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Variations in oxygen concentration cause differential antioxidant response and expression of related genes in Beauveria bassiana a a   Paul Misael GARZA-LOPEZ , Gerardo SUAREZ-VERGEL , b a, Aida HAMDAN-PARTIDA , Octavio LOERA *

noma Metropolitana Iztapalapa, Departamento de Biotecnologıa, San Rafael Atlixco 186, Universidad Auto Col. Vicentina, C. P. 09340, Mexico D. F., Mexico b Universidad Aut onoma Metropolitana Xochimilco, Departamento de Sistemas Biologicos, Calz. del Hueso 1100, Col. Villa Quietud, C. P. 04960, Mexico, D. F., Mexico a

article info

abstract

Article history:

The entomopathogenic fungus Beauveria bassiana is widely used in pest biocontrol strate-

Received 28 October 2014

gies. We evaluated both the antioxidant response mediated by compatible solutes, treha-

Received in revised form

lose or mannitol, and the expression of related genes using oxygen pulses at three

17 December 2014

oxygen concentrations in solid state culture (SSC): normal atmosphere (21 % O2), low

Accepted 23 December 2014

oxygen (16 % O2) and enriched oxygen (26 % O2). Trehalose concentration decreased 75 %

Available online 9 January 2015

after atmospheric modifications in the cultures, whereas mannitol synthesis was three-

Corresponding Editor:

fold higher under the 16 % O2 pulses relative to normal atmosphere (100 and 30 mg mannitol

O. Loera

mg1 biomass, respectively). Confirming this result, expression of the mpd gene, coding for mannitol-1-P dehydrogenase (MPD), increased up to 1.4 times after O2 pulses. The expres-

Keywords:

sion of the bbrgs1 gene, encoding a regulatory G protein related to conidiation, was ana-

Compatible solutes

lysed to explain previously reported differences in conidial production. Surprisingly,

Differential gene expression

expression of bbrgs1 decreased after atmospheric modification. Finally, principal compo-

Entomopathogenic fungus

nent analysis (PCA) indicated that 83.39 % of the variability in the data could be explained

Oxidant conditions

by two components. This analysis corroborated the positive correlation between mannitol

Principal component analysis

concentration and mpd gene expression, as well as the negative correlation between conidial production and bbrgs1 gene expression. This study contributes to understanding of antioxidant and molecular response of B. bassiana induced under oxidant conditions. ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction The entomopathogenic fungus, Beauveria bassiana, is used worldwide in biological control as an alternative to chemical pesticides because of its low environmental impact and high specificity, and because insects rarely develop resistance to

it (Zimmermann 2007). Conidia are its most efficient infective propagules, and are produced mainly in solid state culture (SSC) on agricultural products and by-products such as rice  pez et al. 2012). (Neves & Alves 2000; Ye et al. 2006; Garza-Lo Beauveria bassiana is an aerobic organism that produces reactive oxygen species (ROS) through cellular metabolism such

* Corresponding author. Tel.: þ52 55 58 04 64 08; fax: þ52 55 58 04 64 07. E-mail address: [email protected] (O. Loera). http://dx.doi.org/10.1016/j.funbio.2014.12.012 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

 pez et al. P. M. Garza-Lo

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as mitochondrial respiration and NADPH oxidase activity (Aguirre et al. 2005). Cells operate both enzymatic and nonenzymatic antioxidant defence mechanisms, such as mannitol and trehalose (compatible solutes) biosynthesis, which prevent oxidation induced by ROS in biomolecules (Crowe et al. 1984; Smirnoff & Cumbes 1989; Singer & Lindquist 1998). In addition, oxidising states favour cytodifferentiation in fungal species of the genera Neurospora, Aspergillus, and Metarhizium (Miller et al. 2004; Aguirre et al. 2005). Recently, studies on the effect of oxidising states induced by O2 pulses in B. bassiana, Metarhizium anisopliae, and Isaria fumosorosea have shown strain-related differential effects on conidial production and on quality and infectivity of conidia  pez et al. 2012; (Tlecuitl-Beristain et al. 2010; Garza-Lo  ndez et al. 2014). Specifically, in B. bassiana, Miranda-Herna hypoxic pulses caused oxidative stress, increasing conidial production but with diminished conidial germination (Garza pez et al. 2012). Lo Quantification analysis of gene expression in entomopathogenic fungi has improved understanding of the infective processes (Fang & Bidochka 2006). In this sense, Wang et al. (2011) studied the relationship between mannitol synthesisrelated genes (mpd and mtd ) and multi-stress resistance in B. bassiana. The regulation of conidiation-related genes in filamentous fungi, including entomopathogenic fungi, is controlled by a G protein-mediated signalling pathway (Hamm 1998; Fang et al. 2007, 2008). Signalling through an a subunit (Ga) plays a role in regulating the balance between hyphal vegetative growth and conidiation. Regulatory G proteinsignalling (RGS) proteins interact with Ga, deactivating the signal and promoting the conidiation process (Wang et al. 2013). Disruption of orthologous genes cag8 and bbrgs1, which encode RGS proteins in M. anisopliae and B. bassiana respectively, reduced conidiation and virulence in these species (Fang et al. 2007, 2008). The aim of this study was to determine the effects of different concentrations of O2 pulses on the antioxidant response associated with trehalose or mannitol, in addition to the differential expression of related genes (mpd and bbrgs1) in B. bassiana.

