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MrMid2, encoding a cell wall stress sensor protein, is required for conidium production, stress tolerance, microsclerotium formation and virulence in the entomopathogenic fungus Metarhizium rileyi
Fungal Genetics and Biology 134 (2020) 103278
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MrMid2, encoding a cell wall stress sensor protein, is required for conidium production, stress tolerance, microsclerotium formation and virulence in the entomopathogenic fungus Metarhizium rileyi
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Caiyan Xina, Xiaorui Xinga, Fen Wanga, Jiexing Liub, Zhuonan Ranb, Wenbi Chena, ⁎ Guangxi Wanga, Zhangyong Songa, a b
School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, People’s Republic of China Department of Geriatrics, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Metarhizium rileyi Transmembrane protein Microsclerotium formation Conidiation
Transmembrane proteins as sensors encoded by fungal genes activate specific intracellular signal pathways in response to stress cues to help the fungus survive in a changing environment. To better understand the role of the cell wall integrity (CWI) pathway in the entomopathogenic fungus Metarhizium rileyi, an ortholog encoding the transmembrane protein Mid2, MrMid2, was identified and characterized functionally. Transcriptional analysis indicated that MrMid2 was involved in dimorphic transition, conidiation, and microsclerotium formation. After a targeted deletion of MrMid2, all three traits were impaired. Compared with the wild-type strain, the △MrMid2 mutants were hypersensitive to thermal stress, and cell wall and oxidative stress. Insect bioassays revealed that △MrMid2 mutants had decreased virulence levels following topical (22.5%) and injection bioassays (38.7%). Furthermore, transcription analysis showed that other genes of the CWI pathway, with the exception of another major sensor protein encoding gene, MrWsc1, were down-regulated in △MrMid2 mutants. These results suggest that MrMid2 plays important roles in dimorphic transition, conidiation, the stress response, virulence, and microsclerotium development in M. rileyi.
1. Introduction Metarhizium rileyi is a well-known entomopathogenic fungus with yeast-like hyphal bodies and a filamentous growth phase, and it is used as a biological control agent against lepidopterous pests (Fronza et al., 2017; Song 2018). The melanized microsclerotia (MS) induced in liquid medium exhibit longer effective pest control in the field compared to conidial products (Goble et al., 2017; Jackson and Jaronski 2009; Song et al., 2014). Therefore, the MS product can be used as a mycoinsecticide for insect control. The development of viable MS formulations will promote the mass production and commercialization of M. rileyi for use in pest management (Fronza et al., 2017; Song 2018). To increase M. rileyi MS production, the molecular basis of MS development has already been investigated (Song et al., 2013, 2018a, b; Song 2018; Wang et al., 2018). A number of M. rileyi genes have been shown involved in MS development. Moreover, the changing culture conditions during MS development activate several conserved intracellular signal pathways. Previous investigations had reported that the high osmolarity glycerol (HOG) and cell wall integrity (CWI)
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pathways cooperate to regulate MS development (Song et al., 2016). To sense the environmental stresses, MS development is controlled by interplay between signaling pathways, which occur in concert with complex signaling events (Song et al., 2018a, b; Song 2018). We have investigated the function and adaptation of transmembrane proteins of the HOG pathway and found that these proteins act differently in response to exogenous culture stress (Song et al., 2015). However, little is known about the roles of the transmembrane proteins in the CWI signal pathway during M. rileyi MS development. Mid2 is a transmembrane protein that acts as the main sensor protein in the CWI pathway (Green et al., 2003; Jin et al., 2013; Nishida et al., 2014; Philip and Levin 2001; Straede and Heinisch 2007; Wang et al., 2016). The Mid2 orthologs have been characterized in Saccharomyces cerevisiae (Straede and Heinisch 2007), Beauveria bassiana (Kim et al., 2016), Fusarium oxysporum (Thatcher et al., 2012), and Purpureocillium lilacinum (Wang et al., 2016). This transmembrane protein contains a transmembrane domain, a cytosolic tail and an extracellular serine/threonine-rich region that is highly mannosylated during secretion and which mediates clustering in the sensor in order to sense the
Corresponding author. E-mail address: [email protected] (Z. Song).
https://doi.org/10.1016/j.fgb.2019.103278 Received 30 March 2019; Received in revised form 24 August 2019; Accepted 9 October 2019 Available online 11 October 2019 1087-1845/ © 2019 Elsevier Inc. All rights reserved.
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generation of deletion and complementation mutants were listed in Table S1.
extracellular stress. The cytoplasmic tail interacts with a small Ras-like GTPase (Rho1) and then presumably activates the CWI pathway to transmit extracellular signals to the nucleus to modulate gene expression, although there is more than one transmembrane protein that can sense cell wall stress. Previous investigations have confirmed that different sensors employ different mechanisms to sense cell wall stress (Bermejo et al., 2010; Dichtl et al., 2012). MrMid2 of M. rileyi was shown to be up-regulated during MS formation in our earlier transcriptomic analysis (Song et al., 2013). These results indicated that MrMid2 may have a related function in the regulation of MS development. However, this possible function has not been studied in any detail. In the current study, we employed bioinformatics and an experimental strategy to analyze the function of MrMid2. The MrMid2 gene was deleted by homologous recombination strategy (Song et al., 2016). Our functional studies showed that MrMid2 is needed for dimorphic transition, conidiation, stress tolerance, virulence, and MS formation. Furthermore, investigation of gene expression patterns showed that MrMid2 mediates regulation of target genes involved in MS development in M. rileyi.
2.4. Phenotypic assays The difference in conidial production among the tested strains was assessed by incubation on SMAY plates under continuous light at 25 °C for 12 d. The morphology of colonies was investigated using a digital camera (60-mm Macro lens, Canon Inc., Japan). Conidium yields were determined as described previously (Song et al., 2015). Briefly, the conidium yield of each strain was assessed by taking three 6-mm-diameter samples from different plates, performing conidium counts in a hemocytometer under a microscope to determine the concentration of the suspension, and converting the concentration to number of conidia cm−2. For cell wall and oxidative stress assays, all strains were grown on SMAY medium amended with different stress agents for 12 d according to previously described methods (Song et al., 2018a). The conidium yield of each strain was determined as described above. The conidium yield inhibition rate was calculated relative to untreated controls, where inhibition rate (%) = (number of untreated colonies – number of treated colonies) / the number of untreated colonies × 100. In vitro, M. rileyi in yeast-cell form was cultured for 2–4 d on SMAY to transform it into the hyphal form. To analyze the effect of MrMid2 on this dimorphic transition, tested strains were investigated as previously described (Song et al., 2018a). Briefly, 100 simple yeast cells were pipetted onto SMAY medium and grown at 25 °C. Percentage switching rates at indicated time points were recorded. Median transition time required for 50% transition of yeast cells to hyphae (TT50) was calculated. For the MS yield assay, the tested strains were inoculated into liquid amended medium (AM) (Song et al., 2014) and cultured for 6 d. The morphology of samples was investigated using the digital camera technique described earlier. Biomass and MS yield were determined as described previously (Jackson and Jaronski 2009). For MS yield measurements, the culture broth was diluted in order to count MS under a microscope. For biomass evaluation, the liquid fermentation products were collected by vacuum filtration in a Buchner funnel to remove the culture solution. The dry weight accumulations were determined to a constant weight prior to measurement. Each investigation was repeated three times to generate independent biological replicates. To assess the role of MrMid2 in conidial germination, all strains were investigated according to previously described methods (Song et al., 2016). Briefly, the conidial suspensions were inoculated onto SMAY plates at 25 °C. After inoculation for 14 h, germination was assessed every 2 h, and the number of germination conidia was counted under microscopy (400×). Conidial thermotolerance was quantified as the germination rate after exposure to 45 °C (in a water baths) for 30 or 60 min as described previously (Song et al., 2014). To assess the clonal growth rate, a hyphal mass disk (6-mm diameter) was taken from the 3-d-old SMAY cultures and placed centrally onto a SMAY plates (90-mm diameter) and incubated for 12 d at 25 °C. The vegetative growth diameter was measured by vernier calipers every 2 d. To assess hyphal sensitivity to cell wall perturbation, the hyphal growth rate was measured after treatment with Congo red (1.0 mg mL−1), according to a previously described method (Zhou et al., 2018). Briefly, the hyphal mass disks were placed on a SMAY plates supplemented with Congo red, and the vegetative growth diameter was determined by vernier calipers every 2 d. The growth inhibition rate was calculated relative to untreated controls, where inhibition rate (%) = (diameter of untreated colonies – diameter of treated colonies) / diameter of untreated colonies × 100. Each treatment was replicated independently for three times.
