Expression analysis of the genes involved in the virulence of Beauveria bassiana

Accepted Manuscript Expression analysis of the genes involved in the virulence of Beauveria bassiana

Charbel Al Khoury, Georges Nemer, Jacques Guillot, Afif Abdel Nour, Nabil Nemer PII: DOI: Article Number: Reference:

S2352-2151(19)30014-5 https://doi.org/10.1016/j.aggene.2019.100094 100094 AGGENE 100094

To appear in: Received date: Revised date: Accepted date:

18 April 2019 17 July 2019 18 July 2019

Please cite this article as: C. Al Khoury, G. Nemer, J. Guillot, et al., Expression analysis of the genes involved in the virulence of Beauveria bassiana, , https://doi.org/10.1016/ j.aggene.2019.100094

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ACCEPTED MANUSCRIPT Expression analysis of the genes involved in the virulence of Beauveria bassiana Charbel Al Khourya,b [email protected], Georges Nemerc [email protected], Jacques Guillotb [email protected], Afif Abdel Noura [email protected], Nabil

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Department of Agricultural Sciences, Faculty of Agricultural and Food Sciences, P.O. Box 446

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a

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Nemera,* [email protected]

Jounieh, Lebanon.

Department of Biochemistry and Molecular Genetics, American University of Beirut, Riad El

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c

EA Dynamyc, UPEC, EnvA, 7 Avenue du General de Gaulle, 94700 Maisons-Alfort, France.

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Solh, Beirut, Lebanon; *

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Corresponding author.

Abstract

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The entomopathogenic fungus Beauveria bassiana is used as a natural pest killer in many

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agricultural systems due to its power of overcoming the host immune system. This is partially due to its genome content that expresses key proteins involved in its virulence. In this study, we

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used Real-Time PCR technique to analyze the relative expression at 48 and 72 h post inoculation of the Hyd1, Hyd2, Bbchit1, Cdep1, Bbbeas, Bbbsls and Vlp4 genes implicated in the pathogenesis process of an indigenous strain (LTB) and a commercial strain (IND) of B. bassiana. This analysis revealed a higher induction of Bbbeas, Bbbsls and Vlp4 genes by the LTB strain when comparing to the IND strain and the calibrator. Furthermore, a more rapid repression of Hyd1 and Hyd2 gene was notable by LTB strain. No significant difference was recorded by the expression of the Cdep1 gene between the test samples and the control. Finally, a

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ACCEPTED MANUSCRIPT significant difference was recorded with the expression of Bbchit1 gene by the IND strain 72 h post-inoculation. In conclusion, these results suggest that B. bassiana strains have differential expression of virulence genes that could reflect adaptation to their geographical environment and

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could help classifying their entomopathogenic efficacy accordingly.

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Keywords: Entomopathogenic fungi, Beauveria bassiana, relative expression, R-T PCR,

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virulence genes.

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1. Introduction Entomopathogenic fungi can kill their host by mechanical and chemical forces. Cuticle penetration and release of mycotoxin inside the hemocoel are the used techniques to overpower

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the host immune system. The use of entomopathogenic fungi is widely known in organic farming

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techniques and, nowadays, it's being incorporated as a major part in integrated pest management

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for conventional farming techniques to provide a unique approach to a sustainable agricultural.

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Beauveria spp. is a cosmopolite entomopathogenic fungus that is found in different species attacking a wide variety of insects (700 species in 15 orders) and mites (13 species)

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(Zimmerman, 2007). In recent years, taxonomic studies of entomopathogenic fungi have evolved from classical taxonomical and classification approaches based on conidiogenesis into a modern

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phylogenetic approach based on multilocus DNA sequencing. These loci include the intergenic

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nuclear block region and three protein-coding genes, the elongation factor-1alpha (TEF 1-α), the

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large subunit of the RNA polymerase II (RPB1), and the second major subunit RNA polymerase II (RPB2) (Rehner et al., 2011). Based on these sequencing results, twelve different species of

