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The cyclase-associated protein UvCap1 is required for mycelial growth and pathogenicity in the rice false smut fungus
Phytopathology Research volume 3, Article number: 5 (2021)
Rice false smut caused by Ustilaginoidea virens is one of the widespread rice diseases across the globe in recent years, however, we know little about its molecular mechanism of infection. The cAMP signaling pathway functions directly in the development and formation of infectious structures to regulate the infection process in many pathogenic fungi. In order to investigate the role of the cAMP signaling pathway in U. virens, UvCap1, a cyclase-associated-protein homologous to Saccharomyces cerevisiae Srv2 was identified. Three targeted deletion mutants of the UvCAP1 gene were obtained with gene replacement strategy assisted with CRISPR-Cas9 system. The UvCAP1 deletion mutants showed defects in mycelial growth and conidial production. Inoculation experiments demonstrated that ΔUvcap1 exhibited defects in pathogenicity. Compared with the wild-type strain, ΔUvcap1 showed decreased tolerance to sorbitol and H2O2, and increased tolerance to NaCl, CFW and SDS, and the intracellular cAMP level was significantly reduced in ΔUvcap1. Yeast two-hybrid assay identified the interactions of UvCap1 with UvAc1 (adenylase cyclase), two Ras proteins (UvRas1 and UvRas2) and UvSte50. Taken together, as a component of cAMP signaling pathway, UvCap1 plays important roles in the development and pathogenicity of U. virens.
Rice false smut has become one of the most prevalent fungal diseases of rice in recent years, and is currently seriously threatening food security (Zhou et al. 2008; Sun et al. 2020). This disease is caused by the ascomycete fungus Ustilaginoidea virens (Cooke) Takahashi (teleomorph: Villosiclava virens), which initially infects the stamen filaments and then extends to all floral organs of rice to produce false smut balls in spikelets (Tang et al. 2013; Song et al. 2016). The occurrence of rice false smut not only seriously affects the yield and quality of rice, but also produces a large number of mycotoxins that are harmful to humans and animals (Tsukui et al. 2015). Therefore, there is an ongoing and urgent need for research into prevention of the disease. As a nonobligate biotrophic pathogenic fungus, the infection process of U. virens is significantly different from some other pathogenic fungi such as Magnaporthe oryzae. M. oryzae forms appressorium, a specialized infection structure that accumulates enormous turgor pressure to penetrate the host tissue (Talbot 2003). U. virens, in contrast, does not form specialized infection structures: after the mycelium penetrates the host epidermis, it cannot penetrate the plant cell wall and enter the host cell, but only grows in the intercellular space (Tang et al. 2013). Elucidating the unique infection mechanism of U. virens at the molecular level is of great theoretical significance for understanding the infection process of this fungus.
The cAMP signaling pathway is essential for normal development of many eukaryotes. In plant pathogenic fungi, the conserved cAMP-PKA cascade regulates various developmental and infection processes (Adachi and Hamer 1998; Kang et al. 1999). Cyclase-associated proteins (CAPs), as important components of the cAMP signaling pathway, are well conserved in eukaryotes from yeast to humans (Hubberstey and Mottillo 2002). CAPs have been identified in Saccharomyces cerevisiae, Candida albicans, Cryptococus neoformans, Ustilago maydis and M. oryzae (Kawamukai et al. 1992; Rocha et al. 2001; Bahn et al. 2004; Zhou et al. 2012). In S. cerevisiae, a CAP gene named as SRV2 was identified in the Ras-responsive adenylyl cyclase complex (Field et al. 1990; Mintzer and Field 1994; Yu et al. 1999). The N-terminus of Srv2 interacts with Ras2 and Cyr1 (adenylyl cyclase), and the C-terminus of Srv2 interacts with actin monomers (Quintero-Monzon et al. 2009). In C. albicans, Cap1 also interacts with adenylyl cyclase and Ras proteins to influence the intracellular cAMP level, and the CAP1 mutant is defective in bud-hypha transitions under specific environmental conditions (Bahn and Sundstrom 2001; Zou et al. 2010). In C. neoformans, the CAPs homologous protein Aca1 interacts with the C-terminus of adenylyl cyclase to regulate cell fusion, capsule formation and pathogenesis (Bahn et al. 2004). In U. maydis, Cap1 is an important component of the cAMP signaling pathway that interacts with adenylate cyclase Uac1 and regulates morphogenesis and pathogenesis (Takach and Gold 2010). In M. oryzae, CAP1 functions directly in the activation of adenylate cyclase, and regulates appressorium formation and plant infection. CAP1 is also involved in the feedback inhibition of the Ras2 signaling pathway in M. oryzae (Zhou et al. 2012). Although the cAMP signaling pathway is conserved and has essential functions in numerous plant pathogenic fungi, there is still no advancing research on CAP genes in U. virens.
