Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS

Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS

Journal Pre-proof Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS Lei Qiu, Sheng-Xi...

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Journal Pre-proof Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS

Lei Qiu, Sheng-Xin Nie, Shun-Juan Hu, Shou-Juan Wang, JuanJuan Wang, Kai Guo PII:

S0048-3575(20)30227-3

DOI:

https://doi.org/10.1016/j.pestbp.2020.104732

Reference:

YPEST 104732

To appear in:

Pesticide Biochemistry and Physiology

Received date:

13 February 2020

Revised date:

17 September 2020

Accepted date:

12 October 2020

Please cite this article as: L. Qiu, S.-X. Nie, S.-J. Hu, et al., Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS, Pesticide Biochemistry and Physiology (2019), https://doi.org/10.1016/ j.pestbp.2020.104732

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© 2019 Published by Elsevier.

Journal Pre-proof

Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS Lei Qiu1,* [email protected], Sheng-Xin Nie1, Shun-Juan Hu1, Shou-Juan Wang1, Juan-Juan Wang2,* [email protected], Kai Guo3 1

State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of

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Technology, Shandong Academy of Sciences, Jinan, China School of Biological Science and Technology, University of Jinan, Jinan, China

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Biology Institute, Qilu University of Technology, Shandong Academy of Sciences, Jinan,

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China *

Shandong 250022, PR China. *

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Correspondence to: School of Biological Science and Technology, University of Jinan, Jinan,

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Correspondence to : State Key Laboratory of Biobased Material and Green Papermaking,

PR China.

Abstract

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Qilu University of Technology, Shandong Academy of Sciences, Jinan, Shandong 250353,

Beauveria bassiana is a promising biocontrol agent due to its entomopathogenic activities and residue-free characteristics. However, its susceptibility to abiotic stresses and naturally low virulence limit the effective application of this fungus. To effectively obtain fungal strains with high biocontrol potential, fluorescence-activated cell sorting (FACS) was used to screen mutant libraries generated by atmospheric and room temperature plasma (ARTP). Among about 8000 mutants obtained by ARTP mutagenesis, six candidate mutants were

Journal Pre-proof selected according to the forward scatter (FSC) signal readings of FACS. B6, with a 37.4% higher FSC reading than wild-type (WT), showed a 32.6% increase in virulence. It also presented a 13.5% decrease in median germinating time (GT50) and a 12.1% increase in blastospore production. Comparative analysis between insect transcriptional responses to B6 and WT infection showed that the immune response coupled with protein digestion and absorption progress was highly activated in B6-infected Galleria mellonella larvae, while fatty acid synthesis was suppressed after 3 days of infection. Our results confirmed the

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feasibility of sorting B. bassiana with high biocontrol potential via the combination of ARTP

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and FACS and facilitated the understanding of insect-pathogen interactions, highlighting a

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new strategy for modifying entomopathogenic fungi to improve the efficiency of biological

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control.

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Abbreviations

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Keywords: ARTP; FACS; Beauveria bassiana; Biological control; Transcriptome

FACS

fluorescence-activated cell sorting

ARTP

atmospheric and room temperature plasma

1. Introduction Atmospheric and room temperature plasma (ARTP), a potent technique to induce biological mutation, has made succeed in obtaining strains with desirable characteristics among bacteria, fungi and microalgae in recent years (Zhang et al., 2019a; Zhang et al., 2019b). The ARTP mutation system generates a plasma jet, which can cause damage to DNA and induce biological cells to initiate SOS repair mechanisms at low temperatures under atmospheric

Journal Pre-proof pressure (Zhang et al., 2014). ARTP is relatively more efficient due to its rapid mutation, high safety and operational flexibility (Kong et al., 2016). This technique can induce increased DNA injury in single living cells, which may result in an increased mutation rate relative to traditional

approaches

including

4-nitroquinoline-1-oxide,

UV

or

N-methyl-N’-nitro-N-nitrosoguanidine (Zhang et al., 2015). Traditional screening methods, such as the detection of metabolic levels and observation of colony morphology, have achieved fruitful results (Chen et al., 2019; da Rocha et al., 2019). However, they were

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limited due to the poor efficiency and hard to satisfy the requirement of rapid mutagenesis

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methods. Therefore, in the process of strain development, high-throughput screening

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represents an indispensable link (Dietrich et al., 2010).

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Fluorescence-activated cell sorting (FACS), one of the high-throughput approaches derived from flow cytometry, was adopted in the present work for screening target mutant

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strains, which allowed to rapidly sort and collect cell subsets according to forward scatter

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(FSC, associated with cell size), side scatter (SSC, associated with intracellular granularity), and auto-fluorescence signals (Bleichrodt and Read, 2019). According to fluorochrome

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signals of propidium iodide and diacetate, the FACS was used to conduct a morphological analysis of Penicillium chrysogenum (Ehgartner et al., 2017). By detecting the high fluorescence signal generated by fatty acyl-CoA reductase (FAR) and green fluorescent protein (GFP) fusion protein, this high-throughput screening method has also been successfully applied to screening the strains of Trichoderma reesei with high FAR (Wang et al., 2018). As a biocontrol agent with entomopathogenic activities and residue-free characteristic, Beauveria bassiana has drawn extensive attention (Peng et al., 2019; Ortiz-Urquiza et al., 2016; Kim et al., 2016; Ortiz-Urquia et al., 2015). The conidia of B. bassiana germinate and secrete enzymes on the attached insect body wall to digest the cuticle (Keyhani, 2018;

Journal Pre-proof Ortiz-Urquia and Keyhani, 2013). Following cuticle penetration, the fungi produce blastospores that easily escape attacks by the defense system, grow on the basis of nutrients existing within hemolymph, and cause insect death (Vertyporokh et al., 2019; Zhang et al., 2017). However, it has low natural virulence and is susceptible to abiotic stresses, which have restricted the efficient B. bassiana application (Lovett and St Leger, 2018). The infection progress of B. bassiana is affected by complex factors, such as the production of spores, secretion of cuticle-degrading enzymes and resistance to ultraviolet

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radiation (Tahir et al., 2019; Lee et al., 2018; Vega et al., 2018). Our previous study revealed

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that the virulence of B. bassiana presented a high linear correlation with blastospore size (Qiu

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et al., 2015). In this study, mutants from ARTP libraries with high biocontrol potential were

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obtained by FACS according to blastospore size. Besides, the transcriptional differences between Galleria mellonella larvae infected with wild type (WT) and the mutant with high

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biocontrol potential were analyzed.