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the genus Beauveria with different host range, habitat, and geographical distribution were

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documented (Rehner et al., 2011). The importance of species identification has gained momentum over the recent years because studies of entomopathogenic fungi have progressed from simple observation of the pathogenicity into a deep understanding of the infection process. Although B. bassiana and B. brongniartii, are used as commercial mycoinsecticides for the biological control of pest insect, molecular studies measuring the expression of virulence genes of Beauveria genus are very limited. Virulence is the absolute quantifiable value of pathogenesis and it might be measured with a given lethal dose (LD) and lethal time (LT). However, this

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ACCEPTED MANUSCRIPT definition is not universal and several different factors can interfere with the virulence of the fungi like parasite host range, aggressiveness, infectivity, and fitness, which encompass environmental, ecological, and evolutionary interactions. More importantly, the combinatorial interaction of different genes and the physical interaction between their encoded protein products

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is the fundamental aspect that would describe virulence in a given time at a given place. This has

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been highlighted in recent studies whereby the sequencing of the whole genome of B. bassiana

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in showed that more than 2000 genes might be implicated in the pathogenicity (Xiao et al. , 2012) Despite these breakthroughs little is known about each bassiana species in the current

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global weather changing conditions which might affect gene expression by acquired epigenetic

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modifications. An indigenous strain of B. bassiana was found in the Tannourine cedars forest nature reserve naturally infecting the prenymphal stages of C. tannourinensis (Abdo et al.,

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2008). The cedar web-spinning sawfly is considered the main defoliator to the emblematic tree

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and its damage cause a state of stress on the cedars and induce the formation of summer buds, subjects of secondary infections. Furthermore, the cedars of Lebanon are protected areas and the

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establishment of a biological treatment should be considered to reduce the proliferation of this

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forest pest due to the climate change (Nemer and Nasr, 2004; Sattout and Nemer, 2008). Therefore, the main objective of this study is to investigate the expression of several genes

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involved in virulence of the indigenous B. bassiana and cross-compare it to the commercial

2. Material and methods 2.1 B. bassiana strains

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ACCEPTED MANUSCRIPT A commercial strain of B. bassiana (Srujan Nisarg, India) has been purchased in a formulation of 22% wettable powder. The Lebanese LTB01 strain initially originated from the corpses of cedar sawflies (C. tannourinensis) collected from the natural reserve in Tannourine.

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The strains were cultivated on Potatoes Dextrose Agar (PDA) medium at 25°C for 14

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days. The white colonies of B. bassiana that develop on the culture medium were rubbed by a

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sterile flamed loop while adding 15 to 20 mL of sterile water to prepare a suspension of conidia. Each petri dish was agitated for 1 min to ensure the dissociation of the conidia in the solution.

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The suspension of conidia was then filtered using an autoclaved funnel covered by 6-8 layers

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Cheesecloth to remove the fragments of mycelium. The solution was then poured into a Falcon tube. For each strain, the final volume obtained was about 10 mL. The concentration of the

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suspension solution was measured by Neubauer-improved bright line hemocytometer. Several

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dilutions were performed on the mother suspension to have a suitable concentration of the recommended dose which is 2x108 conidia/mL. Tween 80 (0.1% of the volume) was added to

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avoid the formation of clusters of conidia.

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The viability of the conidia in the solutions was assessed on (PDA) medium according to the protocol described by Goettel & Inglis (1997). A random volume of the conidia solution was

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pipetted and spread over a Petri dish containing the PDA medium and incubated at 26° C for 24 h. Conidia were examined under an optical microscope at 400 × magnification. All conidia with an apparent germ tube were considered viable. The counting was carried out at four different microscopic fields per petri dish in triplicate for each of the two strains of the entomopathogenic fungi. In each field, all conidia were counted and the percentage of germination was calculated. The Lebanese LTB01 strain of the entomopathogen fungus B. bassiana recorded a viability of

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ACCEPTED MANUSCRIPT 97% of the conidia while the Indian strain of this fungus recorded a viability of 98% of the spores.