In this study, we identified and characterized a CAP-coding gene, UvCAP1, in U. virens. UvCap1 was found to interact with UvAc1, UvRas1, UvRas2 and UvSte50 in a yeast two-hybrid assay. ΔUvcap1, the deletion mutant of UvCAP1 was defective in mycelial growth, conidial production, some stress responses and pathogenicity. Collectively, our results demonstrate that UvCap1 plays important roles in the development and pathogenicity of the rice false smut fungus.
Identification of CAP ortholog in U. virens
Srv2 is a CAP in the Ras-responsive adenylyl cyclase complex in the budding yeast S. cerevisiae (Yu et al. 1999). In this study, we identified UvCAP1 (KDB15885.1, UV8b_3197), a gene homologous to the yeast SRV2 gene in the U. virens genome by searching the National Center for Biotechnology Information (NCBI) database (accession number: GCA_000687475.1). UvCap1 was the only CAP ortholog we found in U. virens, and pfam analysis indicated that this protein contains two domains, CAP_N (11–377 aa) and CAP_C (399–557 aa) (Fig. 1a). A phylogenetic analysis of protein sequences showed that UvCap1 shares the highest similarity with the adenylate CAP from the filamentous fungi Claviceps purpurea (Fig. 1b).
UvCap1 is required for mycelial growth and conidiation in U. virens
To investigate the function of UvCAP1 in U. virens, we generated the UvCAP1 deletion mutant in the wild-type strain P-1 by replacing the open reading frame (ORF) of UvCAP1 with the hygromycin resistance gene (HYG) through gene replacement strategy assisted with CRISPR-Cas9 system (Liang et al. 2018). The replacement plasmid was created by homologous recombination (Additional file 1: Figure S1a) (Zheng et al. 2016), and the Cas9-gRNA vector was constructed as described previously (Liang et al. 2018). The resulting vectors were co-transformed into the protoplasts of strain P-1. HYG-resistant transformants were first screened by PCR and further confirmed by sequencing. The results showed that the HYG gene replaced the ORF of UvCAP1 in ΔUvcap1 (#11, #14 and #29) (Additional file 1: Figure S1b). The copy number of HYG in ΔUvcap1 (#11, #14 and #29) was close to 1.0, demonstrating that the HYG gene was inserted as a single copy. These three mutants showed similar phenotypic characteristics, so ΔUvcap1 (#11) was selected for subsequent study. The complemented transformants Uvcap1-c (#1 and #2) were obtained by complementing the ΔUvcap1 (#11) mutant with the UvCAP1 gene driven by its native promoter, and were confirmed by RT-PCR and phenotypic analysis (Additional file 1: Figure S1c).
To explore if UvCap1 is involved in mycelial growth and colony morphology in U. virens, the wild-type strain P-1, the mutant strains ΔUvcap1 (#11, #14) and the complemented strain Uvcap1-c (#1) were cultured on PSA and TB3 medium plates for 20 days. ΔUvcap1 (#11, #14) had a reduced mycelial growth compared with those of P-1 and Uvcap1-c (#1) (Fig. 2a, b). On PSA plate, the average colony diameter for P-1 was 47.6 mm, while those for ΔUvcap1 (#11) and ΔUvcap1 (#14) were 23.4 mm and 24.0 mm, respectively. The colony sizes of ΔUvcap1 (#11, #14) in PSB were also significantly smaller than those of P-1 and Uvcap1-c (#1) (Fig. 2a, b). There was no significant difference in conidial morphology between ΔUvcap1 (#11, #14) and P-1, however, ΔUvcap1 (#11, #14) produced significantly fewer conidia than P-1, with ΔUvcap1 (#11, #14) having only 28–35% of the number of conidia produced by P-1 (Fig. 2c). In Uvcap1-c (#1), the defects in growth and conidiation were restored to the level of the wild-type strain (Fig. 2). These results indicate that UvCap1 is required for mycelial growth and conidial production in U. virens.