2. Materials and methods 2.1. Fungal strains and culture conditions The wild-type (WT) M. rileyi strain CQNr01, obtained from the Engineering Research Center for Fungal Insecticides, Chongqing, China, was used in this study. The WT and all genetic mutants generated in this study were single-spore purified and were cultured on solid Sabouraud maltose agar medium fortified with 1% (w/v) yeast extract (SMAY). Appropriate antibiotics were added for selection of mutants according to previously described methods (Song et al., 2018a). 2.2. Bioinformatics analysis The MrMid2 sequence from our previous transcriptomics analysis (Song et al., 2013) was used as a query to search through the M. rileyi genome database (NCBI accession No. AZHC00000000.1) (Shang et al., 2016). Multiple sequence alignments were conducted with DNAMAN software (http://www.lynnon.com). The potential signal peptide and transmembrane domains were predicted by the SignalP server (Bendtsen et al., 2004). A phylogenetic tree was constructed using MEGA 6.0 software with the Neighbor-Joining method (Tamura et al., 2013). 2.3. Deletion of MrMid2 and mutant complementation Plasmid pPZP-Hph-Knock (Shao et al., 2015), containing the selectable marker hygromycin phosphotransferase gene (hph), was used as the backbone with which to construct the MrMid2 gene deletion plasmid. The 5′ and 3′ flanking sequences were amplified from the WT genome via PCR and inserted independently into pPZP-Hph-Knock. For complementation of the deletion mutant colonies, plasmid pPZP-SurKnock containing the sulfonylurea resistance gene (sur) was used as the complementation vector. A fragment containing the putative promoter, coding sequence, and downstream sequence of MrMid2 was amplified and inserted into pPZP-Sur-Knock. Deletion and complementation vectors were transformed independently into Agrobacterium tumefaciens AGL-1 (Invitrogen, Shanghai, China). Putative MrMid2 deletion mutants were able to grow without check on SMAY plates containing hygromycin B and cephalosporin. After subculture of resistant colonies for five generations, PCR screening was performed with primers homologous to the hph and genomic sequence outside the flanking regions (MrMid2F/hph-R and MrMid2F/hph-F) (Table S1) and the amplicons were sequenced. The positive gene deletions were then confirmed by PCR with primers MrMid2OF/MrMid2OR. All primers used for the
2.5. Virulence assays Virulence assays were performed on Spodoptera litura larvae using immersion into or injection with conidial suspension methods, 2
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Fig. 1. Biological roles of MrMid2 in the asexual cycle. (A) Relative expression levels of MrMid2 during conidiation at different time points. Stages of conidial development included conidia inoculated onto the plate at the start of incubation (day 0), blastospores (day 2), hyphal period (day 4), conidiation initiation (day 6), and the start of conidium maturation (day 8). (B) Colony morphology of tested strains inoculated on SMAY plates and cultured under continuous light at 25 °C for 3, 6, 9, and 12 d. (C) TT50 of WT, complementation strains, and △MrMid2 mutants were compared. (D) Statistical analysis of conidial yields. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences from the WT or from the results at 0 days of incubation.
mortality rate was recorded daily to determine the mean lethal time (LT50) values.
according to previously described methods (Song et al., 2016). Briefly, cultures of the tested strains were incubated on SMAY plates for 12 d at 25 °C. Conidia were harvested and resuspended to 5 μL suspension of 1 × 106 conidia mL−1 in cottonseed oil or 5 μL of 1 × 106 conidia mL−1 in sterile water containing 0.01% Tween 80. Sham controls were treated with pure cotton seed oil or sterile water containing 0.01% Tween 80 only, without conidia. The assay was repeated three times and 30 larvae were tested in each replicate. After treatment, larvae were reared as described previously (Song et al., 2015). The larval
2.6. Quantitative real-time PCR (qPCR) For time-specific expression patterns during conidiation, 3 μL suspension aliquots of 1 × 107 WT conidia mL−1 were pipetted onto SMAY plates, followed by incubation under continuous light at 25 °C for 8 d. Total RNA was isolated during the following stages: conidia at the start 3
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Fig. 2. Deletion of MrMid2 resulted in an altered germination rate, clonal growth rate, and response to stress. (A) Germination of conidia from tested strains and (B) conidial tolerance to heat stress at 45 ℃. A conidium was considered to be germinated if the germ-tube length was at least half that of the conidium. (C) Statistical analysis of fungal growth inhibition rates. (D) Statistical analysis of conidial yield inhibition rates under cell wall and oxidative stress. Values were the mean of three independent experiments. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences when compared with the results observed in the WT.
sampled at different time points. The gene expression profile was assessed in samples of WT, △MrMid2, or complementation hyphae cultured in AM for 60 h. Total RNA was collected according to previous methods (Song et al., 2015). qPCR was performed using SYBR® Premix Ex TaqTM II (TaKaRa, Dalian, China), following the manufacturer’s instructions. β-tubulin (Mrtub) and translation elongation factor (Mrtef) genes were used as internal standards. The 2−ΔΔCt method was used to evaluate relative transcript levels (Vandesompele et al., 2002).
of incubation (day 0), blastospores (day 2), hyphal period (day 4), conidiation initiation (day 6), and the start of conidium maturation (day 8). To assess the possibility of whether changing culture conditions affected the expression of MrMid2, a set of experiments was set up in which the incubated liquid AM or minimal medium (MM, i.e. AM without basal salts) was supplemented with either exogenous iron (1 mM) or H2O2 (3 mM) as described previously (Song et al., 2015). For analyses of gene expression of MrMid2 at different stages of MS development in the WT, samples were prepared in AM culture and sub4
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of △MrMid2 mutants than in the WT and complementation strains during a 2-d incubation period after treatment at 45 ℃ (Fig. 2B). The △MrMid2 mutants exhibited a significant decrease in germination rate over the 24-h incubation period when exposed to thermal stress for 30 min (65.3% decrease) or 60 min (81.9% decrease), compared with decreases in germination rate in the WT over the 24-h incubation period of 51.3% (30 min) or 71.8% (60 min) and those of the complementation strain of 50.9% (30 min) or 65.9% (60 min) (P < 0.001). This thermotolerance study showed that MrMid2 was needed in response to thermal stress. Compared to the WT and complementation strains, the cross-section of mycelial growth of the △MrMid2 mutants was reduced after a 4-day incubation (data not shown) in the absence of stress. Furthermore, the △MrMid2 mutants also displayed defects in vegetative growth, with colony sizes being 15% smaller compared with the WT and complementation strains, after a 12-d incubation on the SMAY medium (Fig. S3). Furthermore, △MrMid2 mutants showed increased sensitivity to cell wall stress. When exposed to cell wall stress, the growth inhibition rate of △MrMid2 mutants was increased by 18.5%, compared with that of the WT and complementation strains (Fig. 2C). Compared to the WT and complementation strains, the conidial yields of the △MrMid2 mutants cultured under stressful conditions were severely affected. Increases in inhibition rates of the △MrMid2 mutants in comparison to WT or complementation is of 18.2 – 71.2% under different stressful conditions (Fig. 2D).
2.7. Statistical analysis Statistical analyses were performed using SPSS 16.0 software (IBM, Armonk, NY, USA). Graphs were prepared using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) was performed, followed by Duncan’s multiple range tests to determine significant differences between samples, with P = 0.05 being the threshold significance level. 3. Results 3.1. Generation of MrMid2 mutants Orthologs of MrMid2 were obtained (NCBI accession No. MK408430), which encodes a protein of 278 amino acid residues and contains a conserved Pfam domain (residues 57–99) (Fig. S1A). In addition, the amino acid sequence of MrMid2 was similar to that of Mid2 protein (70% identity) in Metarhizium brunneum and Mid2 protein (67% identity) in Metarhizium guizhouense (Hu et al., 2014). Phylogenetic analysis from other fungi demonstrated that MrMid2 is relatively conserved among fungal species (Fig. S1B). To examine the biological function of MrMid2 in M. rileyi, MrMid2 deletions were generated by homologous recombination and complementation strains were constructed by heterologous insertion. The positive gene knockout and complementation strains were screened and confirmed by PCR and qPCR analysis (Fig. S2). Taken together, the results achieved deletion of MrMid2 (strain △MrMid2) and mutant complementation with MrMid2 to produce strain △MrMid2 + mid2.