Materials and methods Microorganisms The Bb 882.5 strain of Beauveria bassiana was used, previously isolated and described by Robledo-Monterrubio et al. (2009). This strain belongs to the fungal collection of the Universidad  noma Metropolitana Iztapalapa and was deposited in the Auto  xico, D. F., Me  xico) culture collection with the ENCB-IPN (Me identification name ENCB-MG-80. Propagation was carried out in 250 ml Erlenmeyer flasks containing 50 ml of modified Sabouraud maltose agar (2 %), previously sterilised at 15 PSI  pez et al. 2012). The flasks were incubated for 15 min (Garza-Lo for 8 d at 28  1  C.

Solid state culture Serological bottles (total volume 75 ml) were used as experimental units, each containing 5 g of pre-cooked rice sterilised

at 15 PSI for 15 min. Conidial suspensions were prepared, adding 20 ml of a sterile Tween 80 (Amresco, USA) (0.05 %) solution to the Erlenmeyer flasks used in the propagation. The conidial count was determined under a microscope (BM-180, Boeco, Germany) using a Neubauer chamber (Marienfeld-Superior, Germany), and the suspension was diluted to obtain a concentration of 5  106 conidia per millilitre (con ml1). Finally, 1 ml of this suspension was added to each bottle to obtain a final concentration of 1  106 conidia per gram of initial dry substrate (con gds1) and the necessary amount of a sterile solution of yeast extract (0.5 g l1) to obtain 40 % of initial moisture content.

Modified atmospheres Three atmospheric conditions were used: normal atmosphere (NA, 21 % O2), low oxygen (16 %) and enriched oxygen (26 %). The gaseous mixtures (16 % O2 and 26 % O2) were manufactured and standardised by the Praxair Company (Mexico). All experiments began with a pre-stationary phase in which the experimental units were loosely closed with cotton plugs to allow continuous gas exchange with the external environment. After 3 d of culture, hermetic rubber seals were placed on the bottles to be subjected to the 16 % and 26 % O2 pulses, then the air in the bottles was completely flushed and replaced with the appropriate atmospheric treatment. This procedure was carried out as previously described by Garza pez et al. (2012). Three replicates of each treatment were Lo sampled every 24 h. Biomass and conidial samples were collected after adding 20 ml of Tween 80 (0.05 %) to the serological bottles and stirring using a magnetic stirrer for 10 min.

Determination of trehalose and mannitol concentration Compatible solutes were extracted according to the methods described by Hallsworth & Magan (1994), adding 1 ml of biomass and conidial suspension, obtained as described previously, into 1.5 ml Eppendorf tubes. Samples were centrifuged using 0.5 mm microfilters (Millipore, USA) for 5 min at 8000 g (MiniSpin, Eppendorf, Germany) to eliminate protein residues, then analysed using high performance liquid chromatography (HPLC), with an Aminex HPX-42A column (Bio-Rad, USA) attached to an Agilent 1260 chromatograph (Agilent, USA). Retention times were 15.7 min and 21.5 min for trehalose and mannitol, respectively. Concentrations of both solutes, trehalose and mannitol, were expressed as micrograms of solute per milligram of biomass (mg solute mg1 biomass). Biomass was determined by difference of dry weight obtained by filtration, using 0.45 mm nylon membranes (Millipore, USA). According to the results obtained, we chose to analyse the differential expression of the encoding gene for mannitol-1-phosphate dehydrogenase (mpd gene).