UvCap1 is required for full virulence of U. virens
We next investigated the role of UvCap1 in regulating the virulence of U. virens. Suspensions of shattered hyphae and conidia from P-1, ΔUvcap1 (#11) and Uvcap1-c (#1) were inoculated into Liangyoupeijiu panicles at booting stage (5–7 days before heading). At 30 days post inoculation (dpi), the false smut balls produced on rice panicles were counted to evaluate the virulence of the inoculated strains. The average number of false smut balls per panicle for P-1, Uvcap1-c (#1) and ΔUvcap1 (#11) were 26.5, 27.3 and 19.0, respectively (Fig. 3a, b). Additionally, mycelial expansion of these strains inside the spikelets of rice was observed at 10, 14 and 18 dpi, and the results demonstrated that mycelial expansion was slower in spikelets inoculated with ΔUvcap1 (#11) than with P-1 and Uvcap1-c (#1) (Fig. 3c). To further observe the difference in the infection process, P-1, ΔUvcap1 (#11) and Uvcap1-c1 (#1) were tagged with GFP, and used to inoculate rice plants. Results of fluorescence microscopic examination showed that there were fewer GFP-tagged hyphae inside the spikelets infected by ΔUvcap1 (#11) than by the wild-type and complemented strains (Fig. 3d). These results indicate that UvCap1 affects the growth of infection hyphae to regulate the pathogenicity of U. virens.
UvCap1 regulates stress responses in U. virens
The cAMP signaling pathway is essential in sensing and response to changes of extracellular environments. ΔUvac1 shows increased tolerance to sodium chloride (NaCl) and decreased tolerance to Congo red (CR) (Guo et al. 2019). Here we investigated if UvCap1 is involved in responses of U. virens to osmotic, oxidative and cell wall stresses. The wild-type strain P-1, the ΔUvcap1 (#11) mutant and its complemented strain Uvcap1-c (#1) were cultured on PSA medium plate supplemented with different stress agents. Compared with P-1 and Uvcap1-c (#1), the growth inhibition rate of ΔUvcap1 (#11) was significantly different. Under 0.6 M sorbitol and 0.05% H2O2 treatment, ΔUvcap1 (#11) showed decreased tolerance (Fig. 4a, b). ΔUvcap1 (#11) exhibited increased tolerance to 0.5 M NaCl, 500 μg/mL CFW and 0.05% SDS. On PSA medium plate with 100 μg/mL Congo red (CR), the growth inhibition rate of ΔUvcap1 (#11) showed no difference from P-1 and Uvcap1-c (#1) (Fig. 4a, b). These results suggest that UvCap1 is required for regulating the responses of U. virens to osmotic, oxidative and cell wall stresses.
UvCAP1 was highly expressed during the early infection stage of U. virens
According to previous studies on the infection process of U. virens, conidia germinate to form hyphae to extend to the interior of spikelets through the gap between lemma and palea at 1–2 dpi. The hyphae then infect flower organs including filaments, anthers and stigma at 3–7 dpi, and fungal mycelia develop quickly and enclose the entire flower organs, filling the whole spikelets by 8–15 dpi (Song et al. 2016). In order to gain a deeper understanding of the function of UvCAP1 in the pathogenicity of U. virens, RT-qPCR was used to detect the relative expression level of UvCAP1 during the infection process of U. virens. The inoculated rice spikelets at 0, 1, 2, 3, 5, 7 and 14 dpi were collected for RT-qPCR assay. The results showed that the expression level of UvCAP1 had an upward trend at the initial stage of inoculation, reaching the highest value at 3 dpi, which was 5.14 times the expression level at the time of initial inoculation (Fig. 5). After that, the expression level of the gene showed a downward trend (Fig. 5).
UvCap1 interacts with UvAc1, Ras proteins and UvSte50
Previous studies found that the CAPs function as a component of the cAMP signaling pathway, interacting with adenylate cyclase and regulating morphogenesis and pathogenesis (Bahn et al. 2004; Quintero-Monzon et al. 2009; Takach and Gold 2010; Zou et al. 2010; Zhou et al. 2012). In S. cerevisiae and C. albicans, Cap1 interacts with Ras and adenylyl cyclase proteins (Quintero-Monzon et al. 2009; Zou et al. 2010). In U. maydis and M. oryzae, Cap1 interacts with adenylate cyclase to positively regulate adenylyl cyclase (Takach and Gold 2010; Zhou et al. 2012). Here, we used yeast two-hybrid assays to identify the interactions of UvCap1 with UvAc1, Ras proteins (U. virens has two Ras proteins in the genome database) and UvSte50. The results showed that UvCap1 interacts with UvAc1 (the C-terminal region), UvRas1, UvRas2 and UvSte50 (Fig. 6).