3.4. Deletion of MrMid2 delays MS formation Expression levels of MrMid2 during MS development were analyzed by qPCR. Compared with the vegetative growth stage (36 h), the expression of MrMid2 was up-regulated when MS formation took place (60 – 96 h) (Fig. 3A). Our preliminary experiments had revealed that iron and oxidative stress are critical factors that induce MS differentiation (Song 2018). To assess the effect of exogenous iron and oxidative stress on MrMid2 expression levels, AM was supplemented with exogenous 1 M iron or 3 mM H2O2 as previously described (Song et al., 2018a). The results showed that MrMid2 was up-regulated when exposed to exogenous iron or H2O2 for MS induction (Fig. 3B-3D), indicating that MrMid2 might be involved in the regulation of MS formation. After incubation in liquid AM culture, MS produced by WT and complementation strains matured and were accompanied by secondary mycelial growth, whereas the MS yield in △MrMid2 mutants was significantly lower (Fig. 4A). Compared with the WT strain, the MS yield of △MrMid2 mutants was reduced by approximately 97% (Fig. 4B). With few mycelia in the △MrMid2 mutants, the biomass of the △MrMid2 mutants was decreased by 39% (Fig. 4C). These results indicated that MrMid2 contributed to MS development. To investigate the genes regulated by MrMid2 during MS formation, candidate genes were selected from transcriptome libraries of MS formation (Song et al., 2013, 2018b) and examined by transcriptional analysis. The following genes were selected: several chitin synthase genes (Mrch1, Mrch2, and Mrch4, for class I, II, and IV chitin synthases, respectively), a cell wall biosynthesis-related gene (Mrfks, for β-1,3glucanase synthetase), components of the CWI pathway (namely, the Mrrho1, Mrpks1, Mrbck1, Mrmkk1,2, Mrslt2, and Mrswi6 genes) and another transmembrane protein of the CWI pathway (the Mrwsc1 gene). It was found that the downstream component of the CWI pathway was down-regulated, whereas the Mrwsc1 gene was up-regulated in the △MrMid2 mutants during MS formation (Fig. 5). Compared with the WT, Mrfks was significantly up-regulated and Mrch2 and Mrch4 were significantly down-regulated during MS development in the △MrMid2 mutants (Fig. 5).
3.2. Loss of MrMid2 results in delayed yeast-to hyphae transition and decreased conidial yield Compared with the initial results at day 0, expression of MrMid2 was found to be up-regulated during blastospore formation (day 2) and down-regulated during hyphal vegetative growth (day 4) (Fig. 1A). This result indicated that MrMMid2 may function in the yeast-to-hyphae transition. Intriguingly, compared to WT, at day 3, the △MrMid2 mutants displayed a delay in the yeast-to-hyphae transition (Fig. 1B), and further dimorphic transition analyses showed that a significant difference is between △MrMid2 mutants (TT50 = 8.5 ± 0.4 d) in comparison to WT (TT50 = 7.4 ± 0.1 d) (P < 0.05) (Fig. 1C). These results suggested that MrMid2 was involved in the yeast-to-hyphae transition. Further expression analyses showed that MrMid2 was up-regulated during the conidiation initiation (day 6) and at the start of conidium maturation (day 8) (Fig. 1A). This result indicated that MrMMid2 may also function in the conidiation process. Further investigations showed that the △MrMid2 mutants displayed decreased conidial yield compared to the WT and complementation strains (P < 0.001) (Fig. 1B and 1D). Taken together, these gene expression and phenotypic analyses suggested that MrMid2 was involved in conidium production. 3.3. MrMid2 is essential to the stress response Compared to the WT, the △MrMid2 mutants displayed defects in conidial germination, thermal stress, and cell wall and oxidative stress (Fig. 2). The rate of conidial germination was tested on SMAY and the results are shown in Fig. 2A. The germination rate of conidia of △MrMid2 mutants was reduced compared with that of the WT and complementation strains. After 14 h, the germination rate (32.2 ± 3.6%) for conidia of the △MrMid2 mutants was significantly lower than that of the WT (63.3 ± 4.5%) and complementation strains (60.2 ± 2.5%) (P < 0.001). After 24 h, the germination rate (82.2 ± 1.8%) for conidia of the △MrMid2 mutants was also significantly lower than that of the WT (97.4 ± 1.9%) and complementation strains (95.6 ± 2.4%) (P < 0.001). Overall, there was a significantly slower germination rate of conidia 5
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Fig. 3. qPCR analysis of gene expression levels. Relative expression level of MrMid2 during (A) MS development or following treatment with (B) H2O2 or (C) iron cation in MM or treatment with (D) iron cation in AM. Stages of MS development included blastospore period (36 h of incubation), hyphal elongation and MS initiation period (60 h), MS formation (72 h), mass MS formation (96 h), and MS maturation (120 h). Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared to the results at 36 h or 0 min.
shift (Boucias et al., 2016; Pendland and Boucias 1997; Song et al., 2016, 2018a, b). MrMid2 belongs to a group of transmembrane proteins, which senses cell wall stress and activates the CWI pathway. Interestingly, in this study, △MrMid2 mutants resulted in a delay in the yeast-to-hyphae transition (Fig. 1). This result was similar to that exhibited by the △MrSlt2 and △Mrswi6 mutants (Song et al., 2016; Wang et al., 2019). Like S. cerevisiae (Mathumathi et al., 1999), the vegetative growth defects of the △MrMid2 mutants were exhibited on SMAY medium. Such yeast-to-hyphae and vegetative growth defects of the △MrMid2 mutants may be attributable to decreased conidium production. Previously, it was reported that deletion of Mid2 from S. cerevisiae resulted in defects in terms of the thermal and stress response (Jin et al., 2013; Philip and Levin 2001; Straede and Heinisch 2007). The role of MrMid2 in M. rileyi was similar to that in the yeast (Fig. 2), although its role in the thermal stress response was unlike that in △AgMid2 in Ashbya gossypii, the growth of which (relative to the WT) showed no significant difference at different temperatures (Lengeler et al., 2013). The exact molecular mechanism by which the plasma membrane sensors detect the extracellular stress remains elusive. There are two small families of sensors of the CWI pathway in the yeast and fungi, namely the cell wall stress component-type sensors Wsc1, Wsc2 and Wsc3 on the one hand, and Mid2 and Mtl1 on the other (Jendretzki et al., 2011; Rodicio and Heinisch 2010). Genetic analyses indicated
3.5. MrMid2 is required for virulence Bioassays showed that virulence of △MrMid2 mutants were significantly lower than that exhibited by the WT and complementation strains (Fig. 6). The LT50 values for the WT strain were 6.2 ± 0.3 d in the topical bioassay and 4.3 ± 0.4 d in the injection bioassay. The LT50 values of the complementation strain were 6.3 ± 0.4 d in the topical bioassay and 4.9 ± 0.3 d in the injection bioassay. Compared with these data, the LT50 of △MrMid2 mutants was delayed by 1.4 days in the topical bioassay and 1.9 days in the injection bioassay. These results showed that MrMid2 was required for virulence. 4. Discussion In the present study, the function of MrMid2 was characterized in M. rileyi. Our results showed that deletion of MrMid2 resulted in delayed morphological shift, conidial production and MS formation, and hypersensitivity to thermal stress, and cell wall and oxidative stress. In addition, the △MrMid2 mutants also exhibited reduced virulence. Morphological shift is essential for the life cycle and virulence of dimorphic fungi (Gauthier 2015). The M. rileyi exhibits a yeast-to-hyphae transition lifestyle and recent investigations confirmed that multiple signaling pathways are related to the regulation of morphological 6
