 1.5B
80.8 85.7 81.3 80.8 80.0
± ± ± ± ±
1.3B 1.8A 2.3B 1.6B 2.4B
± SD was calculated from three independent experiments, and different letters indicate statistically significant differences (p < 0.01).
4. Discussion
also found a PdeL protein that shows 50% amino acids identity to MoPdeL, indicating U. virens possesses two similar phosphodiesterases to M. oryzae. The most conserved function of the cAMP signaling pathway in yeast and filamentous fungi is the modulation of intracellular cAMP homeostasis and sensing of surface signals (Li et al., 2012; Yin et al., 2018b). Similar to the situation in other organisms, UvMac1 and UvPdeH play opposing roles in maintaining intracellular cAMP levels by biosynthesis or degradation, indicating them to be key regulators of the cAMP signaling pathway. In several pathogens, including M. oryzae, B. cinerea, and F. graminearum, the deletion of homologs of UvAc1 or UvPdeH causes defects in appressorium differentiation and conidial germination (Choi et al., 1997; Guo et al., 2016; Harren et al., 2013; Jiang et al., 2016; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). The ΔUvac1 and ΔUvpdeH mutants also had a defect in conidial germination. Moreover, UvAc1 and UvPdeH rescued appressorium formation in the corresponding mutants in M. oryzae. Therefore, UvAc1 and UvPdeH might also play conserved roles in germination and surface signal recognition, as in other fungi. Conidial production was decreased in the ΔUvac1 and ΔUvpdeH mutants, similar to the corresponding mutants in M. oryzae, F. graminearum, A. flavus, B. cinerea, and F. verticillioides (Choi et al., 1997, 2010; Harren et al., 2013; Hu et al., 2014; Ramanujam and Naqvi, 2010; Yang et al., 2017, 2016; Zhang et al., 2011a). The cAMP pathway likely has conserved roles in conidiation in a subset of filamentous fungi. A pdeH mutant in B. cinerea exhibited abnormal conidial morphology. Indeed, ΔUvpdeH also produced abnormal conidia, indicating that UvPdeH functions in cell wall integrity; this conjecture is supported by findings in M. oryzae, C. albicans, and the budding yeast Saccharomyces cerevisiae (Jung et al., 2005; Tomlin et al., 2000; Zhang et al., 2011a). Besides, the cAMP pathway may participate in environmental stress responses. An A. flavus acyA mutant showed
In this study, we used the CRISPR/Cas9 system to generate UvAC1 and UvPDEH deletion mutants in the rice false smut fungus U. virens. The gene replacement frequency exceeded 55% for each gene, similar to the report by Liang et al. (2018). Our findings confirm that the CRISPR/Cas9 system can be used for gene replacement in U. virens. Despite some potential limitations, such as selectable makers or offtarget mutations, the CRISPR/Cas9 system will promote additional molecular genetic studies of this important pathogen. The cAMP pathway plays crucial roles in the development and virulence of fungal pathogens. The Ac1 and PdeH are two key components of the cAMP pathway that function to maintain the intracellular cAMP level and are involved in proper infection morphogenesis in a number of pathogens (Choi et al., 2010; Harren et al., 2013; Hu et al., 2014; Jung et al., 2005; Li et al., 2012; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). Here, in U. virens we identified and characterized UvAc1 and UvPdeH, which are homologs of MoMac1 and MoPdeH in the rice blast fungus, and found them play opposing roles in modulating intracellular cAMP levels and are important for the development and infection by this pathogen. These results are consistent with the fact that Ac1 and PdeH also play important roles in fungal development and virulence (Choi et al., 1997; Hicks et al., 2005; Ramanujam and Naqvi, 2010; Yang et al., 2017, 2016, 2018; Zhang et al., 2011a). In addition, UvAc1 and UvPdeH show a high level of sequence identity to their Magnaporthe homologs and could restore the phenotypic defects of the corresponding mutants in M. oryzae. Therefore, we conclude that the Ac1 and PdeH were conserved in terms of their sequence and function during evolution. M. oryzae also has a low-affinity cAMP phosphodiesterase (MoPdeL) that playing roles in asexual development and conidial morphology, as well as a minor role in regulating cAMP levels (Ramanujam and Naqvi, 2010; Zhang et al., 2011a). In U. virens, we
Fig. 9. Exogenous IBMX caused ΔUvpdeHlike defects in the wild type. (A) Effect of exogenous IBMX (1.5 mM) on conidiation of the wild type U. virens. DMSO: solvent control. ± SD was calculated from three independent experiments, and asterisk indicates statistically significant differences (p < 0.01). (B) Effect of exogenous IBMX on conidial germination of the wild type. Photographed were taken at 10 h post-inoculation. (C) Culture filtrate toxicity of the wild type strain treated or untreated with IBMX.
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decreased sensitivity to temperature and hyperosmotic stress, and increased sensitivity to oxidative stress (Yang et al., 2016), while a C. albicans pde2 mutant showed increased sensitivity to cell wall- and membrane-perturbing agents (Jung et al., 2005). Here, ΔUvpdeH and ΔUvac1 also showed different responses to cell wall-perturbing agents, and osmotic and oxidative stresses. Thus, the cAMP pathway likely plays an important role in osmoregulation in various pathogens. Several components of the cAMP signaling pathway are important for infection (Li et al., 2012). We proved that UvAc1 and UvPdeH are conserved pathogenicity-related components of the cAMP pathway. Additionally, the deletion of AcyA and PdeH decreased aflatoxin production in A. flavus (Yang et al., 2017, 2016). The deletion of FgCap1 decreased, while the deletion of FgPde2 increased, deoxynivalenol (DON) production in F. graminearum (Jiang et al., 2016; Yin et al., 2018a). These findings indicated that the cAMP signaling pathway has conserved roles in toxin biosynthesis. In F. graminearum, DON is a virulence factor; mutants with reduced DON production showed defects in virulence (Proctor et al., 1995). In A. flavus, toxin production is related to virulence (Yang et al., 2017, 2016). In U. virens, culture filtrates of the Uvhog1 mutant were less inhibitory to rice seed germination (Zheng et al., 2016). Although ustiloxin production was not detected in ΔUvpdeH and ΔUvac1, the culture filtrates of both mutants showed less toxicity to rice seeds. Therefore, UvAc1 and UvPdeH are likely involved in toxin production, thus contributing to the virulence of U. virens. In summary, we characterized UvAc1 and UvPdeH in the rice false smut fungus using the CRISPR/Cas9 system and obtained evidence that UvAc1 and UvPdeH are key components of the cAMP signaling pathway. A phenotypic analysis revealed that UvAc1 and UvPdeH are important for the development and pathogenicity of this plant pathogen. These findings will improve our understanding of the pathogenetic mechanisms of U. virens.