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2. Materials and methods

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2.1 Strains and culture conditions

The B. bassiana wild strain ARSEF 2860, was provided by RW Holley Center for Agriculture and Health (Ithaca, NY, USA) and grown in the SDAY plates supplemented with 4% glucose, 1.5% agar, 1% peptone, together with 1% yeast extract, for obtaining the mature conidia. Conidial suspensions at a concentration of 107 conidia/ml (the same below unless specified) were incubated in nitrogen-limited broth (NLB) containing 4% glucose, 0.4% NH4NO3, 0.3% KH2PO4 and 4% MgSO4 at 25°C with shaking (110 rpm) for blastospores (Ying and Feng, 2006) and in GB medium (2% sucrose and 0.5% peptone) for germination assessment. 2.2 ARTP mutagenesis of the WT strain Mutagenesis was performed using the ARTP biological mutagenesis system (Wuxi Yuanqing Tianmu Biological Technology Co., Ltd., Wuxi, China). The flow rate of working helium gas

Journal Pre-proof was set at 10 standard L/min, and the power of radio frequency was set at 100 W (Yang et al., 2019; Zhang et al., 2018). For determining the optimal treatment time, the conidial suspension (106 cells/ml) was spread onto the 8-mm disinfected stainless steel plates, followed by 0-120 s exposure to ARTP jet every 10 s, with the non-treated strain being the reference. After ARTP treatment, the steel plates were shaken and eluted with sterile 0.02% Tween-80. The cells in the eluate were diluted to the proper concentration before spreading on SDAY plates. After incubation for 3 days under 25°C, the numbers of control (a) as well

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as survival clones (b) were determined, respectively, to calculate the mortality rate by (a-b)/a

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× 100%.

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To obtain sufficient mutants, 10 μl aliquots of conidial suspension at a concentration of

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106 cells/ml were spread on 8 sterilized stainless steel plates respectively for exposure to ARTP jet for 90 s. After ARTP treatment, 8 steel plates were shaken and eluted with 1 ml

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sterile 0.02% Tween-80. After centrifugation, part of the supernatant was removed to leave

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400 μl suspension, which containing about 8000 alive conidia to spread on SDAY. All conidia of mixed mutants were eluted from the plate using 3 ml 0.02% Tween-80 after 10-day growth

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and the conidial suspension was diluted to the concentration of 107 cells/ml. 100 μl of the conidial suspension which containing approximately 106 conidia of mixed mutants was incubated in 50 ml of NLB at 25°C to obtain blastospores (Fig. 1). 2.3 FACS for mutant libraries based on FSC readings To obtain the unicellular cell, the blastospores suspension was filtered several times with sterile degreasing cotton until no hyphae were observed under a microscope. Before analysis with a MoFlo XDP flow cytometry (Beckman), the suspension was centrifuged and resuspended in phosphate buffer saline to a concentration of 107–108 cells/ml. The FSC and SSC detectors were used, respectively, to assess blastospore size and complexity in the presence of a 488 nm laser according to specific instructions (Puttikamonkul et al., 2010).

Journal Pre-proof The WT signals were analyzed first, and the gate (FSC > 135.5) was marked to collect cells with high FSC readings. In the first round of FACS, 500 particles were captured in a new tube from a 1 ml sample suspension. After the second round of sorting, 15 particles were selected and then cultivated on SDAY plates to acquire candidate mutants, and single colony was picked for purification (Fig. 1). The blastospore size and granularity of WT and mutants were reanalyzed through flow cytometry.

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2.4 Phenotypic characterization of B. bassiana mutant isolates

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To estimate the blastospore yield, 100 μl of conidial suspension of each candidate mutant was

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incubated in 50 ml of NLB. The number of candidate mutant and WT blastospores was

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counted with a microscope at 12 h intervals from 2 days onwards. Colony diameters of candidate mutants were measured by cross-measuring colony

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diameters (Qiu et al., 2019; Qiu et al., 2014). To estimate the conidial yield, 100 μl aliquots of

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the conidial suspension were added onto the cellophane-covered SDAY plates for incubation under 25°C and a 12/12 h light/dark cycle. From the 4th day, the hyphae were removed from

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the plate with a hole puncher (diameter of 6 mm) daily and washed off with 1 ml of 0.02% Tween-80 with ~10 min vibration. Conidial numbers were counted under a microscope with a haemocytometer and converted to conidia per square centimeter of the medium. For estimating conidial viability, 100 μl aliquots of the conidial suspension were inoculated in 1 ml GB, followed by incubation under 25°C and shaking at 140 rpm. Thereafter, the number of germinated together with ungerminated conidia was determined at 2 h intervals until all strains germinated completely. Meanwhile, to estimate the conidial thermotolerance, 2 ml of the conidial suspension were treated with the 45°C hot water for a period of 90 min. Meanwhile, 100 μl solutions were transferred into new tubes at intervals of 15 min, followed by the addition of 1 ml GB for determining germination ratio. The

Journal Pre-proof germination ratio data were converted to median germination time (GT50) and median lethal time (LT50), which are indicators of conidial viability and thermotolerance, respectively (Zhu et al., 2017; Zhao et al., 2019c). 2.5 Insect bioassays The virulence of candidate mutants was determined using fourth instar larvae of G. mellonella by topical inoculation (natural route of infection). Larvae were immersed in conidial suspension (immersed in sterile 0.02% Tween-80 as a control) for 10 s and then

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cultured in transparent plastic boxes at 25°C. From 2 days onwards, the larvae were counted

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for mortality every 12 h until all insects died. Dead larvae were removed to a new plate with

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cotton saturated with sterile water daily for further observation. 30 insects were treated with

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each strain, and all experiments were conducted for thrice. Kaplan-Meyer curves were

utilized as the virulence index.