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that Mid2 and Wsc1 act as the main sensor proteins in the CWI pathway in yeast and filamentous fungi (Futagami et al., 2011; Green et al., 2003; Jin et al., 2013; Nishida et al., 2014; Philip and Levin 2001; Straede and Heinisch 2007). Two transmembrane sensors of the HOG pathway of M. rileyi were investigated and important roles in sensing external culture stress during MS development were identified (Song et al., 2015). The external culture stress also gave rise to up-regulated expression of MrWsc1 and MrMid2 during MS development (Song et al., 2013). However, targeted gene knockout of MrWsc1 has not been successful despite many attempts at transformation. In the current study, the roles of MrMid2 were determined and MrMid2 was found to be critical for MS formation in M. rileyi. As with the defective MS formation in the △MrSlt2 and △Mrswi6 mutants, △MrMid2 mutants produced only a few MS (Fig. 4) (Song et al., 2016; Wang et al., 2019). Preliminary experiments had revealed that the fungus is exposed to various stresses, including pH and iron cation concentration, in the culture medium during MS formation (Song et al., 2015). To sense the extracellular stress, the extracellular domain of transmembrane proteins mediate sensor clustering (Francois et al., 2013). Moreover, the fungi must remodel the cell wall structure during vegetative growth and morphogenesis. Cell wall remodeling and biosynthesis is regulated by the CWI signaling pathway. As with other fungi, MrMid2 deletion led to down-regulation of the expression of the downstream components of the CWI pathway (Dichtl et al., 2012; Green et al., 2003; Jin et al., 2013). Interestingly, as in other fungi (Dichtl et al., 2012; Philip and Levin 2001), the expression of MrWsc1 in the MrMid2 mutants was upregulated, indicating that the functions of the two genes for MS development are partially redundant (Fig. 4). Additionally, the chitin synthase and 1,3-glucanase synthetase genes exhibited differential expression in response to extracellular stress during MS development. To model the cell wall structure during MS development, there must be the other direct downstream interaction partners and effectors of MrWsc1 in the △MrMid2 mutants. Further experiments are needed to identify and elucidate the roles of these genes. The HOG and CWI pathways are known to contribute to the regulation of pathogenesis in yeast and filamentous fungi (Zhao et al., 2007). In the present study, △MrMid2 mutants were found to be significantly less pathogenic following either topical or injection assays (Fig. 6). Similar results have also been observed in the △MrSlt2 and △Mrswi6 mutants (Song et al., 2016; Wang et al., 2019) and in the △BbMid2 mutants of B. bassiana (Kim et al., 2016) and △Mid2 mutants of F. oxysporum (Thatcher et al., 2012). The △MrMid2 mutants exhibited hypersensitivity to various stresses and defective vegetative growth in response to various stresses. As a result, △MrMid2 exhibited decreased
Fig. 4. Microsclerotium development in AM. (A) Morphology of microsclerotia in AM after 144 h of incubation. MS yield (B) and biomass (C) of the tested strains. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared with WT.
Fig. 5. qPCR analysis of gene expression in △MrMid2 mutants after 60 h incubation in AM medium. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared with WT. 7
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Fig. 6. Insect bioassays. Insect survival after (A) topical application or (C) injection of conidia from specific strains. Mean lethal time in (B) topical infection or (D) injection application assays. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared with WT.
virulence. In summary, a gene homologous to Mid2 was functionally characterized in M. rileyi in this study. Phenotypic analyses revealed that MrMid2 has important functions in dimorphic transition, stress response, and conidium production, and is required for virulence and MS formation. Taken together, the data presented in the current study could facilitate future evaluation of cell wall integrity and the development of conidia and MS in M. rileyi.
profiling of wsc1 and mid2 deletion strains and chimeric sensors in Saccharomyces cerevisiae. OMICS 14 (6), 679–688. Boucias, D., Liu, S., Meagher, R., Baniszewski, J., 2016. Fungal dimorphism in the entomopathogenic fungus Metarhizium rileyi: detection of an in vivo quorum-sensing system. J. Invertebr. Pathol. 136, 100–108. Dichtl, K., Helmschrott, C., Dirr, F., Wagener, J., 2012. Deciphering cell wall integrity signaling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases. Mol. Microbiol. 83 (3), 506–519. Francois, J.M., Formosa, C., Schiavone, M., Pillet, F., Martin-Yken, H., Dague, E., 2013. Use of atomic force microscopy (AFM) to explore cell wall properties and response to stress in the yeast Saccharomyces cerevisiae. Curr. Genet. 59, 187–196. Fronza, E., Specht, A., Heinzen, H., de Barros, N.M., 2017. Metarhizium (Nomuraea) rileyi as biological control agent. Biocontrol. Sci. Tech. 2, 1–22. Futagami, T., Nakao, S., Kido, Y., Oka, T., Kajiwara, Y., Takashita, H., Omori, T., Furukawa, K., Goto, M., 2011. Putative stress sensor WscA and WscB are involved in hypo-osmotic and acidic pH stress tolerance in Aspergillus nidulans. Eukaryot. Cell 10 (11), 1504–1515. Gauthier, G.M., 2015. Dimorphism in fungal pathogens of mammals, plants, and insects. PLOS Pathog. 11 (2), e1004608. Goble, T.A., Gardescu, S., Jackson, M.A., Hajek, A.E., 2017. Evaluating Metarhizium brunneum F52 microsclerotia in hydromulch formulations using different tackifiers under forest and orchard conditions. Biocontrol 62 (6), 769–778. Green, R., Lesage, G., Sdicu, A.M., Ménard, P., Bussey, H., 2003. A synthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, and identification of a Mid2p-interacting protein, Zeo1p, that modulates the PKC1-MPK1 cell integrity pathway. Microbiology 149, 2487–2499. Hu, X., Xiao, G.H., Zheng, P., Shang, Y.F., Su, Y., Zhang, X.Y., Liu, X., Zhan, S., St Leger, R.J., Wang, C.S., 2014. Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation. Proc. Natl. Acad. Sci. USA 111, 16796–16801. Jackson, M.A., Jaronski, S.T., 2009. Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects. Mycol. Res. 113, 842–850. Jendretzki, A., Wittland, J., Wilk, S., Straede, A., Heinisch, J.J., 2011. How do I begin? Sensing extracellular stress to maintain yeast cell wall integrity. Eur. J. Cell Biol. 90, 740–744. Jin, C.Y., Parshin, A.V., Daly, I., Strich, R., Cooper, K.F., 2013. The cell wall sensors Mtl1, Wsc1, and Mid2 are required for stress-induced nuclear to cytoplasmic translocation of cyclin c and programmed cell death in yeast. Oxid. Med. Cell. Longev. 2013, 320823. Kim, S., Lee, S.J., Nai, Y.S., Yu, J.S., Lee, M.R., Yang, Y.T., Kim, J.S., 2016.
Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments This research was supported financially by National Science Foundation of the People's Republic of China (No. 31701127), Science and Technology Project of Sichuan (2019YJ0407) and Luzhou (No. 2018-JYJ-32), and Foundation of Southwest Medical University (No. 2017-ZRZD-016, No. 2017-ZRQN-102). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fgb.2019.103278. References Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795. Bermejo, C., García, R., Straede, A., Rodríguez-Peña, J.M., Nombela, C., Heinisch, J.J., Arroyo, J., 2010. Characterization of sensor-specific stress response by transcriptional
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Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007. Appl. Microbiol. Biotechnol. 100 (20), 8889–8900. Lengeler, K.B., Wasserstorm, L., Walther, A., Wendland, J., 2013. Analysis of the cell wall integrity pathway of Ashbya gossypii. Microbiol. Res. 168, 607–614. Mathumathi, R., Bevin, P., Benjamin, M.B., Beverly, E., David, E.L., 1999. Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae. Mol. Cell. Biol. 19 (6), 3969–3976. Nishida, N., Jing, D., Kuroda, K., Ueda, M., 2014. Activation of signaling pathways related to cell wall integrity and multidrug resistance by organic solvent in Saccharomyces cerevisiae. Curr. Genet. 60 (3), 149–162. Philip, B., Levin, D.E., 2001. Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol. Cell. Biol. 21 (1), 271–280. Pendland, J.C., Boucias, D.G., 1997. In vitro growth of the entomopathogenic hyphomycete Nomuraea rileyi. Mycologia 89, 66–71. Rodicio, R., Heinisch, J.J., 2010. Together we are strong - cell wall integrity sensors in yeasts. Yeast 27, 531–540. Shang, Y.F., Xiao, G.H., Zheng, P., Cen, K., Zhan, S., Wang, C.S., 2016. Divergent and convergent evolution of fungal pathogenicity. Genome Biol. Evol. 8 (5), 1374–1387. Shao, C.W., Yin, Y.P., Qi, Z.R., Li, R., Song, Z.Y., Wang, Z.K., 2015. Agrobacterium tumefaciens mediated transformation of the entomopathogenic fungus Nomuraea rileyi. Fungal Genet. Biol. 83, 19–25. Song, Z.Y., 2018. Fungal microsclerotia development: essential prerequisite, influence factors, and molecular mechanism. Appl. Microbiol. Biotechnol. 102 (23), 9873–9880. Song, Z.Y., Shen, L., Yin, Y.P., Tan, W.Y., Shao, C.W., Xu, J.M., Wang, Z.K., 2015. Role of two Nomuraea rileyi transmembrane sensors Sho1p and Sln1p in adaptation to stress due to changing culture conditions during microsclerotia development. World J. Microbiol. Biotechnol. 31, 477–485. Song, Z.Y., Yang, J., Xin, C.Y., Xing, X.R., Yuan, Q., Yin, Y.P., Wang, Z.K., 2018a. A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation. Microbiol. Biotechnol. 11 (6), 1157–1169. Song, Z.Y., Yin, Y.P., Lin, Y.L., Du, F., Ren, G.W., Wang, Z.K., 2018b. The bZip transcriptional factor activator protein-1 regulates Metarhizium rileyi morphology and mediates microsclerotia formation. Appl. Microbiol. Biotechnol. 102 (10), 4577–4588.
Song, Z.Y., Yin, Y.P., Jiang, S.S., Liu, J.J., Wang, Z.K., 2014. Optimization of culture medium for microsclerotia production by Nomuraea rileyi and analysis of their viability for use as a mycoinsecticide. Biocontrol 59, 597–605. Song, Z.Y., Yin, Y.P., Jiang, S.S., Liu, J.J., Chen, H., Wang, Z.K., 2013. Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi. BMC Genomics 14, 411. Song, Z.Y., Zhong, Q., Yin, Y.P., Shen, L., Li, Y., Wang, Z.K., 2016. The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi. Sci. Rep. 6, 38765. Straede, A., Heinisch, J.J., 2007. Functional analyses of the extra- and intracellular domains of the yeast cell wall integrity sensors Mid2 and Wsc1. FEBS Lett. 581, 4495–4500. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Thatcher, L.F., Gardiner, D.M., Kazan, K., Manners, J.M., 2012. A highly conserved effector in Fusarium oxysporum is required for full virulence on Arabidopis. Mol. Plant Microbe. Interact. 25 (2), 180–190. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Roy, N.V., Paepe, A.D., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. Res. 3, 0034. Wang, G., Liu, Z., Lin, R., Li, E., Mao, Z., Ling, J., Yang, Y., Yin, W.B., Xie, B., 2016. Biosynthesis of antibiotic leucinostatins in bio-control fungus Purpureocillium lilacinum and their inhibition on Phytophthora revealed by genome mining. PLOS Pathog. 12 (7), e1005685. Wang, Z.K., Song, Z.Y., Zhong, Q., Du, F., Yin, Y.P., 2018. Adaption to stress via Pbs2 during Metarhizium rileyi conidial and microsclerotia development. World J. Microbiol. Biotechnol. 34, 107. Wang, Z.K., Yang, J., Xin, C.Y., Xing, X.R., Yin, Y.P., Chen, L., Song, Z.Y., 2019. Regulation of conidiation, dimorphism transition, and microsclerotia formation by MrSwi6 transcription factor in dimorphic fungus Metarhizium rileyi. World J. Microbiol. Biotechnol. 35, 46. Zhao, X.H., Mehrabi, R., Xu, J.R., 2007. Mitogen-activated protein kinase pathways and fungal pathogenesis. Eukaryot. Cell 6 (10), 1701–1714. Zhou, G., Ying, S.H., Hu, Y., Fang, X., Feng, M.G., Wang, J., 2018. Roles of three HSF domain-containing protein in mediating heat-shock protein genes and sustaining asexual cycle, stress tolerance, and virulence in Beauveria bassiana. Front. Microbiol. 9, 1677.
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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi
MrMid2, encoding a cell wall stress sensor protein, is required for conidium production, stress tolerance, microsclerotium formation and virulence in the entomopathogenic fungus Metarhizium rileyi
T
Caiyan Xina, Xiaorui Xinga, Fen Wanga, Jiexing Liub, Zhuonan Ranb, Wenbi Chena, ⁎ Guangxi Wanga, Zhangyong Songa, a b
School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, People’s Republic of China Department of Geriatrics, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Metarhizium rileyi Transmembrane protein Microsclerotium formation Conidiation
Transmembrane proteins as sensors encoded by fungal genes activate specific intracellular signal pathways in response to stress cues to help the fungus survive in a changing environment. To better understand the role of the cell wall integrity (CWI) pathway in the entomopathogenic fungus Metarhizium rileyi, an ortholog encoding the transmembrane protein Mid2, MrMid2, was identified and characterized functionally. Transcriptional analysis indicated that MrMid2 was involved in dimorphic transition, conidiation, and microsclerotium formation. After a targeted deletion of MrMid2, all three traits were impaired. Compared with the wild-type strain, the △MrMid2 mutants were hypersensitive to thermal stress, and cell wall and oxidative stress. Insect bioassays revealed that △MrMid2 mutants had decreased virulence levels following topical (22.5%) and injection bioassays (38.7%). Furthermore, transcription analysis showed that other genes of the CWI pathway, with the exception of another major sensor protein encoding gene, MrWsc1, were down-regulated in △MrMid2 mutants. These results suggest that MrMid2 plays important roles in dimorphic transition, conidiation, the stress response, virulence, and microsclerotium development in M. rileyi.
1. Introduction Metarhizium rileyi is a well-known entomopathogenic fungus with yeast-like hyphal bodies and a filamentous growth phase, and it is used as a biological control agent against lepidopterous pests (Fronza et al., 2017; Song 2018). The melanized microsclerotia (MS) induced in liquid medium exhibit longer effective pest control in the field compared to conidial products (Goble et al., 2017; Jackson and Jaronski 2009; Song et al., 2014). Therefore, the MS product can be used as a mycoinsecticide for insect control. The development of viable MS formulations will promote the mass production and commercialization of M. rileyi for use in pest management (Fronza et al., 2017; Song 2018). To increase M. rileyi MS production, the molecular basis of MS development has already been investigated (Song et al., 2013, 2018a, b; Song 2018; Wang et al., 2018). A number of M. rileyi genes have been shown involved in MS development. Moreover, the changing culture conditions during MS development activate several conserved intracellular signal pathways. Previous investigations had reported that the high osmolarity glycerol (HOG) and cell wall integrity (CWI)
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pathways cooperate to regulate MS development (Song et al., 2016). To sense the environmental stresses, MS development is controlled by interplay between signaling pathways, which occur in concert with complex signaling events (Song et al., 2018a, b; Song 2018). We have investigated the function and adaptation of transmembrane proteins of the HOG pathway and found that these proteins act differently in response to exogenous culture stress (Song et al., 2015). However, little is known about the roles of the transmembrane proteins in the CWI signal pathway during M. rileyi MS development. Mid2 is a transmembrane protein that acts as the main sensor protein in the CWI pathway (Green et al., 2003; Jin et al., 2013; Nishida et al., 2014; Philip and Levin 2001; Straede and Heinisch 2007; Wang et al., 2016). The Mid2 orthologs have been characterized in Saccharomyces cerevisiae (Straede and Heinisch 2007), Beauveria bassiana (Kim et al., 2016), Fusarium oxysporum (Thatcher et al., 2012), and Purpureocillium lilacinum (Wang et al., 2016). This transmembrane protein contains a transmembrane domain, a cytosolic tail and an extracellular serine/threonine-rich region that is highly mannosylated during secretion and which mediates clustering in the sensor in order to sense the
Corresponding author. E-mail address: [email protected] (Z. Song).
https://doi.org/10.1016/j.fgb.2019.103278 Received 30 March 2019; Received in revised form 24 August 2019; Accepted 9 October 2019 Available online 11 October 2019 1087-1845/ © 2019 Elsevier Inc. All rights reserved.