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Acknowledgements We thank Dr. Jin-Rong Xu at Purdue University for the pCas9-tRpgRNA vector. We also thank Dr. Yongfeng Liu at Jiangsu Academy of Agricultural Sciences for providing the U. virens field isolate P1. This research was supported by the National Key Research and Development Program of China (Grant No: 2016YFD0300700/2016YFD0300706), the Fundamental Research Funds for the Central Universities (Grant No: KYT201805), the Outstanding Youth Foundation of Jiangsu Province (Grant No: BK20160074), and Innovation Team Program for Jiangsu Universities (2017). References Arazoe, T., et al., 2015. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotech. Bioeng. 112, 2543–2549. Bruno, K.S., et al., 2004. Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryot. Cell 3, 1525–1532. Cai, X.J., et al., 2017. The atypical guanylate kinase MoGuk2 plays important roles in asexual/sexual development, conidial septation, and pathogenicity in the rice blast fungus. Front. Microbiol. 8, 2467. Choi, W.B., et al., 1997. The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9, 1973–1983. Choi, Y.E., et al., 2010. The cAMP signaling pathway in Fusarium verticillioides is important for conidiation, plant Infection, and stress responses but not fumonisin production. Mol. Plant-Microbe Interact. 23, 522–533. Colicelli, J., et al., 1990. Mutational mapping of Ras-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol. Cell. Biol. 10, 2539–2543. Cong, L., et al., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. Daniel, P.B., et al., 1998. Cyclic AMP signaling and gene regulation. Annu. Rev. Nutr. 18, 353–383. Durrenberger, F., et al., 1998. Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis. Proc. Nat. Acad. Sci. USA 95, 5684–5689. Guo, L., et al., 2016. Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F-verticillioides. Mol. Plant Pathol. 17, 196–209.
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Magnaporthe oryzae. PLoS One 6, e17241. Zhang, H., et al., 2011b. Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae. PLoS Pathog. 7, e1002450. Zhang, Y., et al., 2014. Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics. Nat. Commun. 5, 3849. Zheng, D.W., et al., 2016. UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens. Sci. Rep. 6, 24824. Zheng, M.T., et al., 2017. Low-affinity iron transport protein Uvt3277 is important for pathogenesis in the rice false smut fungus Ustilaginoidea virens. Curr. Genet. 63, 131–144. Zhou, X.Y., et al., 2012. The cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae. PLoS Pathog. 8, e1002911. Zhou, Y.L., et al., 2008. Genetic diversity of rice false smut fungus, Ustilaginoidea virens and its pronounced differentiation of populations in North China. J. Phytopathol. 156, 559–564.
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The adenylate cyclase UvAc1 and phosphodiesterase UvPdeH control the intracellular cAMP level, development, and pathogenicity of the rice false smut fungus Ustilaginoidea virens Weiwen Guo, Yixin Gao, Zhaomeng Yu, Yuhan Xiao, Zhengguang Zhang, Haifeng Zhang
T
⁎
Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Adenylate cyclase Phosphodiesterase cAMP level Fungal development Pathogenicity
The cyclic adenosine monophosphate (cAMP) signaling pathway plays pleiotropic roles in regulating development and pathogenicity in eukaryotes. cAMP is a second messenger that is important for the activation of downstream pathways. The intracellular cAMP level is modulated mainly by its biosynthesis, which is catalyzed by adenylate cyclases (ACs), and hydrolysis by phosphodiesterases (PDEs). Here, we identified the AC UvAc1 and the cAMP high-affinity PDE UvPdeH in the rice false smut fungus Ustilaginoidea virens; these enzymes are homologs of MoMac1 and MoPdeH in Magnaporthe oryzae (rice blast fungus). A heterogenous complementation assay revealed that UvAc1 and UvPdeH partially or completely rescued the defects in ΔMomac1 and ΔMopdeH mutant M. oryzae. UvAc1 and UvPdeH play important roles in the development and virulence of U. virens. ΔUvac1 and ΔUvpdeH mutant fungi showed defects in conidial production, morphology, and germination; reduced toxicity against germinating rice seeds; and reduced virulence on rice panicles. ΔUvac1 exhibited increased sensitivity to Calcofluor White (CFW) and sodium chloride (NaCl), and decreased sensitivity to Congo Red (CR), while ΔUvpdeH showed increased sensitivity to sodium dodecyl sulfate, CR, sorbitol, and hydrogen peroxide, and decreased sensitivity to CFW and NaCl. High-performance liquid chromatography revealed that the intracellular cAMP level was significantly increased in ΔUvpdeH and decreased in ΔUvac1. Taken together, our results demonstrate that UvAc1 and UvPdeH are conservative components of the cAMP pathway that are important for conidiogenesis, stress responses, virulence, and regulation of the intracellular cAMP level in U. virens.