UvCap1 affects the intracellular cAMP level in U. virens
cAMP is a ubiquitous second messenger that plays a critical role in the activation of downstream pathways in both eukaryotic and prokaryotic cells. cAMP is produced from ATP by adenylate cyclase and the intracellular cAMP level is significantly decreased (17.0-fold) in the ΔUvac1 mutant (Guo et al. 2019). Here we assayed the cAMP level in the wild-type strain P-1, the ΔUvcap1 (#11) mutant and its complemented strain Uvcap1-c (#1). Compared with that in P-1, the intracellular cAMP content in ΔUvcap1 (#11) was significantly reduced (2.0-fold) (Fig. 7). These results indicate that just like UvAc1, UvCap1 plays an important role in maintaining the intracellular cAMP level in U. virens.
UvCap1 was distributed as spots in the cytoplasm of hyphae and conidia
In order to explain the possible role of UvCap1 in U. virens, a subcellular localization assay was carried out. The UvCap1:GFP transformant was observed using a confocal laser scanning microscope (CLSM). UvCap1 showed strong fluorescence signals as spots in the cytoplasm of hyphae (especially in the apical regions of hyphae) and conidia (Fig. 8a, b). The distribution of UvCap1 in hypha of U. virens was similar to that of Cap1 in M. oryzae (Zhou et al. 2012). Western blot analysis was performed to detect UvCap1 expression in the UvCap1:GFP transformant. UvCap1:GFP showed a 86-kDa band against the anti-GFP antibody, which is consistent with the protein size of UvCap1 fused with GFP (Fig. 8c). The results suggest that UvCap1 is constitutively expressed in the cytoplasm.
In this study, the PEG-mediated transformation and CRISPR/Cas9-based targeted gene replacement method was used to obtain the UvCAP1 gene deletion mutants. The CRISPR/Cas9 system significantly promoted gene replacement frequency in U. virens as previously reported by Liang et al. (2018).
CAPs are involved in regulating the development and pathogenesis of many pathogenic fungi. In human pathogens, the mutant of ACA1 (homologous to CAPs) in Cryptococcus neoformans and the CAP1 mutant in Candida albicans are non-pathogenic (Bahn and Sundstrom 2001; Bahn et al. 2004). In M. oryzae, the Δcap1 mutant has defects in invasive growth in host cells, so lesions caused by this mutant are significantly reduced compared with those by the wild-type strain (Zhou et al. 2012). Adenylate cyclase UvAc1 functions directly in the infection process of U. virens: the ΔUvac1 mutant cannot form false smut balls on rice panicles (Guo et al. 2019). Our results showed that UvCap1 is required for full virulence of U. virens (Fig. 3). Compared with ΔUvac1, ΔUvcap1 had a much milder effect on the virulence of U. virens to rice. In the early stage of infection, the growth of infection hyphae of ΔUvcap1 in the diseased spikelets was slower than that of the wild-type strain, and the number of false smut balls produced by ΔUvcap1 was reduced (Fig. 3). ΔUvcap1 showed serious defects in mycelial growth and conidiation (Fig. 2). UvCap1 is also required for regulating the responses of U. virens to multiple stresses; ΔUvcap1 showed increased tolerance to NaCl, CFW and SDS, and decreased tolerance to H2O2 and sorbitol (Fig. 4). UvCap1 thus has regulatory roles in the development and infection process of U. virens.
The localization of CAPs varies slightly in different organisms at different developmental stages. In yeast, CAP/Srv2p is located in cortical actin patches to regulate the cytoskeleton (Yu et al. 1999). In Dictyostelium discoideum, CAP is distributed throughout the cytoplasm and shows enrichment at plasma membrane regions (Gottwald et al. 1996; Noegel et al. 1999). In mouse, Cap1 is widely present in nonmuscle cells, while Cap2 is mainly present in developing striated muscles. Cap1 colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton in cultured NIH3T3 and B16F1 cells (Bertling et al. 2004). Cap1, as an actin shuttle, provides a link from actin cytoskeleton to mitochondria, and in cells induced for apoptosis, Cap1 is rapidly translocated to the mitochondria (Wang et al. 2008). In M. oryzae, Cap1 also has an actin-like localization pattern in hyphae and germ tubes (Zhou et al. 2012). In U. virens, the strain UvCap1:GFP showed strong GFP signals as spots in the cytoplasm of the hyphae (especially in the apical regions of hyphae) and conidia (Fig. 8a, b). Whether these spots are actin-like positioning patterns remains to be determined.