2.2 Growth conditions The two Beauveria strains were grown in liquid minimal medium (MM) (Pontecorvo et

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al., 1953) without glucose and supplemented with arthropod cuticle (MM + cuticle) (Table 1).

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To obtain the cuticle, third-instar larvae of Galleria mellonella (Linnaeus, 1758) were used. The insects were dissected with a sterile scalpel to remove internal material, dried to a constant

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weight in an oven at 80-90°C, and then the exoskeleton was macerated in potassium tetraborate (1%). The resulting powder was sieved and stored frozen at -25°C. To obtain a cuticle

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suspension (1%), the cuticle powder was resuspended in an aqueous solution of potassium

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tetraborate (1%) and the mixture was subjected to flowing steam for 20 min (Andersen, 1980).

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The cuticle extract was then added to sterile liquid MM. Conidia with a concentration of 2x108 conidia/mL, harvested as described above, were inoculated in 20 mL of culture medium and

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incubated at 28°C within a 150-rpm shaker. In liquid cultures B. bassiana produce yeast-like propagules, namely, blastospores (Vega et al., 2003), which are vegetative cells analogous to the

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hyphal bodies formed naturally in the hemolymph. The media were collected at 24 h, 48 h and

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72 h after inoculation, immediately frozen in liquid nitrogen, and maintained for 24 h at -80°C for subsequent extraction of RNA. The entomopathogenic fungus Beauveria is known to produce its natural toxin after breaching the host cuticle. Knowing that insect hemolymph is solute-rich, Beauvericin and Bassianolide expressions were measured using PDB (Potato dextrose agar) as a growing medium. Strains inoculation, incubation, and storage were done as described above and the cultured media were collected at 24 h, and 72 h after inoculation.

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2.3 Total RNA isolation and cDNA synthesis

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Liquid cultures grown and collected as above were ground in liquid nitrogen. 150 mg of

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powdered sample was weighted in 2mL Eppendorf tube. Extraction of RNA was performed

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using Zymo Research, USA kit according to the manufacturer’s instructions. RNA was suspended in 50 µL DEPC-treated water. RNA concentration and purity were examined by

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Nanodrop based on 260/280 nm absorbance, and RNA integrity was verified by electrophoresis

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on a 1% agarose gel. Until further use, purified RNA was stored at -30°C. 2 µg of purified RNA were transcribed, using BIORAD’s kit, into cDNA using the manufacturer’s instructions

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(iScript™ Reverse Transcription Supermix for RT-qPCR, 1708840).

2.4 RT-qPCR

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The average value collected at 24 h after inoculation was considered as calibrator and

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average values collected at 48 h and 72 h after inoculation were considered as test values. SYBR® Green qPCR Sigma-Aldrich used to obtain qPCR products. qPCR reactions were

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performed in 25µL Eppendorf tubes. Each tube contained the following: 12.5 µL of qPCR SYBR, 1 µL each of the forward and reverse primers (each at 25 µM) (Macrogen, Korea), 9.5 µL nuclease-free water and 1 µL cDNA (20 ng/L cDNA in each sample). In order to ensure that qPCR reagents were free from contamination, negative controls (no DNA template) were included for each primer. The assays were done using a CFX96 Real-Time System (BIO-RAD). qPCR assays were made with the following protocol: 1.5 min activation/denaturation step at

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ACCEPTED MANUSCRIPT 95°C, followed by 40 cycles of 15s at 95°C, 30s at 60°C, and 30s at 72°C. Subsequently, the specificity of the amplicons was verified by melting curve analyses done at 72°C to 96°C. Two qPCR assays were performed in duplicate making it a total of 4 technical replicates per sample for three biological replicates, and mean values were calculated for final analysis. For reference,

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cyclophilin A was chosen (accession number HQ610831) due to its expression stability during