1. Introduction
In fungal pathogens, the core components of the cAMP signaling regulatory pathways are well conserved and play critical roles in development and virulence. In the rice blast fungus Magnaporthe oryzae, the AC Mac1 and PDE PdeH play opposite roles in modulating the intracellular cAMP level, and are important for appressorium formation and virulence (Choi et al., 1997; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). The AC-associated protein Cap1 binds to the actin cytoskeleton and is involved in Mac1 activation during appressorium formation and pathogenicity (Zhou et al., 2012). The PKA catalytic subunit CpkA is activated by cAMP and is involved in appressorium formation and virulence (Mitchell and Dean, 1995; Xu et al., 1997). Additionally, several components of the heterotrimeric G-protein signaling pathway, including Rgs1, Rgs3, Rgs4, and Rgs7 (regulators of Gprotein signaling); Gα subunits MagA and MagB; Gβ subunit Mgb1; and Gγ subunit Mgg1, have been characterized and reported to play important roles in the development and pathogenicity of M. oryzae (Liang
Eukaryotic cells possess well-conserved and complex signal transduction pathways, which mediate the responses to physiological and environmental stimuli. Such pathways include the heterotrimeric guanine-nucleotide binding protein (G-protein) signaling pathway, cyclic adenosine monophosphate (cAMP) signaling pathway, and mitogenactivated protein kinase (MAPK) signaling pathway (Li et al., 2012). The cAMP signaling pathway regulates morphogenesis and pathogenesis in fungal pathogens by modulating the intracellular level of cAMP (Kronstad et al., 2011), a ubiquitous second messenger that plays a critical role in activating downstream signaling components, such as by phosphorylating protein kinase A (PKA) (Daniel et al., 1998). The level of cAMP is dependent on the actions of diverse proteins that affect its synthesis and degradation, including adenylate cyclases (ACs) and phosphodiesterases (PDEs) (Colicelli et al., 1990). ⁎
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.fgb.2019.04.017 Received 29 January 2019; Received in revised form 15 April 2019; Accepted 29 April 2019 Available online 04 May 2019 1087-1845/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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study was obtained from Dr. Yongfeng Liu’s lab, Jiangsu Academy of Agricultural Sciences. All the U. virens strains were routinely cultured on potato sucrose agar (PSA, 200 g of peeled potato, 20 g of sucrose, and 15 g of agar in 1 L of distilled water). For vegetative growth, 2 × 2 mm mycelial blocks were cut from the edge of 10–15 day old cultures and placed onto freshly prepared potato dextrose agar (PDA, 200 g of peeled potato, 20 g of dextrose, and 15 g of agar in 1 L of distilled water), and YT (0.1% yeast extract, 0.1% tryptone, and 1% glucose) plates (Tanaka et al., 2011), and cultured in the dark at 25 °C for 14 days. For stress response assay, mycelial blocks were inoculated onto the YT agar plates with 0.4 M NaCl, 0.5 M sorbitol, 0.07% H2O2, 0.03% (w/v) sodium dodecylsulphate (SDS), or 70 μg/ml Congo red (CR), 100 μg/ml calcofluor white (CFW), respectively, and incubated in the dark at 25 °C for 14 days. Conidiation were assayed with 6-day-old liquid YT cultures, and photographed under a microscope. Freshly harvested conidia were resuspended to 1 × 106 conidia/ml in liquid YTS (0.1% yeast extract, 0.1% tryptone, and 1% sucrose) assayed for germination after incubation for 2, 6, 10, 12, 16, 20 and 24 h (Zheng et al., 2016). The M. oryzae Guy11 strains was used as wild type (WT), the ΔMopdeH mutant was acquired in our previous study (Zhang et al., 2011a), and the ΔMomac1 mutant was as a gift from Dr. Jin-Rong Xu’s lab, Purdue University (Zhou et al., 2012). Both mutants were used for protoplast transformation in this study. All M. oryzae strains were cultured on complete medium (CM) at 28 °C. Protoplast preparation and transformation were performed as described previously (Talbot et al., 1993). For vegetative growth, 2 × 2 mm mycelia blocks were cut from the edge of 7-day-old cultures and placed onto freshly prepared CM, straw decoction and corn (SDC, 100 g straw, 40 g corn powder, 15 g agar in 1 L of distilled water) agar plates, followed by incubation in the dark at 28 °C. The radial growth was measured at 7 dai. For conidium production, strains were maintained on SDC agar plates at 28 °C for 7 days in the dark followed by 3 days of continuous illumination under fluorescent light and analyzed as described previously (Zhang et al., 2010).
