MaCts1, an Endochitinase, Is Involved in Conidial Germination, Conidial Yield, Stress Tolerances and Microcycle Conidiation in Metarhizium acridum

Simple Summary Entomopathogenic fungi are promising biocontrol agents. Microcycle conidiation has shown great potential in enhancing the conidial yield and quality of entomopathogenic fungi. Elucidating the regulatory mechanisms underlying the induction of microcycle conidiation will be helpful for genetic improvement of entomopathogenic fungi. This work focused on the connection of an endochitinase, MaCts1, to the biocontrol potential of the entomopathogenic fungus Metarhizium acridum. MaCts1, an endochitinase gene of M. acridum, was shown to be involved in conidial germination, conidial yield and fungal resistance to UV-B irradiation and heat-shock. Interestingly, disruption of MaCts1 led to maintenance of typical conidiation on the microcycle conidiation induction medium, SYA, which may not be dependent on the MOR/RAM pathway. This work provided insight into the regulatory mechanisms governing the shift to microcycle conidiation in M. acridum. Abstract Entomopathogenic fungi are promising biocontrol agents of insect-mediated crop damage. Microcycle conidiation has shown great potential in enhancing the conidial yield and quality of entomopathogenic fungi. Homologs of Cts1, an endochitinase of Saccharomyces cerevisiae, participate in cell separation in several fungal spp. and may contribute to the morphological differences that occur during the shift to microcycle conidiation. However, the precise functions of Cts1 in entomopathogenic fungi remain unclear. Herein, the endochitinase gene, MaCts1, was characterized in the model entomopathogen, Metarhizium acridum. A loss of function line for MaCts1 led to a delay of 1 h in the median germination time, a 28% reduction in conidial yield and significant defects in fungal resistances to UV-irradiation (18%) and heat-shock (15%), while fungal tolerances to cell wall stressors, oxidative and hyperosmotic stresses and virulence remained unchanged. The MaCts1-disruption strain displayed typical conidiation on the microcycle conidiation induction medium, SYA. In contrast, deletion of key genes in the morphogenesis-related NDR kinase network (MOR pathway)/regulation of Ace2 and morphogenesis (RAM pathway) did not affect the SYA-induction of microcycle conidiation. This indicates that MaCts1 makes contributions to the microcycle conidiation, which may not be dependent on the MOR/RAM pathway in M. acridum.


Introduction
Insect pathogenic fungi are considered as promising biocontrol agents due to their lack of toxicity to humans and animals, and their low likelihood to cause host resistance [1,2]. Infection of the insect host involves conidial adherence to the insect epicuticle, followed by conidial germination, and the development of an infection structure (appressorium),
Czapek-Dox medium containing 500 µg/mL glufosinate ammonium (Sigma, St. Louis, MO, USA) was used to select the putative disruption mutants for validation by PCR or Southern blotting. To rescue the MaCts1 in the MaCts1-disruption mutant (∆MaCts1), the promoter and coding regions of the MaCts1 were cloned into the pK2-Sur vector [33] to form the MaCts1 rescuing vector, pK2-Sur-MaCts1, which was inserted into ∆MaCts1 via Agrobacterium-mediated method [34]. The complemented transformants (CP) of MaCts1 were screened on Czapek-Dox medium containing 20 µg/mL chlorimuron ethyl (Sigma, Bellefonte, PA, USA). To confirm the MaCts1 deletion and complementation strains, Southern blotting was carried out as previously described [35]. Genomic DNA (~5 µg/sample) was digested with StuI/EcoRI. All primers used are listed in Table S1.

Fungal Pathogenicity Assays
Conidial suspensions in paraffin oil (5 µL of 1 × 10 7 conidia/mL) of the WT, ∆MaCts1, CP strains were topically inoculated onto fifth-instar stage nymphs of Locusta migratoria manilensis (Meyen) [33]. The surviving number of insects was recorded every 12 h to calculate the half lethality time (LT 50 ) using the Data Processing System program [38], which was used as a measure of the virulence of the fungal strains.

Data Analysis
In this study, the data from each time point (hours or days) were considered as a single replicate. At first, the normality and uniformity of the original data were analyzed using the Shapiro-Wilk test. After confirmation, the means were compared by Tukey's test. Non-normally distributed, data were further analyzed by a Mann-Whitney U test [38,39]. All the datasets in this study were normally distributed, and one-way analysis of variance (ANOVA) was applied to access the phenotypic estimate, including conidial germination rates with different treatments (UV-B, Heat-shock), conidial yield, locust survival and cell length in the conidiation process of WT, ∆MaCts1, CP. Datasets were analyzed with the SPSS 18.0 program (SPSS Inc, Chicago, IL, USA). All experiments were triplicated.