Differential expression of mpd and bbrgs1 genes In addition to mpd gene expression analysis, expression of bbrgs1 gene was also determined. Samples were collected at 3, 4 and 6 d of culture, corresponding to the beginning of atmospheric modification, 24 h later and the time by which Beauveria bassiana had shown signs of oxidative stress as reported by

Gene expression is altered by oxygen concentrations

 pez et al. (2012). Biomass obtained from experimental Garza-Lo units was ground with liquid nitrogen using a mortar and a pestle. Total RNA (tRNA) was isolated using an RNeasy Plant Mini Kit (cat. # 74904, Qiagen, Germany) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesised with a QuantiTect Reverse Transcription Kit (cat. # 205311, Qiagen, Germany). Concentration and purity were determined using a NanoDrop 2000 (Thermo Scientific, USA). Finally, a quantitative polymerase chain reaction (qPCR) was used to determine differential expression of mpd and bbrgs1 genes; the gpd gene, encoding a glyceraldehyde3-phosphate dehydrogenase, was selected as the housekeeping gene (Fang & Bidochka 2006). Reaction mixes were prepared using a Rotor-Gene Probe PCR Kit (cat. # 204372, Qiagen, Germany). Likewise, specific primers and probes were designed for mpd, bbrgs1 and gpd genes using OligoArchitect online software (SigmaeAldrich, USA) and manufactured by IDT (USA) (Table 1). PCR was performed in a Rotor-Gene 6000 (Corbett Life Science, Australia). Data obtained were normalised with the 2DDCT method (Livak & Schmittgen 2001).

Statistical analysis Analysis of variance (ANOVA) All experiments were performed in triplicate and also repeated twice. Data obtained were analysed using one-way ANOVA and the TukeyeKramer multiple comparisons test, with statistical significance set at P < 0.05 (SPSS 21 for Windows, IBM, USA).

Chemometric analysis Exploratory methods are used to observe tendencies in data which might indicate relationships between samples and variables (Brereton 2003). Principal component analysis (PCA) was used to investigate patterns in the standardised data (Ramette 2007) (XLSTAT Version 2014.3.04 for Windows, Addinsoft, USA). The first component (PC1) represents the largest portion of the variability of the data and successively the remaining components (PC2, PC3, etc.) represent the remaining variability. The nomenclature used describes sampling time and O2 concentration (e.g. 4-26, refers to samples obtained at day 4 under 26 % O2 treatment).

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Results Trehalose and mannitol concentration Trehalose concentration in the Beauveria bassiana cultures at different culture times is shown in Fig 1. Concentration of this compatible solute decreased at least 75 % after atmospheric modification, since this disaccharide was not detected under hypoxic conditions (16 % O2) and under the 26 % O2 treatment concentrations were significantly lower than those in the normal atmospheric O2 treatment (P < 0.05). Furthermore, variations in trehalose concentration throughout the culture in the 26 % O2 treatment were minimal. At the beginning of atmosphere modification (day 3), trehalose was not present. In contrast, mannitol concentration increased with modification of O2 concentration after the first atmospheric exchange (day 4) until the end of culture (Fig 2). The maximum concentration of this polyol was observed on day 7 in all treatments. At the end of culture (day 8), mannitol concentration in the 16 % O2 treatment (100 mg mannitol mg1 biomass) was three times greater than the value obtained in the normal atmosphere (NA) treatment (30 mg mannitol mg1 biomass; P < 0.05). In order to explain these differences observed only on mannitol concentration, as a result of modification of O2 concentration, a possible differential expression of a mannitol synthesis-related gene was analysed (mpd gene).

Differential expression of mpd and bbrgs1 genes Expression of the mpd gene, presented as normalised data, is shown in Fig 3. Atmospheric modifications caused an increase in expression relative to normal conditions. Overall, cultures subjected to enriched treatment (26 % O2) showed significantly

Table 1 e List of qPCR primers. Primer name mpd forward primer mpd reverse primer mpd probea bbrgs1 forward primer bbrgs1 reverse primer bbrgs1 probea gpd forward primer gpd reverse primer gpd probea

Sequence (50 -30 )b GCTACGAGGTCGTATTTG GGCAGTGCTAATCTCTTC CTCGTCCTCACAGCCTCCAT CCCAAAGGATCCCTCAAG TCGGCAGACTCAATGAAG CCACCACAACAACCACCACATT GAGGTCGTTTCCACTGAC GCGACGTAGGAGATAAGG ACGGCAACACTAACTCCTCCAT

a TaqMan probes labelled with 50 FAM dye and 30 IBF quencher with ZEN internal quencher (IDT, USA). b All primers were newly designed.