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different development stages, after exposure to hyperosmotic and temperature stress, and under

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different nutritional conditions (Zhou et al., 2012). Transcripts were amplified using forward and reverse primers listed in Table 2. To check amplification specificity, length of PCR products was

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confirmed on gel electrophoresis, in addition to the melt curve analysis. To calculate

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amplification efficiency, standard curves were constructed by plotting the log of the cDNA values against Cq values obtained over the range of dilutions of the template obtained during

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amplification of each dilution. Serial 10-fold dilutions were performed to cDNA samples and

1/slope]

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used as a template for RT-qPCR reactions. The reaction efficiency was calculated as E = 10[, using the slope of the curves Table 2. The relative differences in expression level of

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virulence genes in different samples were calculated using the 2-ΔΔCq (Livak) Method. First, all

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Cq values obtained by the target genes were normalized to those obtained by the reference genes for both the test sample and the calibrator sample. Second, ΔCq of the test sample obtained is

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normalized to the ΔCq of the calibrator. Finally, fold expressions were calculated according to Livak formulation 2-ΔΔCq .

2.5 Statistical analyses

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ACCEPTED MANUSCRIPT The data obtained by the experiments were analyzed by the statistical comparison test of means (ANOVA) using the software 16.0. Thus, we used, at the 5% threshold, the Tukey test, for the separation of means.

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3. Results

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An opportunistic pathogen, B. bassiana does not require any specialized route of entry

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towards susceptible host targets, and an array of depolymerases (proteases, chitinases), and secondary metabolites are sufficient and necessary in the infection process (Charnley & St Leger,

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1991; El-Sayed et al., 1993; Fuguet et al., 2004; Kirkland et al., 2005). These depolymerases and

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secondary metabolites are encoded by the following genes : Cdep1, Bbchit, Bbbeas, and Bbbsls. In addition, we opted to assess the expression of Hyd1, Hyd2, and Vlp4 which are produced in

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the spores and could account for their virulence based on gene inactivation (Zhang, et al 2011).

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We thus, optimized the RT_PCR conditions for each gene after ensuring RNA quality. The same melting temperatures were recorded for each target gene, indicating a unique specific

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amplification during the experiment. PCR amplification efficiency varied from 99.1 to 102.3%

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indicating suitable reaction conditions (Table 2). 3.1 Genes encoding cuticle-degrading enzymes

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Entomopathogenic fungi can produce several enzymes to breach the host cuticle. The degradation of cuticle component is mainly performed by proteases and chitinases (Charnley and St. Leger, 1991). Fang et al., (2005; 2008) proved the involvement of protease and chitinase in cuticle degradation where an overexpression and mixture of these two genes enhanced cuticle degradation and virulence of B. bassiana.

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ACCEPTED MANUSCRIPT In this present study, the Cdep1 (accession number: AY040532) gene was significantly induced at 24 h, 48 h and 72 h (F = 13.84, df = 4, P <0.05) in both B. bassiana strains. 1.08- and 1.56- fold changes were recorded at 48 h, and 72 h after inoculation of the LTB strain when compared to the calibrating expression at 24 h with no significative difference between the 2 test

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samples and the control sample (Figure 1). The relative expression of Cdep1 gene was induced

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by 1.5- and 2- fold after 48 h and 72 h respectively by the IND strain when normalized to the

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expression of the strain at 24 h. No significant difference was recorded between two test samples within each strain. However, a significant difference was notable between the IND strain

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expression at 72 h post-inoculation and the control (Figure 1).

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Different induction levels of Bbchit1 (accession number: AY145440) gene were recorded with both strains of the entomopathogenic fungi with no significant difference between strains

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and during different timing of the essay (F = 19.43, df = 4, P >0.05). The recorded fold

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expressions of the Bbchit1 gene were 1.04 and 1.43 for by LTB strain at 48 h and 72 h when normalized to the expression at 24 h respectively with no significant difference recorded between

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the two test samples and the control sample. An induction of Bbchit1 gene was also recorded

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with IND strain at 48 h and 72 h, recording 1.46- and 1.98- fold change when comparing it to the calibrator (Figure 1). A significant difference was notable between Bbchit1 induction at 72 h

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post-inoculation and the calibrator.