Features of MaCts1
The Cts1 homolog, MaCts1 of M. acridum, was retrieved from the CQMa102 genome. The MaCts1 has a 1401 bp open reading frame, which encodes a predicted 392 amino acids protein (41.27 kDa). Domain prediction using the web resource SMART (http://smart. embl.de/ (accessed on 14 April 2022) showed its content of a glycosyl hydrolase family 18 domain (Glyco_18) and a C-terminal cellulose binding domain (CBM_1) existed in the deduced MaCts1 ( Figure 1A). A phylogenetic analysis of MaCts1 and other fungal Cts1 homologs showed that it was more closely related to M. robertsii, M. rileyi and B. bassiana, with sequence identities of 52.05%, 45.98% and 37.24%, respectively ( Figure 1B).

MaCts1 Is Involved in Conidial Germination and Influences Conidial Yield but Not Virulence
Southern blotting showed that MaCts1 were successfully disrupted and rescued ( Figure S1). Assessment of the conidial germination rates of the WT, ∆MaCts1 and CP strains indicated that ∆MaCts1 exhibited a significant delay relative to the control strains (WT and CP). The calculations of GT 50 showed that ∆MaCts1 (6.43 ± 0.31 h) was significantly longer than the WT (5.10 ± 0.24 h) and CP (5.34 ± 0.21 h) strains (p < 0.05; Figure 2A,B). The yield of conidia from ∆MaCts1 grown on 1/4 SDAY media for 15 days at 28 • C was remarkably decreased by 28% ( Figure 2C). Microscopic observation indicated that the control strains had produced conidiophores at 24 h of incubation, whereas conidiophores emerged in ∆MaCts1 only after 36 h ( Figure 2D). Insect bioassays via topical inoculation showed that MaCts1 made no significant contribution to fungal virulence ( Figure 2E,F).

MaCts1 Is Involved in Conidial Germination and Influences Conidial Yield but Not Virulence
Southern blotting showed that MaCts1 were successfully disrupted and rescued (Figure S1). Assessment of the conidial germination rates of the WT, ΔMaCts1 and CP strains indicated that ΔMaCts1 exhibited a significant delay relative to the control strains (WT and CP). The calculations of GT50 showed that ΔMaCts1 (6.43 ± 0.31 h) was significantly longer than the WT (5.10 ± 0.24 h) and CP (5.34 ± 0.21 h) strains (p < 0.05; Figure 2A,B). The yield of conidia from ΔMaCts1 grown on 1/4 SDAY media for 15 days at 28 °C was remarkably decreased by 28% ( Figure 2C). Microscopic observation indicated that the control strains had produced conidiophores at 24 h of incubation, whereas conidiophores emerged in ΔMaCts1 only after 36 h ( Figure 2D). Insect bioassays via topical inoculation showed that MaCts1 made no significant contribution to fungal virulence ( Figure 2E,F).

Disruption of MaCts1 Impairs Fungal Resistances to UV-B Irradiation and Heat-Shock
In general, the conidial germination rates decreased with longer heat-shock or UV-B irradiation treatments in all strains ( Figure 3A,C). However, the ∆MaCts1 strain displayed significantly higher sensitivity to UV-B irradiation and heat-shock than the control strains. After 3-h UV-B irradiation,~50% of conidia from ∆MaCts1 germinated, compared to~75% of conidia from the control strains (p < 0.01; Figure 3A). The half germination time of inhibition (IT 50 ) of ∆MaCts1 was significantly decreased by 18% (p < 0.01; Figure 3B). After 6 h of heat-shock, only~36.0% conidia of ∆MaCts1 germinated, compared to more than 50% conidia from the control strains (p < 0.01; Figure 3C), with a significant reduction in the IT 50 of ∆MaCts1 of 15% (p < 0.01; Figure 3D). In addition, fungal sensitivities to oxygen stress (menadione), cell wall stress (CR, CFW, SDS) and osmotic stress (SOR, NaCl) were assessed and measured by morphology and diameters of fungal colonies on 1/4 SDAY medium. The results indicated that the colony morphology of ∆MaCts1 showed no obvious differences to that of the control strains ( Figure 3E). Biology 2022, 11, x FOR PEER REVIEW 6 of 13 Error bars indicate the standard deviation from triplicate experiments. One, two and three asterisks indicate a significant difference at p < 0.05, p < 0.01, p < 0.001, respectively. "ns" indicates no significant difference.