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generation of deletion and complementation mutants were listed in Table S1.
extracellular stress. The cytoplasmic tail interacts with a small Ras-like GTPase (Rho1) and then presumably activates the CWI pathway to transmit extracellular signals to the nucleus to modulate gene expression, although there is more than one transmembrane protein that can sense cell wall stress. Previous investigations have confirmed that different sensors employ different mechanisms to sense cell wall stress (Bermejo et al., 2010; Dichtl et al., 2012). MrMid2 of M. rileyi was shown to be up-regulated during MS formation in our earlier transcriptomic analysis (Song et al., 2013). These results indicated that MrMid2 may have a related function in the regulation of MS development. However, this possible function has not been studied in any detail. In the current study, we employed bioinformatics and an experimental strategy to analyze the function of MrMid2. The MrMid2 gene was deleted by homologous recombination strategy (Song et al., 2016). Our functional studies showed that MrMid2 is needed for dimorphic transition, conidiation, stress tolerance, virulence, and MS formation. Furthermore, investigation of gene expression patterns showed that MrMid2 mediates regulation of target genes involved in MS development in M. rileyi.
2.4. Phenotypic assays The difference in conidial production among the tested strains was assessed by incubation on SMAY plates under continuous light at 25 °C for 12 d. The morphology of colonies was investigated using a digital camera (60-mm Macro lens, Canon Inc., Japan). Conidium yields were determined as described previously (Song et al., 2015). Briefly, the conidium yield of each strain was assessed by taking three 6-mm-diameter samples from different plates, performing conidium counts in a hemocytometer under a microscope to determine the concentration of the suspension, and converting the concentration to number of conidia cm−2. For cell wall and oxidative stress assays, all strains were grown on SMAY medium amended with different stress agents for 12 d according to previously described methods (Song et al., 2018a). The conidium yield of each strain was determined as described above. The conidium yield inhibition rate was calculated relative to untreated controls, where inhibition rate (%) = (number of untreated colonies – number of treated colonies) / the number of untreated colonies × 100. In vitro, M. rileyi in yeast-cell form was cultured for 2–4 d on SMAY to transform it into the hyphal form. To analyze the effect of MrMid2 on this dimorphic transition, tested strains were investigated as previously described (Song et al., 2018a). Briefly, 100 simple yeast cells were pipetted onto SMAY medium and grown at 25 °C. Percentage switching rates at indicated time points were recorded. Median transition time required for 50% transition of yeast cells to hyphae (TT50) was calculated. For the MS yield assay, the tested strains were inoculated into liquid amended medium (AM) (Song et al., 2014) and cultured for 6 d. The morphology of samples was investigated using the digital camera technique described earlier. Biomass and MS yield were determined as described previously (Jackson and Jaronski 2009). For MS yield measurements, the culture broth was diluted in order to count MS under a microscope. For biomass evaluation, the liquid fermentation products were collected by vacuum filtration in a Buchner funnel to remove the culture solution. The dry weight accumulations were determined to a constant weight prior to measurement. Each investigation was repeated three times to generate independent biological replicates. To assess the role of MrMid2 in conidial germination, all strains were investigated according to previously described methods (Song et al., 2016). Briefly, the conidial suspensions were inoculated onto SMAY plates at 25 °C. After inoculation for 14 h, germination was assessed every 2 h, and the number of germination conidia was counted under microscopy (400×). Conidial thermotolerance was quantified as the germination rate after exposure to 45 °C (in a water baths) for 30 or 60 min as described previously (Song et al., 2014). To assess the clonal growth rate, a hyphal mass disk (6-mm diameter) was taken from the 3-d-old SMAY cultures and placed centrally onto a SMAY plates (90-mm diameter) and incubated for 12 d at 25 °C. The vegetative growth diameter was measured by vernier calipers every 2 d. To assess hyphal sensitivity to cell wall perturbation, the hyphal growth rate was measured after treatment with Congo red (1.0 mg mL−1), according to a previously described method (Zhou et al., 2018). Briefly, the hyphal mass disks were placed on a SMAY plates supplemented with Congo red, and the vegetative growth diameter was determined by vernier calipers every 2 d. The growth inhibition rate was calculated relative to untreated controls, where inhibition rate (%) = (diameter of untreated colonies – diameter of treated colonies) / diameter of untreated colonies × 100. Each treatment was replicated independently for three times.
2. Materials and methods 2.1. Fungal strains and culture conditions The wild-type (WT) M. rileyi strain CQNr01, obtained from the Engineering Research Center for Fungal Insecticides, Chongqing, China, was used in this study. The WT and all genetic mutants generated in this study were single-spore purified and were cultured on solid Sabouraud maltose agar medium fortified with 1% (w/v) yeast extract (SMAY). Appropriate antibiotics were added for selection of mutants according to previously described methods (Song et al., 2018a). 2.2. Bioinformatics analysis The MrMid2 sequence from our previous transcriptomics analysis (Song et al., 2013) was used as a query to search through the M. rileyi genome database (NCBI accession No. AZHC00000000.1) (Shang et al., 2016). Multiple sequence alignments were conducted with DNAMAN software (http://www.lynnon.com). The potential signal peptide and transmembrane domains were predicted by the SignalP server (Bendtsen et al., 2004). A phylogenetic tree was constructed using MEGA 6.0 software with the Neighbor-Joining method (Tamura et al., 2013). 2.3. Deletion of MrMid2 and mutant complementation Plasmid pPZP-Hph-Knock (Shao et al., 2015), containing the selectable marker hygromycin phosphotransferase gene (hph), was used as the backbone with which to construct the MrMid2 gene deletion plasmid. The 5′ and 3′ flanking sequences were amplified from the WT genome via PCR and inserted independently into pPZP-Hph-Knock. For complementation of the deletion mutant colonies, plasmid pPZP-SurKnock containing the sulfonylurea resistance gene (sur) was used as the complementation vector. A fragment containing the putative promoter, coding sequence, and downstream sequence of MrMid2 was amplified and inserted into pPZP-Sur-Knock. Deletion and complementation vectors were transformed independently into Agrobacterium tumefaciens AGL-1 (Invitrogen, Shanghai, China). Putative MrMid2 deletion mutants were able to grow without check on SMAY plates containing hygromycin B and cephalosporin. After subculture of resistant colonies for five generations, PCR screening was performed with primers homologous to the hph and genomic sequence outside the flanking regions (MrMid2F/hph-R and MrMid2F/hph-F) (Table S1) and the amplicons were sequenced. The positive gene deletions were then confirmed by PCR with primers MrMid2OF/MrMid2OR. All primers used for the
2.5. Virulence assays Virulence assays were performed on Spodoptera litura larvae using immersion into or injection with conidial suspension methods, 2
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Fig. 1. Biological roles of MrMid2 in the asexual cycle. (A) Relative expression levels of MrMid2 during conidiation at different time points. Stages of conidial development included conidia inoculated onto the plate at the start of incubation (day 0), blastospores (day 2), hyphal period (day 4), conidiation initiation (day 6), and the start of conidium maturation (day 8). (B) Colony morphology of tested strains inoculated on SMAY plates and cultured under continuous light at 25 °C for 3, 6, 9, and 12 d. (C) TT50 of WT, complementation strains, and △MrMid2 mutants were compared. (D) Statistical analysis of conidial yields. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences from the WT or from the results at 0 days of incubation.
mortality rate was recorded daily to determine the mean lethal time (LT50) values.