3.2 Expression of the gene encoding the intravacuolar protein A study held by Chu et al., 2017 revealed that a putative secretory protein localized in vacuoles (Vlp4) (accession number: EJP62569) of germinating conidia and hyphae plays several roles interfering with the infection process of entomopathogenic fungi. Essential roles of the

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ACCEPTED MANUSCRIPT VLP4 protein were summarized by blastospores formation, conidial maturation, and dimorphic transition. Different relative expressions were recorded from both strains of B. bassiana at 24, 48 and 72 h for Vlp4 gene (F = 96.71, df = 4, P <0.05). The Vlp4 gene was induced at 48 h by 7.04-

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fold change when compared to its expression at 24 hours for the LTB strain. This latter induction

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recorded a significant difference from the same gene expression at 72 h for the LTB strain where

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it was induced by 12.1-fold change comparing it with the expression of the IND strain at 24 h (Figure 1). The relative expression of the Vlp4 gene by the LTB strain recorded a significant

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difference when comparing it to the expression of the same gene by the IND strain at 48 h

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(Figure 1). The relative expressions at 48 h and 72 h by the IND strain were 3.24- and 4.87-fold

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recorded between the test samples.

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change respectively when comparing them to the calibrator gene with no significant difference

3.3 Host adhesion hydrophobins

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The Hyd1 (accession number: EF452344) and Hyd2 (accession number: EF520285)

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“hydrophobin genes” are responsible for the production of amphiphatic proteins, the principal constituents of the Rodlet layer involved in the hydrophobicity, adherence, and virulence of B.

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bassiana (Zhang et al., 2011).In this study, a significant difference was recorded between different expression level of Hyd1 gene by the LTB and IND strain during a different time of the test (F =15.17, df = 4, P <0.05). A repression of Hyd1 gene was noticed by the LTB strain. A 0.03- and 0.04-fold change was recorded at 48 h and 72 h after inoculation when comparing it to the calibrating test respectively with no significant difference between the two test samples of the LTB strain while a significant repression was recorded between sample test

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ACCEPTED MANUSCRIPT and the control (Figure 1). In contrast, an induction was recorded with the IND strain at 48 h of the test when comparing it to the calibrator where 1.37-fold change was recorded with a significant difference between the LTB and IND strain at 48 h. The IND Strain recorded a lower induction after 72 h where 1.02 fold change was recorded with no significant difference when

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comparing it to the same strain at 24 h and 48 h (Figure 1).

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The recorded expression levels of Hyd2 gene showed a significant difference between

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different strains of B. bassiana (F=3705, df = 4, P <0.05). A 0.06- and 0.02- fold repression were recorded with 48 h and 72 h respectively when comparing the LTB strain to the calibrator

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with no significant difference between the same strain at different time of the test; however, a

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significant difference was recorded between test samples and the control (Figure 1). In contrast, a significant difference was noticed when comparing the expression of Hyd2 gene at 48 h and 72 h

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within the IND strain of B. bassiana recording 0.96- and 0.44 fold change respectively. A

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same time of the test (Figure 1).

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significant difference was also noticed when comparing the two strain of B. bassiana within the

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3.4 Host killing mycotoxins

A study by Gillespie and Claydon, 1989 discovered that entomopathogenic fungi can kill

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its host by toxicosis. B. bassiana is known to produce a wide range of secondary metabolites during the infection process. Being the most produced mycotoxin, Beauvericin was first described for its insecticidal activity by Hamill et al., (1969). The second most famous mycotoxin, Bassianolide, was first discovered by Kanaoka et al., (1978). Studies by Xu et al., (2008; 2009) revealed that knock-out mutant of the two mentioned genes reduced the virulence of the entomopathogenic fungi.