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constructed to analyze survival data, while Probit analysis was used to determine LT 50 and

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The median lethal dose (LD50) of B6 and WT were determined to further explore the virulence change of B6. Larvae were immersed in conidial suspension at the concentration of

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105 cells/ml, 5 × 105 cells/ml, 106 cells/ml, 5 × 106 cells/ml, 107 cells/ml, 5 × 107 cells/ml and 108 cells/ml (immersed in sterile 0.02% Tween-80 as a control) for 10s and then cultured in transparent plastic boxes at 25°C. From 5 days to 7 days, the larvae were counted for mortality every 24 h, and the LD50 was determined by Probit analysis. 2.6 RNA extraction, library preparation, and transcriptome analysis 200 mg G. mellonella bodies infected by WT and B6 for 3days was used to extract total RNA by the use of TRIzol (Invitrogen, Carlsbad, CA, USA) in accordance with specific protocols. Thereafter, the NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA) was utilized to qualify and quantify RNA. Oligo(dT)-adhered magnetic beads were adopted for the purification of mRNA, which

Journal Pre-proof was fragmented into small pieces using fragment buffer. Afterwards, reverse transcriptase was utilized for generating the first-strand cDNA using random primers, and later second-strand cDNA was synthesized. Subsequently, the RNA index and A-tailing mix adapters were joined through the incubation to end repair. The Ampure XP Beads were used to purify cDNA fragment products, while Agilent Technologies 2100 bioanalyzer was adopted to validate the quality. Later, those double-stranded PCR products obtained in the prior step were subjected to heating, denaturation and circularization. After obtaining the

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single-stranded circular DNA, it was amplified using phi29 for making the DNA nanoball

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(DNB). Then, DNBs were added to patterned nanoarray, while the 150 bp paired-end reads

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were produced using the BGISEQ-500 platform (BGI-Shenzhen, China). Raw sequencing

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reads of two libraries were filtered with Trimmomatic. Clean reads were mapped with HISAT

3. Results

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3.1 ARTP mutagenesis and FACS

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to G. mellonella reference genomic sequence. Each experiment was carried out for thrice.

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As the ARTP treatment time increased, WT strain mortality elevated, and the mortality rate reached 100% when the WT was exposed to the ARTP jet for 120 s, resulting in an LT50 of 43 s. It has been demonstrated that a survival rate of 10–20% was beneficial to obtain positive mutants (Kong et al., 2016). Therefore, 90 s was selected as the optimal treatment time in this study, at which time the mortality rate reached 89% (Fig. 2A). About 8000 mutants were produced from ARTP. 15 particles sorted by two rounds of FACS were plated on agar media and six candidate mutants were isolated (Fig. 1). The other 9 particles produced no colony on the SDAY plates, indicating that they may be the drop of phosphate buffer saline, dead cells or cell debris. The diameters of the 7-d-old colonies of these candidate mutants on SDAY fell in a narrow range of 21.3–27.6 mm (Tukey’s HSD, P > 0.05). The FSC and SSC readings of blastospores of six mutants and WT were reanalyzed

Journal Pre-proof through flow cytometry (Fig. 2B−C). The FSC readings of B2 (25.1%), B3 (12.2%) and B6 (37.4%) were significantly higher than that of WT (Tukey’s HSD, P < 0.05). No difference was detected between B4 (141.4), B5 (135.7) and WT (135.4). In comparison to WT, the SSC reading for B2 increased significantly by 16.1%, and the SSC reading for B4 decreased by 17.2% (Tukey’s HSD, P < 0.05). On 2.5 days, blastospore production of B1, B2, and B6 were 2.51 to 3.11-fold greater than that of WT. On day 3.5, the yields of B1 and B6 were still significantly increased 11.3%

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and 12.1%, respectively (Fig. 2D), while the yields of other mutants (B2, B3, B4, and B5)

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were significantly less than that of WT (Tukey’s HSD, P < 0.05).

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3.2 Conidial yield, viability and thermotolerance analysis

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Assessment of conidial yield demonstrated that B2 and B4 produced more conidia than WT on day 4 (Fig. 3A). On day 7, the conidial yield of B4 and B6 showed no significant

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difference from WT and reached a mean (± SD) yield of 145.57 (± 2.8) × 107 conidia/cm2 and

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145.34 (± 5.1) × 107 conidia/cm2, respectively, but the conidial production capacity of other mutants was decreased by 25.3% – 49.3% compared to that of WT.

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The GT50, an indicator of conidial capacity, was decreased by 13% and 16% (Tukey’s HSD, P < 0.05) in B6 and B3 respectively, compared to that of WT, which was 8.7 h (Fig. 3B). The GT50 values of B1, B4 and B5 were notably improved. As an indicator of thermotolerance, the LT50 of the mutants under heat stress exhibited no significant difference from WT (29.9 min), except for B1 (38.8 min) (Fig. 3C). 3.3 Virulence analysis The insecticidal effect was investigated using the fourth instar larvae of G. mellonella. Kaplan-Meyer curves were constructed to analyze survival data (Fig. 4A), while strain virulence was evaluated by LT50 (Fig. 4B). After 144 h of infection, B3, B4 and B6 caused 100% insect mortality, while WT had a survival of 9.3%. In normal infection, the LT50 value