Disruption of MaCts1 Impairs Fungal Resistances to UV-B Irradiation and Heat-Shock
In general, the conidial germination rates decreased with longer heat-shock or UV-B irradiation treatments in all strains ( Figure 3A,C). However, the ΔMaCts1 strain displayed significantly higher sensitivity to UV-B irradiation and heat-shock than the control strains. After 3-h UV-B irradiation, ~50% of conidia from ΔMaCts1 germinated, compared to ~75% of conidia from the control strains (p < 0.01; Figure 3A). The half germination time of inhibition (IT50) of ΔMaCts1 was significantly decreased by 18% (p < 0.01; Figure 3B). After 6 h of heat-shock, only ~36.0% conidia of ΔMaCts1 germinated, compared to more than 50% conidia from the control strains (p < 0.01; Figure 3C), with a significant reduction in the IT50 of ΔMaCts1 of 15% (p < 0.01; Figure 3D). In addition, fungal sensitivities to oxygen Error bars indicate the standard deviation from triplicate experiments. One, two and three asterisks indicate a significant difference at p < 0.05, p < 0.01, p < 0.001, respectively. "ns" indicates no significant difference.
Biology 2022, 11, x FOR PEER REVIEW 7 of 13 stress (menadione), cell wall stress (CR, CFW, SDS) and osmotic stress (SOR, NaCl) were assessed and measured by morphology and diameters of fungal colonies on 1/4 SDAY medium. The results indicated that the colony morphology of ΔMaCts1 showed no obvious differences to that of the control strains ( Figure 3E). Error bars indicate the standard deviation from triplicate experiments. One and two asterisks indicate a significant difference at p < 0.05 and p < 0.01, respectively. "ns" indicates no significant difference. Error bars indicate the standard deviation from triplicate experiments. One and two asterisks indicate a significant difference at p < 0.05 and p < 0.01, respectively. "ns" indicates no significant difference.

MaCts1 Makes Contributions to the Microcycle Conidiation
To understand the roles of MaCts1 during the microcycle conidiation of M. acridum, the conidiation process of the WT, ∆MaCts1 and CP strains grown on SYA medium was microscopically observed. The result was that microcycle conidiation was induced in the control strains, whereas ∆MaCts1 exhibited the pattern of typical conidiation ( Figure 4A). Disruption of MaCts1 also occurred with a significant increase in the apical and sub-apical cell lengths on SYA medium (p < 0.001; Figure 4B-D).
on the full protein sequences of MOR/RAM components from S. cerevisiae, S. pombe [40] and U. maydis [21] to identify the putatively conserved MOR/RAM components from the M. acridum genome (Table S2). The key components of the MOR/RAM, including two essential serine/threonine protein kinase genes, Cbk1 and Kic1, three associated protein genes, Hym1, Mob2 and Tao3, and an additional transcription factor gene, Ace2, were identified and disrupted in M. acridum. All the disrupted mutants were successfully confirmed by PCR ( Figure S2) and, in all, microcycle conidiation was successfully induced on SYA medium, while only ΔMaCts1 exhibited the typical conidiation pattern ( Figure 5). These results indicate that MaCts1 contributes to the microcycle conidiation, but that the regulation of MaCts1 may be independent on the MOR/RAM pathway.  The length of sub-apical hyphal cells in the WT, ∆MaCts1 and CP strains. Error bars indicate standard deviations from triplicate experiments. Three asterisks indicate significant differences at p < 0.001, one asterisks indicate significant differences at p < 0.05 and "ns" indicates no difference.
The MOR/RAM pathway plays an important regulatory role in fungal morphogenesis [26,28], and has been reported to regulate the expression of Cts1 during cell separation in several fungi [22,23]. Thus, we conducted BLASTp sequence similarity searches based on the full protein sequences of MOR/RAM components from S. cerevisiae, S. pombe [40] and U. maydis [21] to identify the putatively conserved MOR/RAM components from the M. acridum genome (Table S2). The key components of the MOR/RAM, including two essential serine/threonine protein kinase genes, Cbk1 and Kic1, three associated protein genes, Hym1, Mob2 and Tao3, and an additional transcription factor gene, Ace2, were identified and disrupted in M. acridum. All the disrupted mutants were successfully confirmed by PCR ( Figure S2) and, in all, microcycle conidiation was successfully induced on SYA medium, while only ∆MaCts1 exhibited the typical conidiation pattern ( Figure 5). These results indicate that MaCts1 contributes to the microcycle conidiation, but that the regulation of MaCts1 may be independent on the MOR/RAM pathway.