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ACCEPTED MANUSCRIPT Bbbeas (accession number: EU886196) gene responsible for the expression of the secondary metabolite Beauvericin was highly induced at 72 h during the experiment with a significant difference recorded between different test samples at 72 h post-inoculation and the control (F=315.58, df = 2, P <0.05). The expression of Bbbeas gene at 72 h recorded 2122- and

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1020- fold change for the LTB and IND respectively when normalizing it to the calibrator gene

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at 24 h (Figure 1).

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The second gene responsible for the expression of another secondary metabolite Bassianolide Bbbsls (accession number: FJ439897) was also highly induced at 72 h during the

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experiment. A significant difference was recorded between the test sample, at 72 h post-

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inoculation for the LTB strain, and the control; However, this difference was not notable for the IND strain. 25.9- and 7.1- fold changes were recorded at 72 h of the LTB and IND strain

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respectively when normalizing it with the calibrator gene (Figure 1).

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4. Discussion

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Valero-Jiménez et al., (2016) provided an overview of our current knowledge of the genes contributing to virulence in B. bassiana. In this present study we discussed the expression

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of some genes involved in different infection steps of the entomopathogenic fungi.

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When B. bassiana are cultured in liquid medium supplemented with insect cuticle, they tend to produce a range of extracellular cuticle-degrading enzymes corresponding to the major components of insect cuticles; proteins, chitins, and lipids (St. Leger et al., 1986). During this experiment, all enzymes were induced even after 72 h since the beginning of the test proving that colloidal chitin was unable to produce catabolite repression (St. Leger et al., 1986). Cdep1 gene was induced in both strains of B. bassiana during different experimental times, indicating thus that the minimal medium supplemented with cuticle is suitable for in vitro testing of this gene

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ACCEPTED MANUSCRIPT expression level. Cq values of reference gene CypA were highly similar in all analyzed cDNA samples within both strains of B. bassiana during all experimental times. Amplification efficiency variation for all primers was less than 5% (99.1 and 101.2%) indication good conditions in experimental reactions. We thus hypothesize that the molecular signature for the

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two strains is indicative of differential virulent properties.

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Expression of the Cdep1 gene was found to gradually increase during the progression of

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the experiment for both strains of the entomopathogenic fungi but with no significant difference. However, knowing that the test samples were normalized to the expression level of the strain at

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24 h, it is hypothesized the Cdep1 gene was already up-regulated at 24 h for the two strains of B.

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bassiana explaining the low value of fold change at 48 h after the test and low values of Cq. 72 h after the inoculation of the strains, the Cdep1 gene is still up-regulated by both strains of B.

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bassiana indicating stability in the carbon sources in all test samples.

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St. Leger et al., (1986a, 1986b) have reported that within the insect exoskeleton the chitin is masked by the protein, therefore, making the chitinase an inducible enzyme. After the

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degradation of exoskeleton protein, the chitinase gene is usually induced because of the newly

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appeared chitin. In the present study, we find as well that the expression of the Bbchit1 gene is correlated with Cdep1. Expression of Bbchit1 gene was also found to gradually increase during

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the progression of the experiment for both strains of B. bassiana. Low values of fold change at 48 h is also hypothesized by high expression of this enzyme at the calibrator sample indicating a rapid production of the chitinase gene (less than 24 h) in minimal medium. A correlation was also found between the induction of the protease and the chitinase gene 72 h after the beginning of the treatment.