according to previously described methods (Song et al., 2016). Briefly, cultures of the tested strains were incubated on SMAY plates for 12 d at 25 °C. Conidia were harvested and resuspended to 5 μL suspension of 1 × 106 conidia mL−1 in cottonseed oil or 5 μL of 1 × 106 conidia mL−1 in sterile water containing 0.01% Tween 80. Sham controls were treated with pure cotton seed oil or sterile water containing 0.01% Tween 80 only, without conidia. The assay was repeated three times and 30 larvae were tested in each replicate. After treatment, larvae were reared as described previously (Song et al., 2015). The larval
2.6. Quantitative real-time PCR (qPCR) For time-specific expression patterns during conidiation, 3 μL suspension aliquots of 1 × 107 WT conidia mL−1 were pipetted onto SMAY plates, followed by incubation under continuous light at 25 °C for 8 d. Total RNA was isolated during the following stages: conidia at the start 3
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Fig. 2. Deletion of MrMid2 resulted in an altered germination rate, clonal growth rate, and response to stress. (A) Germination of conidia from tested strains and (B) conidial tolerance to heat stress at 45 ℃. A conidium was considered to be germinated if the germ-tube length was at least half that of the conidium. (C) Statistical analysis of fungal growth inhibition rates. (D) Statistical analysis of conidial yield inhibition rates under cell wall and oxidative stress. Values were the mean of three independent experiments. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences when compared with the results observed in the WT.
sampled at different time points. The gene expression profile was assessed in samples of WT, △MrMid2, or complementation hyphae cultured in AM for 60 h. Total RNA was collected according to previous methods (Song et al., 2015). qPCR was performed using SYBR® Premix Ex TaqTM II (TaKaRa, Dalian, China), following the manufacturer’s instructions. β-tubulin (Mrtub) and translation elongation factor (Mrtef) genes were used as internal standards. The 2−ΔΔCt method was used to evaluate relative transcript levels (Vandesompele et al., 2002).
of incubation (day 0), blastospores (day 2), hyphal period (day 4), conidiation initiation (day 6), and the start of conidium maturation (day 8). To assess the possibility of whether changing culture conditions affected the expression of MrMid2, a set of experiments was set up in which the incubated liquid AM or minimal medium (MM, i.e. AM without basal salts) was supplemented with either exogenous iron (1 mM) or H2O2 (3 mM) as described previously (Song et al., 2015). For analyses of gene expression of MrMid2 at different stages of MS development in the WT, samples were prepared in AM culture and sub4
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of △MrMid2 mutants than in the WT and complementation strains during a 2-d incubation period after treatment at 45 ℃ (Fig. 2B). The △MrMid2 mutants exhibited a significant decrease in germination rate over the 24-h incubation period when exposed to thermal stress for 30 min (65.3% decrease) or 60 min (81.9% decrease), compared with decreases in germination rate in the WT over the 24-h incubation period of 51.3% (30 min) or 71.8% (60 min) and those of the complementation strain of 50.9% (30 min) or 65.9% (60 min) (P < 0.001). This thermotolerance study showed that MrMid2 was needed in response to thermal stress. Compared to the WT and complementation strains, the cross-section of mycelial growth of the △MrMid2 mutants was reduced after a 4-day incubation (data not shown) in the absence of stress. Furthermore, the △MrMid2 mutants also displayed defects in vegetative growth, with colony sizes being 15% smaller compared with the WT and complementation strains, after a 12-d incubation on the SMAY medium (Fig. S3). Furthermore, △MrMid2 mutants showed increased sensitivity to cell wall stress. When exposed to cell wall stress, the growth inhibition rate of △MrMid2 mutants was increased by 18.5%, compared with that of the WT and complementation strains (Fig. 2C). Compared to the WT and complementation strains, the conidial yields of the △MrMid2 mutants cultured under stressful conditions were severely affected. Increases in inhibition rates of the △MrMid2 mutants in comparison to WT or complementation is of 18.2 – 71.2% under different stressful conditions (Fig. 2D).
2.7. Statistical analysis Statistical analyses were performed using SPSS 16.0 software (IBM, Armonk, NY, USA). Graphs were prepared using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) was performed, followed by Duncan’s multiple range tests to determine significant differences between samples, with P = 0.05 being the threshold significance level. 3. Results 3.1. Generation of MrMid2 mutants Orthologs of MrMid2 were obtained (NCBI accession No. MK408430), which encodes a protein of 278 amino acid residues and contains a conserved Pfam domain (residues 57–99) (Fig. S1A). In addition, the amino acid sequence of MrMid2 was similar to that of Mid2 protein (70% identity) in Metarhizium brunneum and Mid2 protein (67% identity) in Metarhizium guizhouense (Hu et al., 2014). Phylogenetic analysis from other fungi demonstrated that MrMid2 is relatively conserved among fungal species (Fig. S1B). To examine the biological function of MrMid2 in M. rileyi, MrMid2 deletions were generated by homologous recombination and complementation strains were constructed by heterologous insertion. The positive gene knockout and complementation strains were screened and confirmed by PCR and qPCR analysis (Fig. S2). Taken together, the results achieved deletion of MrMid2 (strain △MrMid2) and mutant complementation with MrMid2 to produce strain △MrMid2 + mid2.
3.4. Deletion of MrMid2 delays MS formation Expression levels of MrMid2 during MS development were analyzed by qPCR. Compared with the vegetative growth stage (36 h), the expression of MrMid2 was up-regulated when MS formation took place (60 – 96 h) (Fig. 3A). Our preliminary experiments had revealed that iron and oxidative stress are critical factors that induce MS differentiation (Song 2018). To assess the effect of exogenous iron and oxidative stress on MrMid2 expression levels, AM was supplemented with exogenous 1 M iron or 3 mM H2O2 as previously described (Song et al., 2018a). The results showed that MrMid2 was up-regulated when exposed to exogenous iron or H2O2 for MS induction (Fig. 3B-3D), indicating that MrMid2 might be involved in the regulation of MS formation. After incubation in liquid AM culture, MS produced by WT and complementation strains matured and were accompanied by secondary mycelial growth, whereas the MS yield in △MrMid2 mutants was significantly lower (Fig. 4A). Compared with the WT strain, the MS yield of △MrMid2 mutants was reduced by approximately 97% (Fig. 4B). With few mycelia in the △MrMid2 mutants, the biomass of the △MrMid2 mutants was decreased by 39% (Fig. 4C). These results indicated that MrMid2 contributed to MS development. To investigate the genes regulated by MrMid2 during MS formation, candidate genes were selected from transcriptome libraries of MS formation (Song et al., 2013, 2018b) and examined by transcriptional analysis. The following genes were selected: several chitin synthase genes (Mrch1, Mrch2, and Mrch4, for class I, II, and IV chitin synthases, respectively), a cell wall biosynthesis-related gene (Mrfks, for β-1,3glucanase synthetase), components of the CWI pathway (namely, the Mrrho1, Mrpks1, Mrbck1, Mrmkk1,2, Mrslt2, and Mrswi6 genes) and another transmembrane protein of the CWI pathway (the Mrwsc1 gene). It was found that the downstream component of the CWI pathway was down-regulated, whereas the Mrwsc1 gene was up-regulated in the △MrMid2 mutants during MS formation (Fig. 5). Compared with the WT, Mrfks was significantly up-regulated and Mrch2 and Mrch4 were significantly down-regulated during MS development in the △MrMid2 mutants (Fig. 5).
3.2. Loss of MrMid2 results in delayed yeast-to hyphae transition and decreased conidial yield Compared with the initial results at day 0, expression of MrMid2 was found to be up-regulated during blastospore formation (day 2) and down-regulated during hyphal vegetative growth (day 4) (Fig. 1A). This result indicated that MrMMid2 may function in the yeast-to-hyphae transition. Intriguingly, compared to WT, at day 3, the △MrMid2 mutants displayed a delay in the yeast-to-hyphae transition (Fig. 1B), and further dimorphic transition analyses showed that a significant difference is between △MrMid2 mutants (TT50 = 8.5 ± 0.4 d) in comparison to WT (TT50 = 7.4 ± 0.1 d) (P < 0.05) (Fig. 1C). These results suggested that MrMid2 was involved in the yeast-to-hyphae transition. Further expression analyses showed that MrMid2 was up-regulated during the conidiation initiation (day 6) and at the start of conidium maturation (day 8) (Fig. 1A). This result indicated that MrMMid2 may also function in the conidiation process. Further investigations showed that the △MrMid2 mutants displayed decreased conidial yield compared to the WT and complementation strains (P < 0.001) (Fig. 1B and 1D). Taken together, these gene expression and phenotypic analyses suggested that MrMid2 was involved in conidium production. 3.3. MrMid2 is essential to the stress response Compared to the WT, the △MrMid2 mutants displayed defects in conidial germination, thermal stress, and cell wall and oxidative stress (Fig. 2). The rate of conidial germination was tested on SMAY and the results are shown in Fig. 2A. The germination rate of conidia of △MrMid2 mutants was reduced compared with that of the WT and complementation strains. After 14 h, the germination rate (32.2 ± 3.6%) for conidia of the △MrMid2 mutants was significantly lower than that of the WT (63.3 ± 4.5%) and complementation strains (60.2 ± 2.5%) (P < 0.001). After 24 h, the germination rate (82.2 ± 1.8%) for conidia of the △MrMid2 mutants was also significantly lower than that of the WT (97.4 ± 1.9%) and complementation strains (95.6 ± 2.4%) (P < 0.001). Overall, there was a significantly slower germination rate of conidia 5
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Fig. 3. qPCR analysis of gene expression levels. Relative expression level of MrMid2 during (A) MS development or following treatment with (B) H2O2 or (C) iron cation in MM or treatment with (D) iron cation in AM. Stages of MS development included blastospore period (36 h of incubation), hyphal elongation and MS initiation period (60 h), MS formation (72 h), mass MS formation (96 h), and MS maturation (120 h). Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared to the results at 36 h or 0 min.