et al., 2006; Liu and Dean, 1997; Nishimura et al., 2003; Zhang et al., 2011b). In the saprophytic soil fungus Aspergillus flavus, the AC AcyA and PDE PdeH regulate development, aflatoxin biosynthesis, and virulence (Yang et al., 2017, 2016). In the Fusarium head blight fungus Fusarium graminearum, the cyclase-associated protein FgCap1 and PKA catalytic subunits Cpk1 and Cpk2 regulate development, toxin production, and plant infection (Guo et al., 2016; Hu et al., 2014; Yin et al., 2018a); PDE Pde2 negatively regulates toxin biosynthesis and virulence (Jiang et al., 2016). In the maize pathogen Fusarium verticillioides and the anthracnose fungus Colletotrichum lagenarium, both Cpk1 and the AC Ac1 play multiple roles in fungal development and virulence in cooperation with the MAPK pathway (Choi et al., 2010; Yamauchi et al., 2004). In the gray mold fungus Botrytis cinerea and in the human pathogens Candida albicans and Cryptococcus neoformans, the PDEs BcPdeH, CaPde2, CnPde1, and CaPka1 control development and virulence by modulating the intracellular cAMP level (Harren et al., 2013; Hicks et al., 2005; Jung et al., 2005). In the maize pathogen Ustilago maydis, PKA catalytic subunits Adr1 and Uka1 are required for virulence and fungal morphogenesis (Durrenberger et al., 1998). These reports suggest that the cAMP-PKA signaling pathway is vital in a variety of fungal pathogens; moreover, AC and PDE, as conserved and key components of this pathway, are involved in multiple steps of fungal development and infection. Rice false smut, caused by Ustilaginoidea virens (Cooke) Takah (Teleomorph: Villosiclava virens), is a serious disease that occurs in most rice-growing areas of the world. The major symptom is the conversion of individual grains into smut balls, resulting in floret sterility (Zhou et al., 2008). U. virens is also a producer of toxic secondary metabolites, including ustiloxins, which are toxic to plants and animals, including humans (Tsukui et al., 2015). Although its genome has been sequenced (Zhang et al., 2014), few molecular genetic studies of U. virens have been performed. Only a few genes—including UvHOG1, Uvt3277, UvSUN2, and UvPRO1, which play important roles in the development or virulence of this phytopathogen—have been identified and characterized by deletion or disruption (Lv et al., 2016; Yu et al., 2015; Zheng et al., 2016, 2017). Simultaneously, the targeted gene deletion frequency by conventional gene-replacement approaches or by random insertional mutagenesis was lower than in other plant pathogenic fungi (Liang et al., 2018). Recently, Liang et al. (2018) reported that, compared to Agrobacterium tumefaciens-mediated transformation (ATMT), the targeted gene deletion frequency was greatly increased when using the clustered regularly interspaced short palindromic repeats (CRISPR)associated RNA-guided DNA endonuclease Cas9 system in U. virens. The highest knockout frequency was 90% and 50% for deletion of the USTA ustiloxin and UvSLT2 MAPK genes, respectively. The CRISPR/Cas9 system has been used for gene editing in plants and animals (Cong et al., 2013). It also has been reported to improve the homologous recombination frequency in several ascomycetes, including Trichoderma reesei, M. oryzae, Neurospora crassa, Alternaria alternata, Penicillium chrysogenum, and Aspergillus niger (Arazoe et al., 2015; Kuivanen et al., 2016; Liu et al., 2015; Matsu-Ura et al., 2015; Pohl et al., 2016; Wenderoth et al., 2017). The CRISPR/Cas9 system enables gene deletion in ascomycetes and should greatly accelerate molecular genetic studies of U. virens. Here, we target-deleted two key components (UvPdeH and UvAc1) of the cAMP signaling pathway using the CRISPR/Cas9 system in U. virens and found that UvPdeH and UvAc1 play conserved roles in different plant pathogens. UvPdeH and UvAc1 regulate the intracellular cAMP level in opposite ways, and they control the conidiogenesis, and stress responses, as well as plant infection by U. virens.
2.2. Construction of the replacement vectors and Cas9-gRNA vectors for deletion of UvPDEH and UvAC1 The UvPDEH and UvAC1 gene replacement constructs (pMD19-TUvPDEHKO and pMD19-T-UvAC1KO) were generated according to the homologous recombination principle as described previously (Zhang et al., 2010). For generation of Cas9-gRNA vectors, the spacers were designed with the gRNA designer program for best on-target scores (http://grna.ctegd.uga.edu/) (Table S1). The sense and antisense oligonucleotides of the selected gRNA spacers were synthesized and annealed to generate the corresponding gRNA spacers, and cloned between the two BsmBI sites of pCas9-tRp-gRNA by Golden Gate cloning (NEB) (Arazoe et al., 2015; Ran et al., 2013) to generate the pCas9-tRpgRNA-UvPDEH and pCas9-tRp-gRNA-UvAC1 constructs. Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.fgb.2019.04.017. 2.3. UvPDEH and UvAC1 gene deletion and complementation To obtain the gene deletion mutants, the gene replacement vector and its corresponding pCas9-tRp-gRNA vector were co-transformed into protoplasts of wild type strain P1 according to the Magnaporthe transformation approach (Talbot et al., 1993). Protoplast preparation was performed as described previously with minor modification (Hohn and Desjardins, 1992). Hygromycin-resistant transformants were screened for ΔUvac1 or ΔUvpdeH deletion mutants by PCR, and confirmed by southern blot analysis. For complementation, a fragment containing the entire UvAC1 or UvPDEH gene and its native promoter region (upstream 1.5 kb sequence) was amplified by PCR, and inserted into pYF11 plasmid (bleomycin resistant) using the yeast gap repair approach
2. Materials and methods 2.1. Strains and culture conditions The U. virens wild-type strain P1 (Zheng et al., 2017) used in this 66
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are listed in Table S1.
(Bruno et al., 2004). The resulting constructs pYF11-UvPDEH and pYF11-UvAC1 were sequenced and transformed into the protoplast of the corresponding mutant. Bleomycin resistant transformants were screened by PCR and selected for phenotype analysis. Primers used in this section are listed in Table S1.
2.7. Pathogenicity assay on rice panicles The strains were cultured in liquid PSB medium (PSB, 200 g of peeled potato, 20 g of sucrose in 1 L of distilled water) for 7 days in shaking. The spore and mycelium mixture was then broken into pieces and adjusted to a concentration of 3 × 106 spores/ml. One milliliter of suspensions was inoculated onto each spike and ten spikes for each strain as described previously (Zheng et al., 2017). The diseased spikes were photographed and the number of false smut balls was counted at 21 dai.
2.4. Toxicity assays with culture filtrates Toxicity assays with culture filtrates were performed according to the method described previously (Zheng et al., 2016). Small agar blocks (2 × 2 mm) were cut from the edge of 10–15 day old cultures and placed into fresh liquid YT medium. After 5 days incubation, mycelia were collected by filtration through two layers of Miracloth (EMD Millipore Corporation, Billerica, MA 01821 USA) and grinded into powder, then lyophilized for 24 h for measurement the dry weight. In addition, culture filtrates were then centrifuged at 3500 rpm for 8 min to collect the supernatants. Seeds of rice cultivar (LYP9) were incubated on sterility filter papers soaked with the culture filtrates at 25 °C. The shoot and root growth were measured after incubation for 5 days.