Journal Pre-proof for the WT against G. mellonella larvae was 4.9 days. Compared to WT, B2, B3, B4 and B6 showed lower LT50 values (18.8%, 24.1%, 16.4% and 32.6%, respectively) through normal infection (Tukey’s HSD, P < 0.05). No significant difference was detected between the virulence of B1, B5 and WT. The median lethal dose (LD50) of B6 and WT were determined to further explore the virulence change of B6. Compared to WT, the LD50 of B6 at 5 days, 6 days and 7 days were decreased by 46.7%, 42.1% and 42.8% respectively (Fig. 4C). 3.4 Comparative transcriptomic analysis of infected larvae

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After filtering for high-quality sequences, 42 and 44 M clean reads were obtained from

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B6-infected and WT-infected G. mellonella, respectively. The clean reads were mapped to the

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reference genome sequence (GCF_003640425.1_ASM364042v1_NCBI_20190722), and the

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mean comparison ratio was 80.47%. A total of 13,600 genes were detected in the two samples, with 329 and 501 unique genes expressed in G. mellonella infected by B6 and WT,

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respectively. A total of 1491 differentially expressed genes (DEGs) exhibited obvious

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expression changes (|FC| ≥ 2, Q ≤ 0.001) in B6-infected G. mellonella, including 622 upregulated and 869 downregulated genes (Fig. 5A). The lists of top 20 up-regulatory and top

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20 down-regulatory genes of DEGs in G. mellonella infected by B6 versus WT were shown as Supplemental data (Table S1 and S2). Gene ontology (GO) assignments were used to classify the DEGs according to functions. A total of 341 DEGs with significant expression changes were mapped to 35 secondary GO categories under three main divisions: biological process, cellular component and molecular function (Fig. 5B). Compared to biological process and cellular component (24.2% and 36.3%, respectively), molecular function contained the most DEGs (39.5%). More downregulated genes were observed for most of the GO terms than upregulated genes, suggesting that the majority of the transcriptional activity of the DEGs was inhibited by the infection of the high-virulent strain. The secondary GO categories of cellular process,

Journal Pre-proof metabolic process, cell, binding and catalytic activity were downregulated dramatically. In contrast, GO categories of cellular component organization or biogenesis, extracellular region, multi-organism process, membrane and membrane part contained more upregulated genes than downregulated genes. GO enrichment revealed that 22 terms were significantly enriched (Q ≤ 0.05). Three genes that mapped to phosphopantetheine binding were downregulated, and two of them related to fatty acid synthesis were dramatically downregulated (FC < -7.5). All eight genes

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in the organic acid catabolic process were upregulated. In the term of structural constituent of

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cuticle, 7 of 9 genes related to DNA repair and recombination proteins were upregulated.

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A total of 766 DEGs were mapped to 44 secondary Kyoto Encyclopedia of Genes and

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Genomes (KEGG) categories. DEGs with |FC| ≥ 4 were significantly enriched (Q ≤ 0.05) in 5 pathways (Fig. 5C). The genes that mapped to fatty acid biosynthesis (69.2%), fatty acid

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metabolism (77.3%) and AMPK signaling pathway (73.3%) were downregulated; 56.6% of

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the genes that mapped to RNA polymerase were upregulated, and all genes that mapped

4. Discussion

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hematopoietic cell lineage were upregulated.

Conventional mutagenesis has been successful in microbial mutation breeding. The LT50 (Plutella xylostella as the host) of the Metarhizium anisopliae mutant obtained by UV radiation was half that of the WT (Zhao et al., 2016). A mutant of Isaria fumosorosea induced by the mutagen NaNO2 exhibited 830-fold increased carbendazim resistance compared with the WT (Song et al., 2011). The cordycepin production of Cordyceps militaris mutant treated with ion beam was 72% more than that produced by the control (Das et al., 2008). In our case, the combination of ARTP and FACS was first applied to screen entomopathogenic fungi, and six candidate mutants of B. bassiana were rapidly acquired in this way. The umu test and fluctuation test illustrated that ARTP resulted in much higher levels of DNA damage and

Journal Pre-proof mutation rates against Salmonella typhimurium compared to UV, 4-nitroquinoline-1-oxide, and N-methyl-N’-nitro-N-nitrosoguanidine mutagenesis (Zhang et al., 2015). FACS enables living cells stained with fluorescent reporters to be sorted for downstream experimentation (Bleichrodt and Read, 2019). Lipid-rich Chlamydomonas reinhardtii cells stained with Nile red (the lipophilic stain) were sorted by FACS (Xie et al., 2014). This high-throughput screening method was used to sort the mutagenized TRI5-GFP reporter strain to identify regulators of mycotoxin production, and a mutant over-producing

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mycotoxin under repressive conditions was identified (Blum et al., 2016). Red fluorescence

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protein was adopted to be the reporter molecule in FACS for screening the hyperproducers of

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cellulase in the T. reesei ARTP libraries (Gao et al., 2018). The Corynebacterium glutamicum

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mutant with the highest L-serine yield was screened by the detection of an enhanced yellow fluorescent protein from ARTP libraries (Zhang et al., 2018).

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FACS can distinguish cells according to the scattered signals associated with shape and

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size (Sycuro et al., 2013). According to our previous study, the size of blastospores and virulence present a high linear correlation (Qiu et al., 2015). FSC readings, the representative

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of blastospores size, were used to screen B. bassiana with high biocontrol potential from ARTP libraries. Among them, B2, B3, and B6 showed a larger size and more effective insecticidal activity, further indicating the possibility of obtaining strains with high virulence according to the size of blastospores. Based on cell size, Escherichia coli mutants were grouped by FACS, and these strains were used for the systematic interrogation of cell morphology effect on the physiological properties of bacteria (Shi et al., 2017). In applications of fungi as biological control agents, fungal pathogenicity is affected by the growth and germination conditions of spores, as well as other environmental abiotic factors. Conidiogenesis accounts for the essential condition for the production of highly virulent fungal conidia (Lee et al., 2018). B2 and B3 exhibited a reduced ability to produce

Journal Pre-proof spores compared to WT. Considering that the environment and certain hosts increase body temperature to resist the attack of pathogenic fungi (Rangel et al., 2010), thermotolerance is vital in the application of fungi as biological control agents (Chandra Teja and Rahman, 2016; de Crecy et al., 2009). The LT50 under heat stress of B1 was significantly increased compared with WT, showing a high thermotolerance of B1, while other candidate mutants showed no differences. B4 showed a significant decrease in germinating ability compared with WT, and germination is important to initiate vegetative growth and spore-mediated disease (Ortiz et al.,

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2019). B6, with a higher FSC reading than WT, showed increased virulence. It also presented

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most ideal strain with high biocontrol potential.