CAPs are well conserved in eukaryotes from yeast to mammals (Hubberstey and Mottillo 2002). The Cap1 protein in U. virens has typical structural characteristics of CAPs (Fig. 1a). In S. cerevisiae, the conserved N-terminal and C-terminal ends of CAPs interact with adenylate cyclase and agonist protein, respectively (Gerst et al. 1991; Freeman et al. 1995; Paavilainen et al. 2004). Our results also showed that the C-terminal region of UvAc1 interacted with UvCap1 in U. virens (Fig. 6). Adenylyl cyclase is a key component of the cAMP signaling pathway, and its function is to maintain the intracellular cAMP level and to participate in the proper infection morphogenesis in many pathogens (Choi and Dean 1997). The localization of adenylate cyclase is slightly different in mammals and yeast. In mammalian cells, adenylate cyclase is a transmembrane protein (Taussig and Gilman 1995), while in budding yeast, it is a peripheral membrane protein (Mitts et al. 1990; Huang et al. 1997).
The binding of CAPs to adenylate cyclase is important in regulating the post-translational modification of Ras2, which in turn is critical for the Ras-dependent activation of adenylate cyclase (Shima et al. 1997). Ras proteins are small GTP-binding proteins that recruit effectors to the cytoplasm membrane, and the activation of Ras requires Ras GTP-binding protein (RasGAP) (Shima et al. 2000; Wennerberg et al. 2005). In yeast, Ira1/2 (Ras GTPase activating protein) induces the GTPase activity of Ras1/2, leading to the activation of adenylate cyclase, meanwhile promoting the synthesis of cAMP (Tanaka et al. 1991). The disruption of the IRA1 gene leads to the accumulation of a large amount of adenylate cyclase in the cytoplasm (Mitts et al. 1991). In U. virens, there is no interaction between UvCap1 and UvGap1 (unpublished data), but UvCap1 interacted with UvRas1 and UvRas2 (Fig. 6). The interaction of UvCap1 with two Ras proteins and UvAc1 may also play an important role in the activation of UvAc1.
The adaptor protein Ste50 interacts with multiple upstream components to activate the MAP kinase cascade in M. oryzae (Park et al. 2006). In S. cerevisiae, Ste50 interacts with Ste11 and is involved in the cell wall integrity in vegetative cells (Jansen et al. 2001; Ramezani-Rad 2003; Kwan et al. 2006). In Schizosaccharomyces pombe and U. maydis, Ste4 and Ubc2 (homologous to Ste50 in S. cerevisiae) act directly upstream of the MAP kinase cascade and are involved in mating and other developmental processes (Barr et al. 1996; Mayorga and Gold 2001). Our study showed that UvCap1 also interacted with Uvste50 in U. virens (Fig. 6). UvCap1 may has an important relationship with MAPK signaling pathways to affect the development and infection process of U. virens.
Rice false smut has become one of the most important panicle diseases of rice in recent years. M. oryzae is also a filamentous ascus fungus that infects rice. The Cap1 proteins showed many similar functions in these two pathomycetes. Mycelial growth and conidiation were both reduced in the CAP1 mutants of U. virens (Fig. 2) and M. oryzae (Zhou et al. 2012). CAP1 regulates the appressoria formation and invasive growth in M. oryzae (Zhou et al. 2012). Although U. virens does not form a special appressorium, the expansion of infection hyphae was influenced by UvCap1 in this fungus (Fig. 3). As is the case in M. oryzae, the intracellular cAMP level in the CAP1 deletion mutant of U. virens was reduced compared with that in the wild-type strain (Fig. 7). Previous study demonstrated that deletion of CAP1 suppresses the effects of RAS2DA on appressoria formation in M. oryzae (Zhou et al. 2012). In this study, we found that UvCap1 interacted with UvRas2 in U. virens (Fig. 6). The subcelluar localization of UvCap1 in hyphae was similar to that of Cap1 in M. oryzae, and GFP-tagged UvCap1 was mainly localized in the apical regions of hyphae (Fig. 8).