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ACCEPTED MANUSCRIPT For hydrophobin genes, Hyd1 gene was down-regulated by the LTB strain at 48 h and 72 h since the beginning of the test. A study by Jackson et al., (2010) showed that B. bassiana are able to produce different kind of infective propagules including aerial conidia and blastospores on solid and liquid culture medium respectively. Knowing that samples were inoculated in a

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liquid medium, down-regulation of the hydrophobin gene was expected as blastopores are

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hydrophilic conidia lacking the rodlet layer present in the aerial conidia. Unexpectedly, this

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down-regulation was not notable by the IND strain; Even though a lower fold change was recorded after 72 h when comparing it to 48 h after the inoculation of the IND strain, no

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significant difference was recorded between the two test samples. Fold value relatively close to 1

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indicates “no expression” of the mentioned gene when it is thoroughly down-regulated within the LTB strain. The expressions of the second hydrophobin gene Hyd2 were correlated with the

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expressions of Hyd1 gene by LTB strain where the Hyd2 gene was down-regulated after 48 h and

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72 h as it is involved in the constitution of the rodlet layer lacked in the blastospores. The Hyd2 gene, similarly to the Hyd1 gene, showed “no expression” by the IND strain after 48 h of the

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beginning of the test. However, a repression started to appear 72 h after the inoculation of the

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IND strain with a significant difference between the fold expression between the two test samples. The lower fold change values recorded by the IND strain at 72 h compared to the values

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recorded at 48 h could be hypothesized by the tendency of IND strain to produce blastospores. Moreover, the presence of the “rodlet layer” after inoculation of the strain on a liquid medium may be hypothesized by the lowest capacity of the latter strain producing “bald” blastospores when compared to the LTB strain. Results obtained by Chu et al., (2017) could be utilized to explain the significant difference of fold expression of the Vlp4 gene between LTB and IND strain. As described above,

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ACCEPTED MANUSCRIPT the LTB strain is hypothesized to have a higher production of blastospores when comparing it to the IND strain; therefore, a higher production of VLP4 protein which plays a vital role in the production of the blastospores is confirmed. Despite their vital role in the infection process, expression analysis studies of the

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secondary metabolites are very limited. In the present study, the production of mycotoxin by B.

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bassiana was genetically assessed for the first time. 72 h after the inoculation in the rich

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medium, LTB strain of the entomopathogenic fungi recorded 2122-fold change, a much higher value than in IND strain that recorded 1020- fold change with the same conditions. Expression

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analysis revealed the same results when it comes to Bassianolide production; Recorded fold

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change of Bbbsls gene by the LTB strain was higher than the IND strain. These results indicate a higher and faster production rate of the two most important mycotoxin by the LTB strain when

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comparing it to the IND strain. The higher and faster production of the secondary metabolite

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could be related to higher and faster virulence in vivo. As revealed, the enzymatic activity by the two strains is almost the same during different

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experimental time. However, LTB strain came superior with the production of blastospores,

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mycotoxins, and putative secretory protein.

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5. Conclusion

Expression analysis of the pathogenicity genes can provide thorough insights into hostfungi interactions. In this present study, we have developed a primary model for analysis and comparison for the most important genes involved in virulence. Despite having many more genes involved in the pathogenicity process, this model has revealed the ways of induction and repression of pathogen genes during the infection process. Advanced studies analyzing the

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ACCEPTED MANUSCRIPT expression of virulence genes during in vivo expression should be held measuring the virulence of entomopathogenic fungi against a specific host of interest. These should include the expression of additional genes indirectly involved in the virulence of entomopathogenic fungi like stress management, adaptation to the hemolymph, and nutrient assimilation genes. The

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development of such model in addition to the traditional application treatment of B. bassiana

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infective propagules could give us a better understanding of the infection process.

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ACCEPTED MANUSCRIPT Abbreviations Hyd1: Hydrophobin Hyd2: Hydrophobin Bbchit: Chitinase Cdep1: Subtilisin-like protease

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Bbbeas: Beauvericin toxin

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Bbbsls: Bassianolide toxin

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CypA: Cyclophilin A DNA: Deoxyribonucleic acid

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RNA: Ribonucleic acid cDNA: DNA complementary to RNA

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bp: base pair(s) Cq: Quantification cycle

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ANOVA: Analysis of Variance

RT-qPCR: Quantitative Real Time-polymerase chain reaction

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LD: lethal dose

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LT: lethal time

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DEPC: Diethyl pyrocarbonate

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ACCEPTED MANUSCRIPT Funding: This work was funded by the Lebanese National Council for Scientific Research (CNRS), grant

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name (CNRS-L/USEK) and CEDRE grant number 37349SA.