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a 13.5% decrease in the GT50 and a 12.1% increase in blastospore production; thus, it is the

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Transcriptome analysis showed that the immune response was highly activated in B6-infected larvae. The immune response of G. mellonella involves cellular components and

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humoral components (Trevijano-Contador and Zaragoza, 2018). The cellular immune

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response includes phagocytosis, nodule formation, and encapsulation, and these are regulated via the circulating haemocytes (Ono and Yoshiga, 2018; Fan et al., 2013). Based on GO and

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KEGG analysis of G. mellonella infected by B6 and WT (as control), five genes involved in phagosomes were upregulated. Two genes encoding cathepsin L and B (mapped to lysosomes) were upregulated (Table. 1). The high expression of these two genes inhibited Bactrocera dorsalis larvae growth and development after exposure to azadirachtin (Zhao et al., 2019a). The humoral response contains lytic enzymes, melanin and antimicrobial peptides (AMPs), which were found to be modulated by the Toll and Imd signaling pathways (Park and Kim, 2012). In our DEGs, one gene encoding C-lysozyme was found to be upregulated. The overexpression of Bombyx mori C-lysozyme remarkably suppressed viral replication within cells in the process of infection (Chen et al., 2018). C-lysozyme from Schistocerca gregaria was upregulated against bacterial challenge (Mohamed et al., 2016). Seven genes related to

Journal Pre-proof the Toll and Imd signaling pathways were upregulated, including two antimicrobial peptide genes encoding gallerimycin and galiomicin. These two peptides, which are the most effective against fungal infection, were both activated in G. mellonella infected by B. bassiana (Dekkerova-Chupacova et al., 2018; Vertyporokh and Wojda, 2017). The protein digestion and absorption progress were activated in B6-infected larvae. Twenty-two genes were expressed at a higher level in the protein digestion and absorption pathway, including genes encoding trypsin and carboxypeptidase, which are involved in the

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process of digestion during molting (Zhao et al., 2019b). Consistently, seven genes encoding

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aminopeptidase N (APN) in the haematopoietic cell lineage pathway were upregulated. APN

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plays a role in modulating the development and activities of immune-related cells and

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regulating the functions of inflammatory mediators (Lu et al., 2020). The fatty acid synthesis was suppressed on the third day of B6 infection. Fatty acid

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synthesis is regulated by synthase (FAS) and acetyl-CoA carboxylase (ACC) (Gao et al.,

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2019). Fourteen genes involved in FAS and a gene encoding ACC were downregulated, illustrating that fatty acid synthesis could be inhibited by B6 infection. In Lysiphlebia

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japonica, the expression of most FAS genes on day 3 remarkably decreased within parasitized aphids compared with nonparasitized aphids (Gao et al., 2019). The absence of BgFas1 in female cockroaches results in inhibition of the expression of insect cuticular hydrocarbons, which exert an important part in the protection of insects against ultraviolet radiation and entomopathogen invasion, together with in the resistance of chemical xenobiotics (Pei et al., 2019). In conclusion, the B6 mutant of B. bassiana with a 32.6% increased virulence compared to WT was obtained by the combination of ARTP and FACS according to the FSC readings. Our findings confirmed the feasibility of sorting B. bassiana with high biocontrol potential in this way and provide a new method to improve the efficiency of B. bassiana in biological

Journal Pre-proof control. Comparative analysis of insect transcriptional responses to mutant and original strains facilitated the understanding of insect-pathogen interactions. The following are the supplementary data related to this article. Table S1. A list of top 20 up-regulatory genes of DEGs in G. mellonella infected by B6 versus WT. Table S2. A list of top 20 down-regulatory genes of DEGs in G. mellonella infected by B6

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versus WT.

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Acknowledgments

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Funding of this work was provided by the National Natural Science Foundation of China

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(Grants 31800062 and 31600063), the Foundation (No. ZZ20190315) of State Key

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Laboratory of Biobased Material and Green Papermaking and Youth Doctor Cooperation Fund Project (2019BSHZ0030), Qilu University of Technology, Shandong Academy of

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Sciences. Conflict of interest

The authors declare that they have no conflict of interest. References Bleichrodt, R.J., Read, N.D., 2019. Flow cytometry and FACS applied to filamentous fungi. Fungal Biol. Rev. 33, 1–15. Blum, A., Benfield, A.H., Stiller, J., Kazan, K., Batley, J., Gardiner, D.M., 2016. High-throughput FACS-based mutant screen identifies a gain-of-function allele of the

Journal Pre-proof Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production. Fungal Genet. Biol. 90, 1–11. Chandra Teja, K.N.P., Rahman, S.J., 2016. Characterisation and evaluation of Metarhizium anisopliae (Metsch.) Sorokin strains for their temperature tolerance. Mycology 7, 171– 179. Chen, G.Q., Zhu, Y.S., Zhang, G., Liu, H.B., Wei, Y.X., Wang, P.G., Wang, F., Xian, M.,

of

Xiang, H.Y., Zhang, H.B., 2019. Optimization and characterization of pullulan

ro

production by a newly isolated high-yielding strain Aureobasidium melanogenum. Prep.