Fig 1 e Trehalose concentration in the Bb 882.5 strain in different O2 concentrations (Black filled bars: normal atmosphere, 21 % O2; open bars: 16 % O2; grey filled bars: 26 % O2). Letters distinguish significantly different values on each day (Tukey: P < 0.05). Error bars represent standard deviations.

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Fig 2 e Mannitol concentration in the Bb 882.5 strain in different O2 concentrations (Black filled bars: normal atmosphere, 21 % O2; open bars: 16 % O2; grey filled bars: 26 % O2). Letters distinguish significantly different values on each day (Tukey: P < 0.05). Error bars represent standard deviations.

were no significant differences among modified atmospheres in mannitol concentration (Fig 2). Elsewhere, modification of O2 concentration has been associated with different conidiation patterns in Beauveria bassiana; a 32 % increase in conidial yield was obtained under hypoxia pulses (16 % O2), while no changes were detected un pez der 26 % O2 compared with normal atmosphere (Garza-Lo et al. 2012). The bbrgs1 gene has been related to conidiation in B. bassiana. The expression of bbrgs1 decreased from the beginning of atmospheric exchanges (Fig 4). At day 6 of culture under hypoxic conditions, expression of this gene was significantly lower than the other treatments (P < 0.05). Furthermore, bbrgs1 gene expression progressively decreased during the culture after atmospheric pulses. This is not related to the differences in conidial production obtained with the  pez et al. 2012) since at day 4; consame treatments (Garza-Lo idiation was lower in the normal atmospheric treatment, whereas conidial production was similar in all treatments at day 6 of culture.

Principal component analysis

greater mpd gene expression compared with both other treatments (P < 0.05). There were temporal differences in the relationships among treatments in mpd gene expression and concentration of mannitol. On day 4 the differences among treatments in expression of the mpd gene were similar to differences in mannitol concentration. However, at day 6 with 26 % O2 treatment, the expression of the mpd gene was significantly greater than the other treatments (Fig 3), while there

The PCA method allowed investigation of possible patterns among variables to explain these findings. The analysis indicated that 83.39 % of the variability can be explained by two components PC1 (63.78 %) and PC2 (19.61 %). Mannitol concentration was positively correlated with differential expression of the mpd gene (Fig 5). A negative correlation between conidia production and bbrgs1 gene expression was confirmed. In addition, Fig 5 shows the distribution of sampling times (D) and corresponding variables. The correlation between mpd gene expression and mannitol concentration was stronger at days 4 and 6 with modified atmospheres (referred to as 4-16, 4-26, 6-16 and 6-26), confirming data presented previously.

Fig 3 e Normalised relative expression of mpd gene in the Bb 882.5 strain in different O2 concentrations (Black filled bars: normal atmosphere, 21 % O2; open bars: 16 % O2; grey filled bars: 26 % O2). Letters distinguish significantly different values on each day. (Tukey: P < 0.05). Error bars represent standard deviations.

Fig 4 e Normalised relative expression of bbrgs1 gene in the Bb 882.5 strain in different O2 concentrations (Black filled bars: normal atmosphere, 21 % O2; open bars: 16 % O2; grey filled bars: 26 % O2). Letters distinguish significantly different values on each day (Tukey: P < 0.05). Error bars represent standard deviations.

Gene expression is altered by oxygen concentrations

Fig 5 e Principal components analysis (PC1 vs PC2; 83.39 % of accumulated variance explained) of the determined variables in the Bb 882.5 strain. Open triangles represent the different sampling times and O2 concentrations.

Conversely, the modification of O2 concentration negatively affected both bbrgs1 gene expression and trehalose production (Fig 5).

Discussion Trehalose and mannitol concentrations are useful parameters to determine whether such solutes are produced in response to an oxidative state, and their subsequent capacity to prevent biomolecule oxidation. Trehalose aids stabilisation of proteins and lipids to avoid damage produced by different abiotic stresses (Crowe et al. 1984; Singer & Lindquist 1998; Benaroudj et al. 2001; Elbein et al. 2003). Trehalose concentration increased in Beauveria bassiana under thermal stress conditions (Liu et al. 2009). Likewise, Gocheva et al. (2006, 2009) observed a similar effect in many Penicillium species induced by lower temperatures. The effect of temperature, pH and age of culture on trehalose production has been evaluated in some species of entomopathogenic fungi, including B. bassiana (Hallsworth & Magan 1996). Results from those studies are not consistent with our data, since oxidative stress induced by different O2 concentration pulses decreased trehalose concentration (Fig 1), suggesting that this disaccharide was hydrolysed in order to promote other physiological processes (Thevelein 1984; Singer & Lindquist 1998). In contrast, mannitol is able to scavenge free radicals and is considered as an antioxidant (Smirnoff & Cumbes 1989; Jennings et al. 1998; Ruijter et al. 2003). Modifying temperature and pH increased mannitol concentration in B. bassiana and Metarhizium anisopliae, and different carbon sources caused variations in mannitol concentration (Hallsworth & Magan 1994, 1996). In contrast, mannitol concentration decreased in B. bassiana under long thermal stress conditions (Liu et al.