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ACCEPTED MANUSCRIPT References Abdo, C., Nemer, N., Nemer, G., Abou Jawdah, Y., Atamian, H., Kawar, N. S., 2008. Isolation of Beauveria species from Lebanon and evaluation of its efficacy against the cedar web-spinning sawfly, Cephalcia tannourinensis. BioControl. 53, 341–352.

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https://doi:10.1007/s10526-006-9062-0

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Andersen, S.O., 1980. Cuticular sclerotization. In: Miller TA (ed) Cuticle Techniques in

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Arthropods. Springer-Verlag, New York, pp 185-215.

Charnley, A.K., St Leger, R.J., 1991. The role of cuticle degrading enzymes in fungal

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pathogenesis in insects, in: Cole, G.T., Hoch, H.C. (Eds.), Fungal Spore Products and

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Pathogenesis. Academic Press, San Diego, CA,, pp. 267-286.

Chu, Z.J., Sun, H.H., Zhu, X.G., Ying, S.H., Feng, M.G., 2017. Discovery of a New

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Intravacuolar Protein Required for the Autophagy, Development and Virulence of Beauveria

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ACCEPTED MANUSCRIPT Beauveria bassiana during development, under changing nutrient conditions, and after exposure to abiotic stresses. Appl Microbiol Biot. 93, 679–685. https://doi.org/10.1007/s00253-011-35613. Zimmermann, G., 2007. Review on safety of the entomopathogenic fungi Beauveria

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bassiana and Beauveria brongniartii. Biocontrol Sci Techn. 17, 553-596.

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ACCEPTED MANUSCRIPT Table 1. Ingredients of growing medium for genetic expression

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6g 0.52g 0.52g 1.52g Traces 10g 1% 1L 6.5

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Ingredients Sodium nitrate Potassium chloride Magnesium sulfate Potassium di-hydrogen phosphate Iron and Zinc Dextrose Cuticle powder Water pH

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ACCEPTED MANUSCRIPT Table 2. PCR amplification primer sequence, amplicon size, and amplification efficiency.

Chit-R

TGATTGTCACGCCCTGAATA

Hyd1-F

ATGGTGGAAAGGATCTGCAC

Hyd1-R

GGTGGGAAAGAAGACCATCA

Hyd2-F

ACCATCTTCGCTACCCTCCT

Hyd2-R

GAGATCCAGGTCGCTGAGAA

BeauF

CTGGGGAGTAATGTCCTCTA

BeauR

AATCTTACGCGCAGTCTGGT

BassF

CGAACTCGACCTGATCCATT

BassR

TCGACGAACCTCTTCGACTT

ProtF

TCTGGCACTTCAGATGGTCA

ProtR

CCGTAGCGACAAAGTCCATT

IntraF IntraR

AGAGTCTCCTCCGCCATCTT

CypA-F

ATGGCTAACCCCAAGGTCTT

CypA-R

AACTGGGAGCCGTTGGTGTT

size (bp)

(%)

159

99.1

177

99.2

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ACCTGGACAATGGTCGTCTC

Amplification efficiency

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Chit-F

Amplicon

174

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Primer (5'-3')

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Gene

99.2

101.2

169

100.4

GTATCGTCCCTTGTCGTCGT

243

99.4

287

102.3

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226

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156

101.7

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ACCEPTED MANUSCRIPT Figure 1. Fold expression levels of Cdep1, Bbchit, Vlp4, Hyd1, Hyd2, Bbbeas, and Bbbsls genes in LTB and IND strains of Beauveria bassiana after 48 h and72 h of growth in minimal media.

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Means followed by distinct letters differ by the Tukey test at the 5% probability level.

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