In summary, we analyzed the function of UvCAP1 in the development and pathogenicity of U. virens. Further characterization of the functional relationships among UvCap1, UvAc1, Ras proteins and UvSte50 will provide valuable information to better understand the role of UvCap1 during the infection process of the rice false smut fungus.
We identified a cyclase-associated protein UvCap1, which showed a high expression level during the early infection process stage of U. virens. The deletion of UvCAP1 led to defects in mycelial growth, conidial production and pathogenicity. ΔUvcap1 exhibited more sensitive to sorbitol and H2O2 stresses. The intracellular cAMP level was significantly reduced in ΔUvcap1 compared with the wild-type strain. Yeast two-hybrid assay showed UvCap1 interacted with UvAc1, UvRas1, UvRas2 and UvSte50. Taken together, UvCap1 functions as a component of the cAMP signaling pathway, interacting with UvAc1, UvRas1, UvRas2 and UvSte50 to regulate the develpoment and pathogenicity of U. virens. Further work should focus on the role of UvCap1 in the cAMP signaling pathway and the relationship of UvCap1 with the PMKA signaling pathway during the infection process of the rice false smut fungus.
Strains and culture conditions
The wild-type strain P-1 of U. virens was used as the starting strain for subsequent strain construction; P-1 and the derivative strains were stored in 20–30% glycerol solution at -70 °C. The strains were cultured on potato sucrose agar (PSA) medium (200 g/L potato, 20 g/L sucrose, 15 g/L agar) or TB3 medium (3 g/L yeast extract, 3 g/L acid hydrolyzed casein, 20 g/L sucrose) at 28 °C in the dark for 10–20 days (Tsukui et al. 2015). The rice cultivar Liangyoupeijiu, which is susceptible to strain P-1, was used in the inoculation experiments (Yu et al. 2019). Yeast strain Y2HGold was cultured on YPDA medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 0.3 g/L adenine hemisulfate and 20 g/L agar) at 30 °C.
Phylogenetic analysis of CAP homologous proteins
All the homologous protein sequences of CAP in different fungi were obtained from the NCBI database and their accession numbers are shown as below: Ustilago maydis (XP_011386706.1), Cryptococcus neoformans (XP_012048542.1), Saccharomyces cerevisiae (PTN15174.1), Aspergillus oryzae (XP_001727621.1), Botrytis cinerea (XP_024547956.1), Neurospora crassa (XP_962678.3), Magnaporthe oryzae (MGG_01722), Colletotrichum orbiculare (TDZ25012.1), Colletotrichum gloeosporioides (KAF3811478.1), Verticillium longisporum (CRK25305.1), Verticillium longisporum (CRK26284.1), Beauveria bassiana (KGQ04943.1), Cordyceps javanica (TQV96574.1), Neonectria ditissima (KPM35571.1), Fusarium graminearum (XP_011317774.1), Fusarium fujikuroi (SCV37630.1), Fusarium oxysporum (SCO81237.1), Trichoderma harzianum (PNP52737.1), Trichoderma guizhouense (OPB37971.1), Trichoderma lentiforme (KAF3072262.1), Tolypocladium ophioglossoides (KND86868.1), Purpureocillium lilacinum (OAQ86548.1), Drechmeria coniospora (ODA78517.1), Hirsutella minnesotensis (KJZ78123.1), Claviceps purpurea (CCE30517.1), Ustilaginoidea virens (KDB15885.1), Pochonia chlamydosporia (RZR68491.1), Metarhizium guizhouense (KID90709.1), Metarhizium robertsii (EXV04750.1), Metarhizium anisopliae (KAF5133669.1). A neighbor-joining tree based on the above sequences was built using the Phylogeny.fr platform as described previously (Dereeper et al. 2008) with 1000 bootstrap repeats for distance estimation.
Construction of the UvCAP1 deletion mutant and its complemented strain
Targeted deletion mutants of the UvCAP1 gene were obtained with gene replacement strategy assisted with CRISPR-Cas9 system. First, we created a homologous recombination construct pMD19-UvCAP1 by inserting the HYG cassette between the two flanking sequences of the UvCAP1 gene. The 1025-bp upstream and 1104-bp downstream flanking sequences of UvCAP1 were amplified with primer pairs 1F/2R and 3F/4R, respectively (Additional file 2: Table S1). The up- and downstream flanking sequences and HYG cassette were cloned to T-Vector pMD19 (simple) (TaKaRa, Japan) using a ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing).