Discussion
Chitin is the main polysaccharide component of fungal cell walls and is critical for fungal cell morphogenesis [16,41]. Chitin is degraded by endo-and exochitinases to provide nutrition to fungal cells and determine fungal morphology [40][41][42]. In this study, MaCtst1, a gene encoding the M. acridum homolog of the Cts1 endochitinase, was characterized. Deletion of MaCts1 led to severe defects in conidial germination and conidial yields, with decreased resistances to UV-B irradiation and heat-shock. Interestingly, we found MaCts1 contributes to the microcycle conidiation of M. acridum, as shown by the inability of the MaCts1 strain to produce microcycle conidia on SYA media and the restoration of this ability in CP strains. In S. cerevisiae and other fungi, the Cts1 gene is regulated by the MOR/RAM pathway during cell separation [22,23]. However, our results indicate that the disruption of these pathways in M. acridum had no effect on the induction of microcycle conidiation, suggesting that MaCts1 may not be regulated by the MOR/RAM pathway during microcycle conidiation in M. acridum.
The efficiency of conidial germination is key for the use of entomopathogenic fungi as biological control agents [43]. In this study, the deletion of MaCts1 resulted in delayed conidial germination. Similar phenomena were observed after the disruption of Cts1 homologs in Aspergillus fumigatus [44] and C. albicans [25], while Cts1 deletions had no effect on conidial germination in Trichoderma harzianum [45], T. roseum [46], Talaromyces flavus [47] and Fusarium chlamydosporum [48], indicating that Cts1 plays distinct roles in conidial germination in different fungal species.
Conidial resistances to high temperature and UV irradiation directly determine their efficiency as mycoinsecticides in the field [49]. The fungal cell wall plays an important protective role against external stress [17,19] and chitin is one of the major cell wall