-p

Biochem. Biotech. 49:557–566.

re

Chen, T.T., Tan, L.R., Hu, N., Dong, Z.Q., Hu, Z.G., Jiang, Y.M., Chen, P., Pan, M.H., Lu, C., 2018. C-lysozyme contributes to antiviral immunity in Bombyx mori against

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nucleopolyhedrovirus infection. J. Insect Physiol. 108, 54–60.

na

da Rocha, W.R.V., Costa-Silva, T.A., Agamez-Montalvo, G.S., Feitosa, V.A., Machado, S.E.F., Lima, G.M.D., Pessoa, A., Alves, H.S., 2019. Screening and optimizing fermentation

Jo ur

production of L-asparaginase by Aspergillus terreus strain S-18 isolated from the Brazilian Caatinga Biome. J. Appl. Microbiol. 126, 1426–1437. Das, S.K., Masuda, M., Hatashita, M., Sakurai, A., Sakakibara, M., 2008. A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation. Lett. Appl. Microbiol. 47, 534–538. de, Crecy, E., Jaronski, S., Lyons, B., Lyons, T.J., Keyhani, N.O., 2009. Directed evolution of a filamentous fungus for thermotolerance. BMC Biotechnol. 9, 74. Dekkerova-Chupacova, J., Borghi, E., Morace, G., Bujdakova, H., 2018. Up-regulation of antimicrobial peptides gallerimycin and galiomicin in Galleria mellonella infected with

Journal Pre-proof candida yeasts displaying different virulence traits. Mycopathologia 183, 935–940. Dietrich, J.A., McKee, A.E., Keasling, J.D., 2010. High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu. Rev. Biochem. 79, 563–590. Ehgartner, D., Herwig, C., Fricke, J., 2017. Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry-the fast alternative to microscopic image analysis. Appl. Microbiol. Biot. 101, 7675–7688.

of

Fan, J., Xie, Y., Xue, J., Zhang, Y., Yang, Q., 2013. Cellular apoptosis of hemocytes from

ro

Dendrolimus tabulaeformis Tsai et Liu larvae induced with the secondary metabolites of

-p

Beauveria brongniartii (Sacc.) Petch. PloS One 8, e71600.

re

Gao, F., Hao, Z.Z., Sun, X.H., Qin, L.N., Zhao, T., Liu, W.Q., Luo, H.Y., Yao, B., Su, X.Y.,

lP

2018. A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting. Biotechnol.

na

Biofuels. 11, 13.

Jo ur

Gao, X., Luo, J., Zhu, X., Wang, L., Ji, J., Zhang, L., Zhang, S., Cui, J., 2019. Growth and fatty acid metabolism of Aphis gossypii parasitized by the parasitic wasp Lysiphlebia japonica. J. Agric. Food Chem. 67, 8756–8765. Keyhani, N.O. 2018. Lipid biology in fungal stress and virulence: entomopathogenic fungi. Fungal Biology 122, 420-429. Kim, S., Lee, S.J., Nai, Y.S., Yu, J.S., Lee, M.R., Yang, YT., Kim, J.S., 2016. Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007. Appl. Microbiol. Biot. 100, 8889–8900. Kong, X.P., He, A.Y., Zhao, J., Wu, H., Ma, J.F., Wei, C., Jin, W.Q., Jiang, M., 2016. Efficient acetone-butanol-ethanol (ABE) production by a butanol-tolerant mutant of Clostridium

Journal Pre-proof beijerinckii in a fermentation-pervaporation coupled process. Biochem. Eng. J. 105, 90– 96. Lee, S.J., Lee, M.R., Kim, S., Kim, J.C., Park, S.E., Shin, T.Y., Kim, J.S., 2018. Conidiogenesis-related DNA photolyase gene in Beauveria bassiana. J. Invertebr. Pathol. 153, 85–91. Lovett, B., St Leger, R.J., 2018. Genetically engineering better fungal biopesticides. Pest

of

Manag. Sci. 74, 781–789.

ro

Lu, C.Y., Amin, M.A., Fox, D.A., 2020. CD13/Aminopeptidase N is a potential therapeutic

-p

target for inflammatory disorders. J. Immunol. 204, 3–11.

re

Mohamed, A.A., Zhang, L., Dorrah, M.A., Elmogy, M., Yousef, H.A., Bassal, T.T.M., Duvic,

lP

B., 2016. Molecular characterization of a c-type lysozyme from the desert locust, Schistocerca gregaria (Orthoptera: Acrididae). Dev. Comp. Immunol. 61, 60–69.

na

Ono, M. Yoshiga, T., 2018. Cellular immunity in the insect Galleria mellonella against insect

Jo ur

non-parasitic nematodes. Parasitology 1–8. Ortiz, S.C., Huang, M.W., Hull, C.M., 2019. Spore germination as a target for antifungal therapeutics. Antimicrob. Agents Ch. 63, 16. Ortiz-Urquiza, A., Keyhani, N.O., 2013. Action on the surface: entomopathogenic fungi versus the insect cuticle. Insects 4, 357-374. Ortiz-Urquiza, A., Keyhani, N.O., 2016. Molecular genetics of Beauveria bassiana infection of insects. Adv. Genet. 94, 165-249. Ortiz-Urquiza, A., Z. Luo, Keyhani, N.O., 2015. Improving mycoinsecticides for insect biological control. Appl. Microbiol. Biot. 99, 1057-68.

Journal Pre-proof Park, J.A., Kim, Y., 2012. Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection. J. Invertebr. Pathol. 110, 382-388. Pei, X.J., Chen, N., Bai, Y., Qiao, J.W., Li, S., Fan, Y.L., Liu, T.X., 2019. BgFas1: A fatty acid synthase gene required for both hydrocarbon and cuticular fatty acid biosynthesis in the German cockroach, Blattella germanica (L.). Insect Biochem. Mol. Biol. 112, 103203. Peng, Y.J., Ding, J.L., Feng, M.G. Ying, S.H., 2019. Glc8, a regulator of protein phosphatase

of

type 1, mediates oxidation tolerance, asexual development and virulence in Beauveria

ro

bassiana, a filamentous entomopathogenic fungus. Curr. Genet. 65, 283–291.

