Genetics and Molecular Biology, 38, 1, 86-92 (2015) Copyright © 2015, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/S1415-475738138120140236 Research Article Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis Mariele Porto Carneiro Leão1, Patricia Vieira Tiago1, Fernando Dini Andreote2, Welington Luiz de Araújo3 and Neiva Tinti de Oliveira1 1Departamento de Micologia, Universidade Federal de Pernambuco, Recife, PE, Brazil. 2Departamento de Ciência do Solo, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, SP, Brazil. 3Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil. Abstract The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to differ- ent growth conditions. The identification and characterization of these proteases can provide insight into how the fun- gus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different cul- ture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially ex- pressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher ex- pression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions. Keywords: entomopathogen, Diatraea saccharalis, quantitative RT-PCR, expression pattern. Received: August 5, 2014; Accepted: October 30, 2014. Introduction Metarhizium are collectively able to infect a broad range of In Brazil, sugarcane monoculture forms the basis of insects (Samuels et al., 1989) and are adapted to life in the the sugar export and biofuel industries (Alves et al., 2008). root rhizosphere (Roberts and St. Leger, 2004). However, The sugarcane borer Diatraea saccharalis Fabricius is con- little is known about the molecular and physiological mecha- sidered one of the major sugarcane pests in the Americas. nisms involved in their adaptation to different growth con- Due to its cryptic lifestyle, conventional control measures ditions, and this lack of understanding has prevented the by deploying chemical insecticides targeting the larvae are development of new strategies to improve their effective- ineffective. ness in biological pest control (Zhang et al., 2011). Entomopathogenic fungi are used in biological pest The taxonomy of Metarhizium was reassessed based control as a promising alternative to chemical pesticides on multigenic phylogenetic analysis covering the tef-1 gene due to a number of advantages, including lower environ- a mental impact, lower costs, higher specificity and lesser (translation elongation factor 1- ) rpb1 (large subunit of risk of development of resistance (Samuels et al., 1989; RNA polymerase II) rpb2 (second largest subunit of RNA Frazzon et al., 2000). Species that belong to the genus polymerase II) and b-tub (b-tubulin). This revaluation led to the recognition of different Metarhizium species: M. anisopliae (Mestchnikoff) Sorokin, M. guizhouense Send correspondence to Mariele Porto Carneiro Leão. Departa- Q.T. Chen & H.L. Guo, M. pingshaense Q.T. Chen & H.L. mento de Micologia, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves s/no, Cidade Universitária, 50670-420 Recife, Guo, M. acridum (Driver and Milner) Bischoff, Rehner & PE, Brazil. E-mail: [email protected]. Humber, M. lepidiotae (Driver and Milner) Bischoff, Leão et al. 87 Rehner & Humber and M. majus (Johnston) Bischoff, Growth conditions for expression analysis of the Rehner & Humber (Bischoff et al., 2009). pr1A gene A large and diverse range of enzymes and toxins have Analysis of the pr1A gene expression pattern was been identified in several studies that are thought to be criti- performed after growth of the two fungi species in YPD cal for the ability of the fungus to infect diverse groups of medium (0.2% yeast extract, 1% peptone, 2% dextrose), in insects and ticks, and to grow in different types of substrate liquid minimal medium (MM) (Pontecorvo et al., 1953) (Freimoser et al., 2003, 2005; Wang et al., 2005). In partic- without glucose and supplemented with 1% casein (MM + ular, subtilisin protease PR1A is the predominant protein casein), and in liquid MM without glucose and supple- produced during degradation of insect cuticle (Bagga et al., mented with 1% (w/v) insect cuticle (MM + cuticle). To ob- 2004). Thus, the gene encoding this protein (pr1A) is inves- tain the cuticle, we used third-instar larvae of tigated for its use in the development of advanced engi- D. saccharalis. The insects were crushed to remove inter- neered biopesticides (St. Leger et al., 1989). nal material, dried to a constant weight in an oven at 80 °C, and then the exoskeleton was macerated. The resulting Identifying