Discussion
Chitin is the main polysaccharide component of fungal cell walls and is critical for fungal cell morphogenesis [16,41]. Chitin is degraded by endo-and exochitinases to provide nutrition to fungal cells and determine fungal morphology [40][41][42]. In this study, MaCtst1, a gene encoding the M. acridum homolog of the Cts1 endochitinase, was characterized. Deletion of MaCts1 led to severe defects in conidial germination and conidial yields, with decreased resistances to UV-B irradiation and heat-shock. Interestingly, we found MaCts1 contributes to the microcycle conidiation of M. acridum, as shown by the inability of the ∆MaCts1 strain to produce microcycle conidia on SYA media and the restoration of this ability in CP strains. In S. cerevisiae and other fungi, the Cts1 gene is regulated by the MOR/RAM pathway during cell separation [22,23]. However, our results indicate that the disruption of these pathways in M. acridum had no effect on the induction of microcycle conidiation, suggesting that MaCts1 may not be regulated by the MOR/RAM pathway during microcycle conidiation in M. acridum.
The efficiency of conidial germination is key for the use of entomopathogenic fungi as biological control agents [43]. In this study, the deletion of MaCts1 resulted in delayed conidial germination. Similar phenomena were observed after the disruption of Cts1 homologs in Aspergillus fumigatus [44] and C. albicans [25], while Cts1 deletions had no effect on conidial germination in Trichoderma harzianum [45], T. roseum [46], Talaromyces flavus [47] and Fusarium chlamydosporum [48], indicating that Cts1 plays distinct roles in conidial germination in different fungal species.
Conidial resistances to high temperature and UV irradiation directly determine their efficiency as mycoinsecticides in the field [49]. The fungal cell wall plays an important protective role against external stress [17,19] and chitin is one of the major cell wall structural components imparting matrix rigidity and wall maintenance [19]. Chitin synthases are essential for cell wall synthesis, and the deletion of their genes can lead to reduced cell wall integrity and tolerances to stress [17,19]. Chitinases are involved in fungal cell wall decomposition for nutrition, but can also be targeted during development, such as in mother-daughter cell separation during conidial cytokinesis [40]. In this work, deletion of MaCts1 reduced the fungal resistances to UV-B irradiation, as well as heat-shock, which may be due to the alterations of cell wall structure and composition in ∆MaCts1.
In M. acridum, the microcycle conidiation produces higher yields of conidia with higher quality than those from typical conidiation [13]. In microcycle conidiation, the new conidia are directly separated from the germinated conidia, while in typical conidiation, multicellular mycelia form and extend from the germinated conidia [8,9]. The shift to microcycle conidiation is ultimately regulated by the availability of nutrients and other stress conditions [9]. However, the differential regulation of cell separation during microcycle vs. typical conidiation is an early and essential step in this process. Cts1 has been shown to affect the separation between mother cells and daughter cells in S. cerevisiae, leading to the formation of pseudo hyphae [21,22]. In this work, disruption of MaCts1 led to the maintenance of typical conidiation on SYA medium, indicating that the availability of MaCts1 plays a pivotal role in the shift to microcycle conidiation in M. acridum [13].
In fungi, the morphogenesis-related NDR kinase network (MOR pathway) and the regulation of Ace2 and morphogenesis (RAM) pathway in baker's yeast are involved in morphogenetic regulation during conidiation [26]. The MOR/RAM pathways have very similar components, such as Hym1, Kic1, Tao3, Cbk1 and Mob2, but the RAM pathway involves the zinc finger transcription factor Ace2, which is not conserved in fungi and specific for budding yeast [29,50,51]. Previous studies have shown that the expression of Cts1 depends on genes in the RAM network, including Tao3 and Ace2 [22,23,52]. Both Cts1 and Ace2 are essential to degrade the septum for successfully detaching cells in S. cerevisiae, and their mutants lead to consistent defects in cell separation after mitosis [22,53]. The mutants of Cts1 and other genes in the MOR/RAM pathway, such as Tao3, Hym1 and Kic1, also showed similar sensitivities to oxidative stress [54]. Moreover, in other fungi, the mutants of Cbk1 and Ace2 homologs, essential components in the MOR/RAM pathway, displayed altered colonial morphology, defective cell separation and a decreased expression of CTS1 [30,52,55]. We therefore initially speculated that MaCts1 contributes to the microcycle conidiation and that it is governed by the MOR/RAM pathway in M. acridum. Consequently, we identified the putatively conserved M. acridum homologs of the MOR/RAM pathway. However, deletion of these genes did not impair the ability of M. acridum to induce the microcycle conidiation on SYA medium. Thus, MaCts1 contributes to the microcycle conidiation, which may not be dependent on the MOR/RAM pathway in M. acridum. The identification of regulatory elements in MaCts1 expression is of interest, since this would provide insight into the network of genes governing early steps in the shift to microcycle conidiation in M. acridum. Future work will be directed towards predicted binding sites in the MaCts1 promoter in combination with the analysis of differentially expressed genes in WT vs. ∆MaCts1 during conidiation on SYA media. These results would be helpful to further clarify the molecular mechanisms underlying the microcycle conidiation in M. acridum and other entomopathogenic fungi.

Conclusions
The endochitinase, MaCts1, has been shown to make contributions to the conidial germination, conidiation yield and the resistance to UV-irradiation and heat-shock in M. acridum. Interestingly, MaCts1 also contributes to the microcycle conidiation, which may be not dependent on the MOR/RAM pathway in M. acridum.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/biology11121730/s1, Figure S1: The disruption and complementation of MaCts1. (A) MaCts1 disruption vector was constructed based on pk2-PB vector. (B) MaCts1 complementation vector was constructed based on pk2-Sur vector. (C) The WT, ∆MaCts1 and CP were verified by Southern blotting. The genomic DNA was cut with EcoRI and StuI. The Probe was amplified from genomic DNA by PCR using primer pair MaCts1-TF and MaCts1-TR (Table S1). Figure S2: Disruption of key genes, including MaHym1, MaKic1, MaTao3, MaCbk1 and MaMob2, in MOR pathway and MaAce2 in M. acridum. All mutants were verified by PCR analyses. All primers used in this study were listed in Table S1. Table S1: Primers used in this work. Table S2: Identification of MOR/RAM components in M. acridum.
Funding: This work was supported by the National Natural Science Foundation of China (32172479), Chongqing talent program of China (cstc2021ycjh-bgzxm0313), the Venture and Innovation Support Program for Chongqing Overseas Returnees (cx2019035).