Then the gRNA designer program was used to design UvCAP1-gRNA spacers, and the Cas9 off program was used to screen the high-score spacer to minimize potential off-target effects in the genome (Doench et al. 2014; Guo et al. 2014). The sense and antisense strands of UvCAP1-gRNA spacer (Additional file 2: Table S1) were synthesized and annealed to generate double-stranded gRNA spacers (Arazoe et al. 2015; Liang et al. 2018). Then the double-stranded gRNA spacers were cloned to the two BsmBI sites of pmCas9:tRp-gRNA (Liang et al. 2018) using a ClonExpress II One Step Cloning Kit (Vazyme, Nanjing). The resulting construct was confirmed by sequencing.
The vectors pMD19-UvCAP1 and UvCAP1-gRNA were co-transformed into protoplasts of the wild-type strain P-1 using the PEG-mediated method as described previously (Liang et al. 2018). Hygromycin-resistant transformants were screened for deletion of UvCAP1 by PCR with primers 5F/6R (Additional file 2: Table S1). Overlapping PCR with primers 7F/8R and 9F/10R was used to detect whether UvCAP1 was replaced by the HYG cassette. The ∆Uvcap1 mutants were verified by sequencing analysis (Additional file 2: Table S1).
For complementation assays, the UvCAP1 gene, along with 1025-bp upstream and 365-bp downstream sequences was amplified with primers UvCAP1-comF/comR (Additional file 2: Table S1) and inserted into the pKO1-NEO (G-418 resistance) vector. Then the construct was transformed into ∆Uvcap1 (#11) using the Agrobacterium-mediated transformation (ATMT) method (Lv et al. 2016). The resulting transformants were first screened by recovery of growth defects, followed by RT-PCR amplification, and verified by fully restored phenotype characterization.
Subcellular location of UvCap1
To construct the subcellular location strain UvCap1:GFP, we amplified 1955 bp coding sequence of UvCAP1 with primers UvCAP1-GFPF/GFPR (Additional file 2: Table S1). The resulting fragment was cloned into the BamHI and SmaI sites of vector pKD2-GFP and then transformed into the wild-type strain P-1 by ATMT as described previously (Lv et al. 2016). G-418-resistant transformants were screened for further fluorescence microscopic observation.
Phenotypic analysis of U. virens strains
In the assays for mycelial growth of U. virens strains, the mycelia of the wild-type strain P-1, the ΔUvcap1 (#11, #14) mutant and the complemented Uvcap1-c (#1) were ground with a tissue blender (Waring Commercial Blender 8011S, USA) and diluted with potato sucrose broth (PSB), then individually spread evenly on PSA medium plates, and cultured at 28 °C. A mycelial block (5 mm in diameter) cut from the margin of 5-day-old colony was inoculated on the center of a PSA or TB3 medium plate at 28 °C for 20 days with 5 replicates for each group (Yu et al. 2019; Yong et al. 2020). Colony diameters were measured using the cross-measurement method.
In the conidiation assays, the same method as above-mentioned was used to obtain mycelial blocks. Three mycelial blocks were transferred into 50 mL of YT (1 g/L yeast extract, 1 g/L tryptone and 1 g/L glucose) liquid medium and incubated with shaking (150 rpm) at 28 °C for 6 days. The number of conidia was recorded under a microscope using a hemocytometer. All the experiments were performed three times with three replicates.
Plant infection assays of U. virens strains
To detect if UvCap1 is involved in the infection process of U. virens, the susceptible rice variety Liangyoupeijiu was inoculated as described previously (Tang et al. 2013; Yu et al. 2015). The wild-type strain P-1, the ΔUvcap1 (#11) mutant and its complemented Uvcap1-c (#1) were cultured in PSB with shaking at 150 rpm, 28 °C for 7 days. A tissue blender (Waring Commercial Blender 8011S, USA) was used for the preparation of a mixture of hyphae and conidia, and the conidia were diluted to a concentration of 1 × 106 conidia/mL with PSB. About 5–7 days before heading, 1–2 mL of hyphae and conidia suspensions were injected into the panicles of rice plants using sterilized syringes. Inoculated rice plants were cultured in a humid environment with a water spray system and the number of rice false smut balls per panicle were counted at 30 dpi (Hu et al. 2014; Yong et al. 2020). The expansion of infection hyphae inside the spikelets of the wild-type strain P-1, the ΔUvcap1 (#11) mutant and its complemented Uvcap1-c (#1) were observed at 10, 14 and 18 dpi. GFP-tagged strains of U. virens were also constructed to further ascertain if UvCap1 is involved in the infection process. The binary vector pKD2-GFP was transformed into P-1, ΔUvcap1 (#11) and Uvcap1-c (#1) by ATMT as described previously (Lv et al. 2016). The expansion of infection hyphae inside the spikelets of these GFP-tagged strains was observed at 10 dpi under the stereo fluorescence microscope (Olympus IX71, Japan). The test was repeated three times in total.