2.8. Pathogenicity assay of the M. oryzae strains on rice seedlings Pathogenicity assays were performed according to the method described previously (Zhang et al., 2011b). Conidia were harvested from 10-day-old SDC agar cultures, filtered through one-layer Miracloth (EMD Millipore Crop, USA) and resuspended by 0.2% (w/v) gelatin solution to a concentration of 5 × 104 spores/ml. For spraying assay, two-week-old rice seedlings (CO-39) were sprayed with 5 ml of conidial suspension of each strain. The inoculated plants were kept in a growth chamber at 28 °C with 90% humidity and in the dark for the first 24 h, followed by a 12 h/12 h light/dark cycle. Diseased leaves were photographed at 7 days after inoculation (dai).
2.5. Intracellular cAMP measurement and HPLC analysis The 7-day-old YT cultures were collected and quickly ground into powder with liquid nitrogen and lyophilized for 24 h. Intracellular cAMP extraction was performed according to the established procedures (Liu et al., 2007). The cAMP levels were quantified by HPLC analysis as described previously (Liu et al., 2016). 2.6. Reverse transcription-polymerase chain reaction (RT-PCR) and southern blot analysis
2.9. Appressorium formation of the M. oryzae strains on hydrophobic and hydrophilic surfaces
For detection of the transcription of UvPDEH and UvAC1 gene in wild-type and gene deletion strains, total RNA samples were extracted from 7-day-old liquid YT cultures. The stable expression ACTIN gene (UV_6104) was used as an internal control. For southern blot analysis, genomic DNA was isolated from the wild type P1 and candidate gene deletion mutants. A total of 10 μg of DNA from each strain was digested with the corresponding restriction enzyme, separated by electrophoresis and transferred onto a positively charged nylon membrane as described (Zhang et al., 2010). The gene probe and HPH probe were amplified with primer pairs, respectively. Probe labeling, hybridization, and detection were performed with a DIG High Prime DNA Labeling and Detection Starter Kit (Roche). The experiments were repeated three times with three replicates each time. The primers used in this section
Conidia of the wild type, ΔMopdeH, ΔMopdeH/UvPDEH, ΔMomac1 and ΔMomac1/UvAC1 strains were harvested from 10-day-old SDC agar cultures, filtered through one-layer of Miracloth and washed with double-distilled water for three times. Droplets (20 μl) of conidial suspension (5 × 104 spores/ml) were placed onto hydrophobic (cover glasses, Fisher-brand, UK) or hydrophilic (Gelbond Film, GE Healthcare) surfaces and incubated at 28 °C. Photographs and appressorium formation rate were taken and analyzed at 12 or 24 h post-inoculation (Cai et al., 2017). The experiments were repeated three times with three replicates each time.
Fig. 1. UvPDEH and UvAC1 rescued the growth and conidiogenesis defects of the ΔMopdeH and ΔMomac1 mutants in M. oryzae. (A) Mycelial autolysis and vegetative growth of the indicated strains cultured in CM agar plates for 14 or 7 days. (B) Conidiation, conidial morphology and size of the indicated strains. For conidiation, ± SD was calculated from three independent experiments. For conidial size, values are the mean ± SD from 100 conidia for each strain, and asterisks indicate statistically significant differences (p < 0.01).
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Fig. 2. UvPDEH and UvAC1 rescued appressorium formation, virulence and intracellular cAMP level defects of the ΔMopdeH and ΔMomac1 mutants. (A) Appressorium formation of the indicated strains on hydrophilic or hydrophobic surfaces. Appressoria were pointed by black triangles. (B) Pathogenicity assay. Conidial suspensions of the indicated strains were sprayed onto 2-week-old rice seedlings, and photographed at 7 days post-inoculation. (C) Measurement of the intracellular cAMP level in the indicated strains. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01). Bar = 10 μm.
3. Results
homologous recombination (Fig. S1A), and the pCas9-tRNA constructs were made as described previously (Arazoe et al., 2015; Liang et al., 2018). The resulting constructs were co-transformed into the protoplasts of the wild-type strain P1 (Zheng et al., 2017). Hygromycin Bresistant transformants were first screened by PCR and confirmed by RT-PCR and Southern blot analyses (Fig. S1B and C). UvAC1 or UvPDEH was successfully replaced in the two mutants, and a single copy of each gene is present in the U. virens genome.
3.1. UvAC1 and UvPDEH completely or partially rescued ΔMomac1 and ΔMopdeH mutant in M. oryzae The AC MoMac1 and PDE MoPdeH play crucial roles in conidiation, appressorium formation, and virulence by modulating the intracellular cAMP level in M. oryzae (Choi et al., 1997; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). Here, we identified their homologs (UvAc1 and UvPdeH, respectively) in U. virens by a BLAST_P search; 50% and 66% amino acid sequence identities to MoPdeH and MoMac1, respectively, were revealed. To determine whether Ac1 and PdeH have conserved functions in different fungi, UvAC1-GFP and UvPDEH-GFP complementation constructs were generated and transformed into ΔMomac1 and ΔMopdeH mutant protoplasts, respectively. The resulting transformants were screened by PCR and analyzed for phenotype. The results show that UvAC1 and UvPDEH completely or greatly rescued the defects in ΔMomac1 and ΔMopdeH in terms of colony morphology, conidiation, appressorium formation, virulence, and intracellular cAMP level, respectively (Figs. 1 and 2). These findings suggest that Ac1 and PdeH play similar and conserved roles in M. oryzae and U. virens.
Because MoMac1 and MoPdeH play crucial roles in maintaining the intracellular cAMP level in the rice blast fungus, we first determined whether UvAc1 and UvPdeH have similar roles in U. virens. The intracellular cAMP level of ΔUvac1 and ΔUvpdeH was assayed by highperformance liquid chromatography. The cAMP level was significantly decreased (17-fold) in ΔUvac1 and increased 2.6-fold in ΔUvpdeH compared to wild type (Fig. 3). These results suggest that UvAc1 and UvPdeH play opposing roles in regulating intracellular cAMP levels, which may be important for the development and virulence of U. virens.