genes that are up- or down-regulated in powder was sieved and stored frozen at -25 °C. To obtain a response to a given host insect or growth condition should cuticle suspension (1%), we resuspended the cuticle pow- increase our understanding of the genetic mechanisms in- der in an aqueous solution of potassium tetraborate (1%) volved in host specificity and adaptation (Pathan et al., and subjected the mixture to flowing steam for 20 min 2007; He et al., 2012; Luo et al., 2013; Jin et al., 2014). M. (Andersen, 1980). The cuticle extract was then added to anisopliae has been reported to have a large host range of sterile liquid MM. over 200 insect species (Samuels et al., 1989). In contrast, Conidia were harvested in 0.01% Tween 80 aqueous M. acridum has a very limited host-range and is only known solution, and the conidia suspen-sion was filtered through to attack orthopteran insects (Bischoff et al., 2009). A more glass wool to remove mycelia. Conidia (2 x 108 coni- thorough understanding of this behavioral flexibility may dia/mL) were inoculated in 20 mL of culture medium as de- be obtained by comparatively studying the two species, and scribed above and incubated as shake cultures at 150 rpm, such studies should contribute to the development of more 28 °C. The mycelia were collected at 24 h and 72 h after in- effective pest control. Thus, the objectives of this study oculation, immediately frozen in liquid nitrogen, and main- were to analyze the expression of the pr1A gene in M. tained for 24 h at -80 °C for subsequent extraction of RNA. anisopliae and M. acridum during growth in different cul- The experimental design followeda2x3randomized fac- ture conditions and during the pathogenic process of torial scheme (two fungal strains + three culture medium) D. saccharalis infection. We then went on to correlate the for a total of six treatments with two replicates each. expression of pr1A with the pathogenicity of M. anisopliae and M. acridum, against D. saccharalis. Preparation of insects for expression analysis of the pr1A gene Third-instar larvae of D. saccharalis were infected by Materials and Methods immersion for 1 min in a conidia suspension of2x108 conidia/mL. Each infected larva was then placed in a Petri Fungal culture and identification dish containing a small piece of sugarcane stalk, and the plates were incubated at 28 °C. The process of fungal Metarhizium anisopliae URM 4921, originally iso- pathogenesis is commonly divided into the following five lated from Mahanarva pos-ticata Stal from Brazil, and M. phases: uninfected insect, 20 h after infection, dead in- acridum URM 4412, originally isolated from Austracnis fected insect, emergent mycelia from the insect cadaver, guttulosa Walker from Australia, were obtained from the and insect cadaver mummified with conidia. The insects mycological collection of the Department of Mycology, collected in the phases described above were immediately Federal University of Pernambuco (URM-UFPE). Cultures frozen in liquid nitrogen and maintained for 24 h at -80 °C were grown on potato dextrose agar at 28 °C for 12 days to for subsequent RNA extraction. Two biological replicates obtain conidia. For confirmation of the species identity the were performed for each phase analyzed. Eight insects partial sequence of tef-1 gene (translation elongation factor were used per biological replicate. 1-a) was amplified and sequenced. The primers used for amplification and sequencing were designed for the EF-1a Total RNA isolation and cDNA synthesis intron region: EF1T (5’-ATGGGTAAGGARGACAAG The frozen samples of mycelia grown in different cul- AC) and EF2T (5’-GGAAGTACCAGTGATCATGTT) ture media and the insect samples described above were (Bischoff et al., 2009). Sequence data were manually ad- ground in liquid nitrogen. For each sample, 100-150 mg of justed by Staden software (Staden et al., 1998) and com- powdered sample was placed in a cooled 2-mL tube. RNA pared to the GenBank database using BLASTn (Altschul et was extracted with Trizol reagent (Invitrogen) according to al., 1990). the manufacturer’s instructions. RNA was suspended in 88 pr1A gene expression in Metarhizium 50 mL DEPC-treated water. The purity of the total RNA expression ratios were calculated using a mathematical was determined based on the absorbance at 260/280 nM, model that includes an efficiency correction for real-time and RNA integrity was verified by electrophoresis in a 1% qPCR efficiency of the individual transcripts (Pfaffl, 2001).