shift (Boucias et al., 2016; Pendland and Boucias 1997; Song et al., 2016, 2018a, b). MrMid2 belongs to a group of transmembrane proteins, which senses cell wall stress and activates the CWI pathway. Interestingly, in this study, △MrMid2 mutants resulted in a delay in the yeast-to-hyphae transition (Fig. 1). This result was similar to that exhibited by the △MrSlt2 and △Mrswi6 mutants (Song et al., 2016; Wang et al., 2019). Like S. cerevisiae (Mathumathi et al., 1999), the vegetative growth defects of the △MrMid2 mutants were exhibited on SMAY medium. Such yeast-to-hyphae and vegetative growth defects of the △MrMid2 mutants may be attributable to decreased conidium production. Previously, it was reported that deletion of Mid2 from S. cerevisiae resulted in defects in terms of the thermal and stress response (Jin et al., 2013; Philip and Levin 2001; Straede and Heinisch 2007). The role of MrMid2 in M. rileyi was similar to that in the yeast (Fig. 2), although its role in the thermal stress response was unlike that in △AgMid2 in Ashbya gossypii, the growth of which (relative to the WT) showed no significant difference at different temperatures (Lengeler et al., 2013). The exact molecular mechanism by which the plasma membrane sensors detect the extracellular stress remains elusive. There are two small families of sensors of the CWI pathway in the yeast and fungi, namely the cell wall stress component-type sensors Wsc1, Wsc2 and Wsc3 on the one hand, and Mid2 and Mtl1 on the other (Jendretzki et al., 2011; Rodicio and Heinisch 2010). Genetic analyses indicated
3.5. MrMid2 is required for virulence Bioassays showed that virulence of △MrMid2 mutants were significantly lower than that exhibited by the WT and complementation strains (Fig. 6). The LT50 values for the WT strain were 6.2 ± 0.3 d in the topical bioassay and 4.3 ± 0.4 d in the injection bioassay. The LT50 values of the complementation strain were 6.3 ± 0.4 d in the topical bioassay and 4.9 ± 0.3 d in the injection bioassay. Compared with these data, the LT50 of △MrMid2 mutants was delayed by 1.4 days in the topical bioassay and 1.9 days in the injection bioassay. These results showed that MrMid2 was required for virulence. 4. Discussion In the present study, the function of MrMid2 was characterized in M. rileyi. Our results showed that deletion of MrMid2 resulted in delayed morphological shift, conidial production and MS formation, and hypersensitivity to thermal stress, and cell wall and oxidative stress. In addition, the △MrMid2 mutants also exhibited reduced virulence. Morphological shift is essential for the life cycle and virulence of dimorphic fungi (Gauthier 2015). The M. rileyi exhibits a yeast-to-hyphae transition lifestyle and recent investigations confirmed that multiple signaling pathways are related to the regulation of morphological 6
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that Mid2 and Wsc1 act as the main sensor proteins in the CWI pathway in yeast and filamentous fungi (Futagami et al., 2011; Green et al., 2003; Jin et al., 2013; Nishida et al., 2014; Philip and Levin 2001; Straede and Heinisch 2007). Two transmembrane sensors of the HOG pathway of M. rileyi were investigated and important roles in sensing external culture stress during MS development were identified (Song et al., 2015). The external culture stress also gave rise to up-regulated expression of MrWsc1 and MrMid2 during MS development (Song et al., 2013). However, targeted gene knockout of MrWsc1 has not been successful despite many attempts at transformation. In the current study, the roles of MrMid2 were determined and MrMid2 was found to be critical for MS formation in M. rileyi. As with the defective MS formation in the △MrSlt2 and △Mrswi6 mutants, △MrMid2 mutants produced only a few MS (Fig. 4) (Song et al., 2016; Wang et al., 2019). Preliminary experiments had revealed that the fungus is exposed to various stresses, including pH and iron cation concentration, in the culture medium during MS formation (Song et al., 2015). To sense the extracellular stress, the extracellular domain of transmembrane proteins mediate sensor clustering (Francois et al., 2013). Moreover, the fungi must remodel the cell wall structure during vegetative growth and morphogenesis. Cell wall remodeling and biosynthesis is regulated by the CWI signaling pathway. As with other fungi, MrMid2 deletion led to down-regulation of the expression of the downstream components of the CWI pathway (Dichtl et al., 2012; Green et al., 2003; Jin et al., 2013). Interestingly, as in other fungi (Dichtl et al., 2012; Philip and Levin 2001), the expression of MrWsc1 in the MrMid2 mutants was upregulated, indicating that the functions of the two genes for MS development are partially redundant (Fig. 4). Additionally, the chitin synthase and 1,3-glucanase synthetase genes exhibited differential expression in response to extracellular stress during MS development. To model the cell wall structure during MS development, there must be the other direct downstream interaction partners and effectors of MrWsc1 in the △MrMid2 mutants. Further experiments are needed to identify and elucidate the roles of these genes. The HOG and CWI pathways are known to contribute to the regulation of pathogenesis in yeast and filamentous fungi (Zhao et al., 2007). In the present study, △MrMid2 mutants were found to be significantly less pathogenic following either topical or injection assays (Fig. 6). Similar results have also been observed in the △MrSlt2 and △Mrswi6 mutants (Song et al., 2016; Wang et al., 2019) and in the △BbMid2 mutants of B. bassiana (Kim et al., 2016) and △Mid2 mutants of F. oxysporum (Thatcher et al., 2012). The △MrMid2 mutants exhibited hypersensitivity to various stresses and defective vegetative growth in response to various stresses. As a result, △MrMid2 exhibited decreased
Fig. 4. Microsclerotium development in AM. (A) Morphology of microsclerotia in AM after 144 h of incubation. MS yield (B) and biomass (C) of the tested strains. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared with WT.
Fig. 5. qPCR analysis of gene expression in △MrMid2 mutants after 60 h incubation in AM medium. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared with WT. 7
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Fig. 6. Insect bioassays. Insect survival after (A) topical application or (C) injection of conidia from specific strains. Mean lethal time in (B) topical infection or (D) injection application assays. Error bars represent standard error. * P < 0.05, ** P < 0.01, denote significant differences compared with WT.
virulence. In summary, a gene homologous to Mid2 was functionally characterized in M. rileyi in this study. Phenotypic analyses revealed that MrMid2 has important functions in dimorphic transition, stress response, and conidium production, and is required for virulence and MS formation. Taken together, the data presented in the current study could facilitate future evaluation of cell wall integrity and the development of conidia and MS in M. rileyi.
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Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments This research was supported financially by National Science Foundation of the People's Republic of China (No. 31701127), Science and Technology Project of Sichuan (2019YJ0407) and Luzhou (No. 2018-JYJ-32), and Foundation of Southwest Medical University (No. 2017-ZRZD-016, No. 2017-ZRQN-102). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fgb.2019.103278. References Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795. Bermejo, C., García, R., Straede, A., Rodríguez-Peña, J.M., Nombela, C., Heinisch, J.J., Arroyo, J., 2010. Characterization of sensor-specific stress response by transcriptional
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