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2009). These studies suggest that polyol biosynthesis varies as a response to diverse induced stresses; in our study O2 pulses increased mannitol concentration which may play a role in preventing protein damage under conditions where oxidative stress and lipid oxidation has been corroborated  pez et al. 2012). (Garza-Lo Quantification of mpd and bbrgs1 gene expression helps to determine the relationship between the response to pulses of different oxygen concentrations and increased mannitol concentration and conidia production, respectively. Wang et al. (2011) observed a decrease in mannitol concentration, virulence, and resistance to different types of stress in B. bassiana after suppression of mpd and mtd genes (both genes involved in mannitol biosynthesis). In addition, mpd gene suppression caused stronger effects on those parameters evaluated by Wang et al. (2011). Another report suggested that the most important mannitol synthesis pathway in fungi is one that involves the MPD enzyme  le €z et al. 2007). (mannitol-1-phosphate dehydrogenase) (Ve The MPD enzyme of B. bassiana has been sequenced and characterised (Wang et al. 2010). Recently, it was observed that the expression of mpd and mtd genes is inherent to growth stage in Aspergillus niger (Aguilar-Osorio et al. 2010). The results obtained in our research support the idea that mpd gene expression is closely related to oxidative state induced by different atmospheric concentrations. Nevertheless, there are no reports linking oxidative stress and polyol synthesis-related genes in entomopathogenic fungi. In addition, differential expression of the bbrgs1 gene was not related to conidia production profiles previously reported  pez et al. 2012). Nevertheless, Fang et al. (2008) ob(Garza-Lo served that this gene is related to conidiation and thermotolerance of B. bassiana strain ARSEF 252. Likewise, the orthologous gene cag8 of M. anisopliae was also implicated in processes of conidiation, virulence, and hydrophobin synthesis (Fang et al. 2007). This apparent inconsistency with our data suggests that alternative regulatory mechanisms may be implicated when B. bassiana is subjected to oxidant states under SSCs. The bbrgs1 gene encodes an RGS protein whose function could be different to RGS proteins in other fungi (Fang et al. 2008). In fact, culture medium and conditions induced different conidiation regulatory mechanisms (PalmaGuerrero et al. 2010). In addition, bbrgs1 and cag8 genes have been phylogenetically characterised and are closely related to the flbA gene of Aspergillus nidulans (Fang et al. 2007, 2008). Furthermore, Liu et al. (2013) observed that the transcriptional factor Msn2, orthologous in B. bassiana, is involved in conidiation as well as in virulence and multi-stress response. For a gene to have a key role in conidiation, the temporal expression pattern should be consistent with conidial profiles (Wu et al. 2008). PCA showed that atmospheric modification induced positive correlation between mannitol concentration and expression of mpd gene (Fig 5), although no clear pattern among different treatments or sampling times was observed. The PCA approach has suggested correlation among growth and infectivity variables in many B. bassiana strains (MontesinosMatıas et al. 2011). However, there are few reports on using this statistical tool to enhance understanding of the multifactorial responses of entomopathogenic fungi.

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Conclusions Beauveria bassiana increased mannitol production and expression of the mpd gene under both hypoxic and hyperoxic conditions. However, the opposite was found for trehalose biosynthesis. Furthermore, expression of the bbrgs1 gene was not related to conidial yields previously reported, suggesting that B. bassiana may have alternative regulatory mechanisms for the production of conidia under the conditions analysed. This study supports the idea that the oxidative state, induced by O2 pulses under SSC, caused different antioxidant and genetic responses which can be considered in the production of conidia by B. bassiana aimed at biocontrol strategies.

Acknowledgements This study was financed by the PROMEP project UAM-PTC-447 and the National Council for Science and Technology (CONACYT) for the project CB-2010-01-152420. We wish to thank  pez for her technical assistance in chemoJosefa Espitia-Lo metric analysis.

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Variations in oxygen concentration cause differential antioxidant response and expression of related genes in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is widely used in pest biocontrol strategies. We evaluated both the antioxidant response mediated by co...
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