3.2. Targeted deletion of UvAC1 and UvPDEH using CRISPR/Cas9 in U. virens
3.4. Both UvAc1 and UvPdeH are important for conidiogenesis and conidial germination in U. virens
In the latest study of U. virens, it was reported that the homologous recombination frequency of targeted gene replacement was significantly increased by the CRISPR/Cas9 system (Liang et al., 2018). Here, we target-deleted UvAC1 and UvPDEH in U. virens using the CRISPR/Cas9 system. The replacement constructs were generated by
To investigate whether the alteration of the intracellular cAMP level in ΔUvac1 and ΔUvpdeH related to their phenotypes, we tested the vegetative growth of the indicated strains on yeast extract tryptone (YT) and potato dextrose agar (PDA) plates. After 2 weeks of incubation at 25 °C, the ΔUvpdeH and ΔUvac1 mutant showed similar colony
3.3. UvAc1 and UvPdeH play opposing roles in regulating intracellular cAMP levels
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was evaluated in liquid YTS medium. The ΔUvpdeH mutant formed shorter and thicker germ tubes, while the ΔUvac1mutant formed fewer germ tubes from each conidium compared to wild type and the complemented transformants (Fig. 6). These results suggest that both UvAc1 and UvPdeH regulate conidial production, conidial morphology, and germination in U. virens. 3.5. Culture filtrates of ΔUvac1 and ΔUvpdeH are less toxic to rice seed germination To investigate whether UvAc1 and UvPdeH regulate the production of phytotoxic compounds, we collected YT culture filtrates from 5-dayold wild-type P1, ΔUvac1, ΔUvpdeH mutant cells, and from the complemented strains, and subjected them to rice seed germination assays. The culture filtrates of the wild-type and complemented strains almost blocked rice root growth; very short roots were observed after 5 days. Rice shoot growth was also greatly inhibited by the wild-type and complemented strains. In contrast, rice seeds treated with the ΔUvac1 and ΔUvpdeH mutants showed roots and shoots that were two-fold longer than those treated with the wild-type and complemented strains under the same conditions (Fig. 7A and B).
Fig. 3. UvAc1 and UvPdeH play crucial role in regulating intracellular cAMP levels. Mycelia of the indicated strains were harvested from 2-day old cultures in liquid YT medium, and intracellular cAMP level was measured by HPLC analysis. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01).
3.6. Deletion of UvAC1 and UvPDEH results in defects in plant infection To determine whether UvAc1 and UvPdeH play a role in virulence, we evaluated the infection of rice panicles by inoculating with conidial suspensions of wild-type P1, ΔUvac1, ΔUvpdeH, and the complemented strains. After 3 weeks of incubation, the ΔUvac1 and ΔUvpdeH mutants caused no disease on grains, and no false smut balls were visible on rice panicles. In contrast, approximately 12–20 diseased grains with small false smut balls were found on each spike inoculated with wild-type P1 and the complemented strains (Fig. 8). These results suggest that UvAc1 and UvPdeH play a crucial role in infection by U. virens.
Fig. 4. UvAc1 is involved in pigment biosynthesis. Colony morphology of the wild type P1, ΔUvpdeH and ΔUvac1 mutants, and the complemented transformants ΔUvpdeH/UvPDEH and ΔUvac1/UvAC1 cultured on YT and PDA agar plates. Photographs were taken 14 dai at 25 °C in the dark.
3.7. UvAc1 and UvPdeH are involved in stress responses
morphology and growth rate to the wild-type P1, while the ΔUvac1 mutant showed almost no pigment on both media (Fig. 4), suggesting that UvAc1 plays a role in pigment biosynthesis in U. virens. We then examined the production of conidia and conidial germination in liquid YT medium. After incubation at 25 °C with shaking for 6 days, the ΔUvpdeH and ΔUvac1 mutants exhibited fewer conidia compared to wild type and the complemented transformants (Fig. 5). The number of conidia was decreased by 95.4% in ΔUvpdeH and 93.4% in ΔUvac1 compared to wild type (Fig. 5). Some of the conidia produced by the mutants were abnormal—8.9% of the conidia of ΔUvpdeH and 16.0% of those of ΔUvac1 were larger or longer compared to wild type and the complemented transformants (Fig. 5). Conidial germination
To determine whether UvAc1 and UvPdeH play a role in responding to environmental stresses, wild-type P1, ΔUvac1, ΔUvpdeH, and the complemented strains were inoculated onto YT plates with cell-wall disturbing agents (sodium dodecyl sulfate [SDS], Calcofluor White [CFW], and Congo Red [CR]), osmotic or salt stress (sorbitol and sodium chloride [NaCl]), and oxidative stress (hydrogen peroxide [H2O2]). After incubation for 14 days, the inhibition rate of ΔUvpdeH was significantly increased on the SDS, CR, sorbitol, and H2O2 plates, and decreased on the CFW and NaCl plates in comparison to the wildtype and complemented strains. In comparison, the inhibition rate of ΔUvac1 was significantly increased on CFW, sorbitol, and NaCl plates, and decreased on CR plates (Table 1). Thus, UvAc1 and UvPdeH are involved in the responses of U. virens to environmental stress. Fig. 5. UvAc1 and UvPdeH play an important role in conidial production and morphology. (Upper panel) Conidial morphology of the wild type P1, ΔUvpdeH, ΔUvac1, ΔUvpdeH/UvPDEH and ΔUvac1/ UvAC1 strains. Black triangles indicate abnormal conidia. Bar = 20 μm. (Lower panel) Conidiation and statistical analysis the conidial number of the indicated strains. Photographs were taken from 6-day old YT cultures. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01). Bar = 20 μm.