-p

Puttikamonkul, S., Willger, S.D., Grahl, N., Perfect, J.R., Movahed, N., Bothner, B., Park, S.,

re

Paderu, P., Perlin, D.S., Cramer Jr, R.A., 2010. Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the

lP

human fungal pathogen Aspergillus fumigatus. Mol. Microbiol. 77, 891–911.

na

Qiu, L., Wang, J.J., Chu, Z.J., Ying, S.H., Feng, M.G., 2014. Phytochrome controls conidiation in response to red/far-red light and daylight length and regulates multistress

Jo ur

tolerance in Beauveria bassiana. Environ. Microbiol. 16, 2316–2328. Qiu, L., Wang, J.J., Ying, S.H., Feng, M.G., 2015. Wee1 and Cdc25 control morphogenesis, virulence and multistress tolerance of Beauveria bassiana by balancing cell cycle-required cyclin-dependent kinase 1 activity. Environ. Microbiol. 17, 1119–1133. Qiu, L., Wei, X.Y., Wang, S.J., Wang, J.J., 2019. Characterization of trehalose-6-phosphate phosphatase in trehalose biosynthesis, asexual development, stress resistance and virulence of an insect mycopathogen. Pestic. Biochem. Phys. 163, 185–192.

Journal Pre-proof Rangel, D.E.N., Fernandes, E.K.K., Dettenmaier, S.J., and Roberts, D.W., 2010. Thermotolerance of germlings and mycelium of the insect-pathogenic fungus Metarhizium spp. and mycelial recovery after heat stress. J. Basic Microb. 50, 344–350.

Shi, H., Colavin, A., Bigos, M., Tropini, C., Monds, R.D., Huang, K.C., 2017. Deep phenotypic mapping of bacterial cytoskeletal mutants reveals physiological robustness to cell size. Curr. Biol. 27, 3419.

of

Song, T.T., Ying, S.H., Feng, M.G., 2011. High resistance of Isaria fumosorosea to

ro

carbendazim arises from the overexpression of an ATP-binding cassette transporter (ifT1)

-p

rather than tubulin mutation. J. Appl. Microbiol. 112, 175–184.

re

Sycuro, L.K., Rule, C.S., Petersen, T.W., Wyckoff, T.J., Sessler, T., Nagarkar, D.B., Khalid, F.,

lP

Pincus, Z., Biboy, J., Vollmer, W., Salama, N.R., 2013. Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter

na

pylori morphology. Mol. Microbiol. 90, 869–883.

Jo ur

Tahir, M., Wakil W., Ali, A., Sahi, S.T., 2019. Pathogenicity of Beauveria bassiana and Metarhizium anisopliae isolates against larvae of the polyphagous pest Helicoverpa armigera. Entomol. Gen. 38, 225–242. Trevijano-Contador, N., Zaragoza, O., 2018. Immune Response of Galleria mellonella against Human Fungal Pathogens. J. Fungi 5, 3. Vega, F.E., 2018. The use of fungal entomopathogens as endophytes in biological control: a review. Mycologia 110, 4–30. Vertyporokh, L., Hulas-Stasiak, M., Wojda, I., 2019. Host-pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana. Insect Sci. 00, 1–11.

Journal Pre-proof Vertyporokh, L., Wojda, I., 2017. Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana. Acta. Biochim. Pol. 64, 273–278. Wang, G.K., Jia, W.D., Chen, N., Zhang, K., Wang, L.X., Lv, P., He, R.L., Wang, M., Zhang, D.Y., 2018. A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi. Biotechnol. Biofuels. 11, 232.

of

Xie, B., Stessman, D., Hart, J.H., Dong, H.L., Wang, Y.J., Wright, D.A., Nikolau, B.J.,

ro

Spalding, M.H., Halverson, L.J., 2014. High-throughput fluorescence-activated cell

-p

sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants. Plant

re

Biotechnol. J. 12, 872–882.

Yang, M., An, Y., Zabed, H.M., Guo, Q., Yun, J., Zhang, G., Awad, F.N., Sun, W., Qi, X.,

lP

2019. Random mutagenesis of Clostridium butyricum strain and optimization of

284, 188–196.

na

biosynthesis process for enhanced production of 1,3-propanediol. Bioresour. Technol.

Jo ur

Ying, S.H., Feng, M.G., 2006. Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana. Appl. Microbiol. Biot. 72, 206–210. Zhang, H., Chen, R., Zhang, J., Bu, Q., Wang, W., Liu, Y., Li, Q., Guo, Y., Zhang, L., Yang, Y., 2019a. The integration of metabolome and proteome reveals bioactive polyphenols and hispidin in ARTP mutagenized Phellinus baumii. Sci. Rep. 9, 16172. Zhang, K., Mohsin, A., Dai, Y., Chen, Z., Zhuang, Y., Chu, J., Guo, M., 2019b. Combinatorial effect of ARTP mutagenesis and ribosome engineering on an industrial strain of Streptomyces albus S12 for enhanced biosynthesis of salinomycin. Front. Bioeng. Biot. 7, 212.

Journal Pre-proof Zhang, W., Chen, J.H., Keyhani, N.O., Jin, K., Wei, Q.L., Xia, Y., 2017. Central nervous system responses of the oriental migratory, Locusta migratoria manilensis, to fungal infection. Sci. Rep. 7, 10340. Zhang, X., Zhang, C., Zhou, Q.Q., Zhang, X.F., Wang, L.Y., Chang, H.B., Li, H.P., Oda, Y., Xing, X.H., 2015. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis. Appl.

of

Microbiol. Biot. 99, 5639–5646.

ro

Zhang, X., Zhang, X., Xu, G., Zhang, X., Shi, J., Xu, Z., 2018. Integration of ARTP

-p

mutagenesis with biosensor-mediated high-throughput screening to improve L-serine

re

yield in Corynebacterium glutamicum. Appl. Microbiol. Biot. 102, 5939–5951. Zhang, X., Zhang, X.F., Li, H.P., Wang, L.Y., Zhang, C., Xing, X.H., Bao, C.Y., 2014.

lP

Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool.

na

Appl. Microbiol. Biot. 98, 5387–5396.

