November 2014  Vol. 24  No. 11 J. Microbiol. Biotechnol. (2014), 24(11), 1495–1502 http://dx.doi.org/10.4014/jmb.1404.04044 Research Article jmb Review Correlation between pr1 and pr2 Gene Content and Virulence in Metarhizium anisopliae Strains Ninfa M. Rosas-García 1 * , Osvaldo Ávalos-de-León 1 , Jesús M. Villegas-Mendoza 1 , Maribel Mireles-Martínez 1 , J. E. Barboza-Corona 2 , and J. C. Castañeda-Ramírez 2 1 Laboratorio de Biotecnología Ambiental, Centro de Biotecnología Genómica-Instituto Politécnico Nacional, Reynosa 88710, México 2 University of Guanajuato, Life Science Division, Food Department, Irapuato, Guanajuato 36500, México Introduction Metarhizium anisopliae is a deuteromycete that belongs to family Moniliaceae. Owing to the great number of insect species that it attacks, it has become an important entomopathogenic fungus. M. anisopliae has been studied widely as a model to understand virulence and pathogenicity processes against insect pests since many years ago. Such studies involve the relation with the host that includes adhesion, germination, appressorium formation, penetration, colonization, and pathogen release. An important part of the study of the initial pathogenic process has been focused on proteins with enzymatic activities that intervene in the degradation of the insect cuticle. It has been considered since past decades that the main M. anisopliae virulence factors lie in such proteins. In the penetration process, once the appressorium is formed, enzymes such as proteases, chitinases, lipases, phospholipases, esterases, phosphatases, and others degrade the insect cuticle. Such enzymes lead the way for the fungus to penetrate the insect cuticle and obtain nutrients from the hemolymph, causing an imbalance in the insect due to the production of different metabolites synthesized by the fungus [6, 7]. As insect cuticle contains a chitin filament matrix with lipids embedded in proteins, protease enzymes degrade or weaken the physical barrier between the insect and the environment [16]. Among many enzymes that participate in this process, proteases are considered the most important enzymes for cuticle degradation. Pr1 proteases, coded by pr1 genes, are subtilisin-like enzymes, and Pr2 proteases, coded by pr2, are trypsin-like enzymes. These enzymes are two different types of proteases called isoforms as they can be distinguished Received: April 24, 2014 Revised: June 12, 2014 Accepted: July 18, 2014 First published online July 22, 2014 *Corresponding author Phone: +52-899-9243627, ext. 87721; E-mail: nrosas@ipn.mx, ninfarosasg@yahoo.com.mx pISSN 1017-7825, eISSN 1738-8872 Copyright © 2014 by The Korean Society for Microbiology and Biotechnology Metarhizium anisopliae is a widely studied model to understand the virulence factors that participate in pathogenicity. Proteases such as subtilisin-like enzymes (Pr1) and trypsin-like enzymes (Pr2) are considered important factors for insect cuticle degradation. In four M. anisopliae strains (798, 6342, 6345, and 6347), the presence of pr1 and pr2 genes, as well as the enzymatic activity of these genes, was correlated with their virulence against two different insect pests. The 11 pr1 genes (A, B, C, D, E, F, G, H, I, J, and K) and pr2 gene were found in all strains. The activity of individual Pr1 and Pr2 proteases exhibited variation in time (24, 48, 72, and 96 h) and in the presence or absence of chitin as the inductor. The highest Pr1 enzymatic activity was shown by strain 798 at 48 h with chitin. The highest Pr2 enzymatic activity was exhibited by the 6342 and 6347 strains, both grown with chitin at 24 and 48 h, respectively. Highest mortality on S. exigua was caused by strain 6342 at 48 h, and strains 6342, 6345, and 6347 caused the highest mortality 7 days later. Mortality on Prosapia reached 30% without variation. The presence of subtilisin and trypsin genes and the activity of these proteases in M. anisopliae strains cannot be associated with the virulence against the two insect pests. Probably, subtilisin and trypsin enzyme production is not a vital factor for pathogenicity, but its contribution is important to the pathogenicity process. Keywords: Entomopathogen, enzymes, virulence, pathogenicity, protease, infection