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Fig. 6. UvAc1 and UvPdeH are involved in conidial germination. Conidia of the indicated strains were cultured in liquid YTS medium. Examination was performed at 2, 6, 10, 12, 16, 20 and 24 h under a microscopy. Bar = 20 μm. Fig. 7. Culture filtrates of the ΔUvac1 and ΔUvpdeH mutants are less toxic to rice seed germination. (A) Seeds of rice cultivar were incubated on filter papers soaked with blank control or filtrates of 5-day-old YT cultures of the indicated strains. Root and shoot growth were examined after incubation at 25 °C for 5 days. CK, liquid YT medium. (B) Statistical analysis the root and shoot growth of the indicated strains. ± SD was calculated from three independent experiments, and asterisks indicate statistically significant differences (p < 0.01).
3.8. Exogenous IBMX caused ΔUvpdeH-like defects in the wild type U. virens The results above showed that the loss of UvPdeH function leads to higher intracellular cAMP level, thus affecting the development and virulence of U. virens. Therefore, we further investigated whether exogenous IBMX (3-isobutyl-1-methylxanthine: a phosphodiesterase inhibitor), which has been extensively used to artificially cause the enhancement of endogenous cAMP levels (Lee et al., 1993; Liu et al., 2007; Mitchell and Dean, 1995; Ramanujam and Naqvi, 2010; Skamnioti et al., 2007) would mimic the ΔUvpdeH mutant. As shown in the Figure, wild type strain treated by IBMX displayed a significant reduction in conidial number and less toxic to rice seed germination, as well as defect in conidial germination, which are similar to the ΔUvpdeH mutant (Fig. 9A–C). These results support our findings that the defects of the ΔUvpdeH mutant are indeed due to increased cAMP levels, caused due to the loss of UvPdeH function in the rice false smut fungus.
Fig. 8. UvAc1 and UvPdeH are important for plant infection. Conidial suspensions collected from the indicated strains were inoculated into rice panicles and photographed at 21 dpi. The diseased grains were separated and displayed.
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Table 1 Sensitivity test of the indicated strains under the treatment of various stressors. Strains
Inhibition rate of colony diameter (%) 100 μg/ml CFW
0.05% SDS Wild type ΔUvpdeH ΔUvac1 ΔUvpdeH/UvPDEH ΔUvac1/UvAC1
30.8 46.4 31.3 32.6 30.8
± ± ± ± ±
B
1.5 1.4A 2.0B 1.9B 2.3B
57.7 53.6 65.5 57.7 57.7
± ± ± ± ±
B
1.2 1.7C 2.1A 1.5B 1.8B
70 μg/ml CR 42.3 57.1 37.5 43.1 42.3
± ± ± ± ±
B
1.5 1.9A 1.9C 2.1B 2.1B
0.5 M Sorbitol 26.9 50.0 37.5 26.9 24.0
± ± ± ± ±
C
1.6 1.8A 1.7B 1.7C 1.6C
0.035% H2O2
0.4 M NaCl 61.5 46.4 81.3 61.5 60.0
± ± ± ± ±
B
1.2 2.0C 1.5A 1.5B 1.5B
80.8 85.7 81.3 80.8 80.0
± ± ± ± ±
1.3B 1.8A 2.3B 1.6B 2.4B
± SD was calculated from three independent experiments, and different letters indicate statistically significant differences (p < 0.01).
4. Discussion
also found a PdeL protein that shows 50% amino acids identity to MoPdeL, indicating U. virens possesses two similar phosphodiesterases to M. oryzae. The most conserved function of the cAMP signaling pathway in yeast and filamentous fungi is the modulation of intracellular cAMP homeostasis and sensing of surface signals (Li et al., 2012; Yin et al., 2018b). Similar to the situation in other organisms, UvMac1 and UvPdeH play opposing roles in maintaining intracellular cAMP levels by biosynthesis or degradation, indicating them to be key regulators of the cAMP signaling pathway. In several pathogens, including M. oryzae, B. cinerea, and F. graminearum, the deletion of homologs of UvAc1 or UvPdeH causes defects in appressorium differentiation and conidial germination (Choi et al., 1997; Guo et al., 2016; Harren et al., 2013; Jiang et al., 2016; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). The ΔUvac1 and ΔUvpdeH mutants also had a defect in conidial germination. Moreover, UvAc1 and UvPdeH rescued appressorium formation in the corresponding mutants in M. oryzae. Therefore, UvAc1 and UvPdeH might also play conserved roles in germination and surface signal recognition, as in other fungi. Conidial production was decreased in the ΔUvac1 and ΔUvpdeH mutants, similar to the corresponding mutants in M. oryzae, F. graminearum, A. flavus, B. cinerea, and F. verticillioides (Choi et al., 1997, 2010; Harren et al., 2013; Hu et al., 2014; Ramanujam and Naqvi, 2010; Yang et al., 2017, 2016; Zhang et al., 2011a). The cAMP pathway likely has conserved roles in conidiation in a subset of filamentous fungi. A pdeH mutant in B. cinerea exhibited abnormal conidial morphology. Indeed, ΔUvpdeH also produced abnormal conidia, indicating that UvPdeH functions in cell wall integrity; this conjecture is supported by findings in M. oryzae, C. albicans, and the budding yeast Saccharomyces cerevisiae (Jung et al., 2005; Tomlin et al., 2000; Zhang et al., 2011a). Besides, the cAMP pathway may participate in environmental stress responses. An A. flavus acyA mutant showed