Zhao, J., Yao, R., Wei, Y., Huang, S., Keyhani, N.O., Huang, Z., 2016. Screening of

Jo ur

Metarhizium anisopliae UV-induced mutants for faster growth yields a hyper-virulent isolate with greater UV and thermal tolerances. Appl. Microbiol. Biot. 100, 9217–9228. Zhao, T., Lai, D., Zhou, Y., Xu, H., Zhang, Z., Kuang, S., Shao, X., 2019a. Azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae by releasing cathepsin in the midgut. Ecotoxicol. Environ. Saf. 183, 109512. Zhao, X.M., Qin, Z.Y., Zhang, J., Yang, Y., Jia, P., Yang, Q., Ma, E.B., Zhang, J.Z., 2019b. Nuclear receptor hormone receptor 39 is required for locust moulting by regulating the chitinase and carboxypeptidase genes. Insect Mol. Biol. 28, 537–549.

Journal Pre-proof Zhao X., Yang X., Lu Z., Wang H., He Z., Zhou G., Luo Z., Zhang Y. 2019c. MADS-box transcription factor Mcm1 controls cell cycle, fungal development, cell integrity and virulence in the filamentous insect pathogenic fungus Beauveria bassiana. Environ. Microbiol. 21, 3392–3416. Zhu X.G., Chu Z.J., Ying S.H., Feng M.G. 2017. Lysyl-tRNA synthetase (Krs) acts a virulence factor of Beauveria bassiana by its vital role in conidial germination and

ro

of

dimorphic transition. Fungal Biol. 121, 956–965.

Fig. 1. The experimental strategy of this study. Candidate mutants of B.bassiana were

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selected according to the FSC signal readings (cell size) of FACS from ARTP mutagenesis

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library.

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Fig. 2. ARTP mutagenesis and characteristics of B. bassiana blastospores screened by FACS.

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(A) Effects of ARTP treatment time on the mortality rate of B. bassiana. (B-C) Size and density (complexity) of blastospores indicated by the FSC and SSC readings from flow

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cytometry. (D) Blastospore yield over the incubation time on NLB medium at 25°C. Different lowercase letters signify obvious differences (Tukey’s HSD, P < 0.05). Error bar: SD of the mean from three replicates.

Fig. 3. Assessment of conidial yield, viability and thermotolerance. (A) Conidial yields quantified during normal cultivation on SDAY plates at 25°C under light/dark cycles of 12/12 h. (B) Length of time which is required for 50% conidial germination (GT50) in GB medium at 25°C. (C) Mean conidial half-lethal time (LT50) under 45°C heat stress. Different lowercase letters mean significant differences (Tukey’s HSD, P < 0.05). Error bar: SD of the mean from three replicates. Fig. 4. Measurement of fungal infection virulence to G. mellonella larvae. (A) Kaplan-Meyer

Journal Pre-proof survival curves of G. mellonella larvae after topical inoculation. (B) Median lethal time (LT50) for the WT and candidate mutants estimated by modelling analysis of the survival trends. (C) The median lethal dose (LD50) of B6 and WT from 5 days to 7 days. Different lowercase letters signify significant differences (Tukey’s HSD, P < 0.05). Error bar: SD of the mean from three replicates. ** P < 0.001 for indicated B6 strain versus the WT in each group. Fig. 5. Comparative transcriptome analysis of larvae infected with B6 and WT. (A)

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Correlation analysis of FC and Q-value. (B) Histogram presentations of Gene ontology (GO)

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classification of differentially expressed genes (DEGs). DEGs were categorized into three groups including biological process, cellular component and molecular function. (C) Kyoto

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Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs with |FC| ≥ 4.

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The pathways (bold type) were enriched pathway terms with Q ≤ 0.05.

Table 1. DEGs described from the analysis of transcriptomes of G. mellonella infected by B6 versus WT. Annotation

Q-Value b

6.37 4.87

cathepsin K/L/S cathepsin K/L

1.31E-20 4.59E-08

3.99 2.72

antifungal peptide gallerimycin mRNA inducible metalloproteinase inhibitor protein-like

0 0

-8.19 -7.89 -7.63

fatty acid synthase-like fatty acid synthase-like fatty acid synthase-like

0 0 0

10.05 9.34 6.68 5.79 4.97 3.02 8.65 7.22 9.18

trypsin beta-like trypsin delta-like trypsin, alkaline A-like carboxypeptidase B-like carboxypeptidase O carboxypeptidase O aminopeptidase N membrane alanyl aminopeptidase-like membrane alanyl aminopeptidase-like

0 3.49E-228 3.98E-94 1.55E-14 2.98E-05 4.64E-15 3.85E-15 6.70E-67 1.13E-53

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FC a

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Category or gene ID Phagosome 113519233 113510561 Antimicrobial peptide 113523440 113518892 Fatty acid synthase 113515169 113515170 113522241 Protease 113515392 113515393 113520339 113509223 113522073 113514532 113515697 113517732 113515584 a.

FC, fold change (log2 ratio) of gene expression.

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Genes with |FC| ≥ 2 and (Q ≤ 0.05) are significantly differentially expressed genes.

Graphical abstract

Highlights Approximately 8000 mutants were produced by ARTP. Candidate mutants with high biocontrol potential were selected according to the FSC signal readings of FACS.

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B6, with a 37.4% higher FSC reading than WT, showed a 32.6% increase in virulence. The immune response coupled with protein digestion and absorption progress was highly activated in B6-infected larvae. Fatty acid synthesis was suppressed in B6-infected larvae.

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