Stress response assays of U. virens strains
In the stress response assays, the same method as above-mentioned was used to obtain mycelial blocks. A mycelial block (5 mm in diameter) cut from the margin of 5-day-old colony was inoculated on the center of a PSA medium plate or PSA supplemented with different concentrations of stress agents, including 0.5 M NaCl, 0.6 M sorbitol, 500 μg/mL CFW, 100 μg/mL Congo red, 0.05% SDS and 0.05% H2O2. The plates were incubated in 28 °C for 20 days, with five replicates for each group (Yu et al. 2019; Yong et al. 2020). Colony diameters were measured using the cross-measurement method. Inhibition rates were calculated as described previously (Xie et al. 2019; Yong et al. 2020).
RT-qPCR analysis of the expression level of UvCAP1
Rice spikelets were collected at 0, 1, 2, 3, 5, 7 and 14 dpi. RNA was extracted using an RNA extraction kit (BioTeKe, China) and cDNA reverse transcription was performed with the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan) as previously described (Yong et al. 2020). qPCR was performed in an ABI Q6 Real-Time System with primers UvCAP1-qF/qR (Additional file 2: Table S1). TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Japan) was used to evaluate the relative abundance of target gene transcripts, and the average threshold cycle (Ct) was normalized to that of the TUBULIN gene (TUBULIN-qF/-qR) (Additional file 2: Table S1).
Yeast two-hybrid assay
Protein-protein interactions were performed using the yeast two-hybrid system (Clontech, USA). The ORF regions of UvCAP1 was amplified from the cDNA of the wild-type strain P-1 with primers UvCAP1-BDF/BDR (Additional file 2: Table S1) and cloned into pGBKT7 vector using the ClonExpress II One Step Cloning Kit (Vazyme, China). The ORF regions of UvRAS1, UvRAS2 and UvSTE50 were individually cloned into the pGADT7 vector to obtain prey constructs. Then the resulting bait and prey constructs were co-transformed into yeast strain Y2HGold. The transformants were grown on SD/-Leu/-Trp, SD/-His/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp medium for 3–5 days at 30 °C.
Availability of data and materials
Confocal laser scanning microscope
Days post inoculation
- HYG :
Hygromycin resistance gene
Green fluorescent protein
National Center for Biotechnology Information
Open reading frame
Potato sucrose agar
Potato sucrose broth
Reverse transcription quantitative PCR
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This work was supported by the National Natural Science Foundation of China (31901838) and Natural Science Foundation of Jiangsu Province (BK20180296 and BK20160588).
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Construction of the UvCAP1 deletion mutant and its complemented strain. a Schematic diagram of the construction of replacement plasmid. b Hygromycin-resistant transformants were screened for deletion of UvCAP1 by PCR. I, bands for the UvCAP1 gene with primers 5F/6R; II and III, bands for overlapping PCR with primers 7F/8R and 9F/10R to detect whether UvCAP1 was replaced by the HYG cassette. IV, bands for β-TUBULIN (as a control). c G-418-resistant transformants were screened for complementation of ΔUvcap1(#11) by RT-PCR. I, bands for the UvCAP1 gene with primers 5F/6R; II, bands for β-TUBULIN (as a control).
Primers used in this study.
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Cao, HJ., Zhang, JJ., Yong, ML. et al. The cyclase-associated protein UvCap1 is required for mycelial growth and pathogenicity in the rice false smut fungus. Phytopathol Res 3, 5 (2021). https://doi.org/10.1186/s42483-021-00083-0
- Rice fungal disease
- Ustilaginoidea virens
- Cyclase-associated protein UvCap1
- Mycelial growth