In this study, we used the CRISPR/Cas9 system to generate UvAC1 and UvPDEH deletion mutants in the rice false smut fungus U. virens. The gene replacement frequency exceeded 55% for each gene, similar to the report by Liang et al. (2018). Our findings confirm that the CRISPR/Cas9 system can be used for gene replacement in U. virens. Despite some potential limitations, such as selectable makers or offtarget mutations, the CRISPR/Cas9 system will promote additional molecular genetic studies of this important pathogen. The cAMP pathway plays crucial roles in the development and virulence of fungal pathogens. The Ac1 and PdeH are two key components of the cAMP pathway that function to maintain the intracellular cAMP level and are involved in proper infection morphogenesis in a number of pathogens (Choi et al., 2010; Harren et al., 2013; Hu et al., 2014; Jung et al., 2005; Li et al., 2012; Ramanujam and Naqvi, 2010; Zhang et al., 2011a). Here, in U. virens we identified and characterized UvAc1 and UvPdeH, which are homologs of MoMac1 and MoPdeH in the rice blast fungus, and found them play opposing roles in modulating intracellular cAMP levels and are important for the development and infection by this pathogen. These results are consistent with the fact that Ac1 and PdeH also play important roles in fungal development and virulence (Choi et al., 1997; Hicks et al., 2005; Ramanujam and Naqvi, 2010; Yang et al., 2017, 2016, 2018; Zhang et al., 2011a). In addition, UvAc1 and UvPdeH show a high level of sequence identity to their Magnaporthe homologs and could restore the phenotypic defects of the corresponding mutants in M. oryzae. Therefore, we conclude that the Ac1 and PdeH were conserved in terms of their sequence and function during evolution. M. oryzae also has a low-affinity cAMP phosphodiesterase (MoPdeL) that playing roles in asexual development and conidial morphology, as well as a minor role in regulating cAMP levels (Ramanujam and Naqvi, 2010; Zhang et al., 2011a). In U. virens, we
Fig. 9. Exogenous IBMX caused ΔUvpdeHlike defects in the wild type. (A) Effect of exogenous IBMX (1.5 mM) on conidiation of the wild type U. virens. DMSO: solvent control. ± SD was calculated from three independent experiments, and asterisk indicates statistically significant differences (p < 0.01). (B) Effect of exogenous IBMX on conidial germination of the wild type. Photographed were taken at 10 h post-inoculation. (C) Culture filtrate toxicity of the wild type strain treated or untreated with IBMX.
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decreased sensitivity to temperature and hyperosmotic stress, and increased sensitivity to oxidative stress (Yang et al., 2016), while a C. albicans pde2 mutant showed increased sensitivity to cell wall- and membrane-perturbing agents (Jung et al., 2005). Here, ΔUvpdeH and ΔUvac1 also showed different responses to cell wall-perturbing agents, and osmotic and oxidative stresses. Thus, the cAMP pathway likely plays an important role in osmoregulation in various pathogens. Several components of the cAMP signaling pathway are important for infection (Li et al., 2012). We proved that UvAc1 and UvPdeH are conserved pathogenicity-related components of the cAMP pathway. Additionally, the deletion of AcyA and PdeH decreased aflatoxin production in A. flavus (Yang et al., 2017, 2016). The deletion of FgCap1 decreased, while the deletion of FgPde2 increased, deoxynivalenol (DON) production in F. graminearum (Jiang et al., 2016; Yin et al., 2018a). These findings indicated that the cAMP signaling pathway has conserved roles in toxin biosynthesis. In F. graminearum, DON is a virulence factor; mutants with reduced DON production showed defects in virulence (Proctor et al., 1995). In A. flavus, toxin production is related to virulence (Yang et al., 2017, 2016). In U. virens, culture filtrates of the Uvhog1 mutant were less inhibitory to rice seed germination (Zheng et al., 2016). Although ustiloxin production was not detected in ΔUvpdeH and ΔUvac1, the culture filtrates of both mutants showed less toxicity to rice seeds. Therefore, UvAc1 and UvPdeH are likely involved in toxin production, thus contributing to the virulence of U. virens. In summary, we characterized UvAc1 and UvPdeH in the rice false smut fungus using the CRISPR/Cas9 system and obtained evidence that UvAc1 and UvPdeH are key components of the cAMP signaling pathway. A phenotypic analysis revealed that UvAc1 and UvPdeH are important for the development and pathogenicity of this plant pathogen. These findings will improve our understanding of the pathogenetic mechanisms of U. virens.
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Acknowledgements We thank Dr. Jin-Rong Xu at Purdue University for the pCas9-tRpgRNA vector. We also thank Dr. Yongfeng Liu at Jiangsu Academy of Agricultural Sciences for providing the U. virens field isolate P1. This research was supported by the National Key Research and Development Program of China (Grant No: 2016YFD0300700/2016YFD0300706), the Fundamental Research Funds for the Central Universities (Grant No: KYT201805), the Outstanding Youth Foundation of Jiangsu Province (Grant No: BK20160074), and Innovation Team Program for Jiangsu Universities (2017). References Arazoe, T., et al., 2015. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotech. Bioeng. 112, 2543–2549. Bruno, K.S., et al., 2004. Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryot. Cell 3, 1525–1532. Cai, X.J., et al., 2017. The atypical guanylate kinase MoGuk2 plays important roles in asexual/sexual development, conidial septation, and pathogenicity in the rice blast fungus. Front. Microbiol. 8, 2467. Choi, W.B., et al., 1997. The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9, 1973–1983. Choi, Y.E., et al., 2010. The cAMP signaling pathway in Fusarium verticillioides is important for conidiation, plant Infection, and stress responses but not fumonisin production. Mol. Plant-Microbe Interact. 23, 522–533. Colicelli, J., et al., 1990. Mutational mapping of Ras-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol. Cell. Biol. 10, 2539–2543. Cong, L., et al., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. Daniel, P.B., et al., 1998. Cyclic AMP signaling and gene regulation. Annu. Rev. Nutr. 18, 353–383. Durrenberger, F., et al., 1998. Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis. Proc. Nat. Acad. Sci. USA 95, 5684–5689. Guo, L., et al., 2016. Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F-verticillioides. Mol. Plant Pathol. 17, 196–209.
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