전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Research article

Molecular Biology and Omics

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

1Laboratorio de Biotecnología Ambiental, Centro de Biotecnología Genómica-Instituto Politécnico Nacional, Reynosa 88710, México, 2University of Guanajuato, Life Science Division, Food Department, Irapuato, Guanajuato 36500, México

Received: April 24, 2014; Accepted: July 18, 2014

J. Microbiol. Biotechnol. 2014; 24(11): 1495-1502

Published November 28, 2014 https://doi.org/10.4014/jmb.1404.04044

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

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

References

  1. Bagga S, Hu G, Screen SE, St. Leger RJ. 2004. Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 324: 159-169.
    Pubmed CrossRef
  2. Barboza-Corona JE, Ortiz-Rodriguez T, de la Fuente-Salcido N, Ibarra J, Bideshi DK, Salcedo-Hernández R. 2009. Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis. Antonie Van Leuwenhoek. 96: 31-42.
    Pubmed CrossRef
  3. Bidochka MJ, Khachatourians GG. 1990. Identification of Beauveria bassiana extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanopus sanguinipes. J. Invertebr. Pathol. 56: 362-370.
    CrossRef
  4. Cha mplin FR, Cheung PYK, Pekrul S, Smith RJ, Burton RL, Grula EA. 1981. Virulence of Beauveria bassiana mutants for the pecan weevil. J. Econom. Entomol. 74: 617-621.
  5. Dhar P, Kaur G. 2010. Production of cuticle-degrading proteases by Beauveria bassiana and their induction in different media. Afr. J. Biochem. Res. 4: 65-72.
  6. Fang W, Feng J, Fan Y, Zhang Y, Bidochka MJ, St. Leger RJ, Pei Y. 2009. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana. J. Invertebr. Pathol. 102: 155-159.
    Pubmed CrossRef
  7. Freimoser FM, Screen S, Bagga S, Hu G, St. Leger RJ. 2003. Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology. 149: 239-247.
    Pubmed CrossRef
  8. Gillespie JP, Bateman R, Charnley K. 1998. Role of cuticledegrading proteases in the virulence of Metarhizium spp. for the desert locust, Schistocerca gregaria. J. Invertebr. Pathol. 71: 128-137.
    Pubmed CrossRef
  9. Raeder U, Broda P. 1985. Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbiol. 1: 17-20.
    CrossRef
  10. Rojas-Aveli zapa LI, Cruz-Camarillo R, Guerrero MI, RodriguezVazquez R, Ibarra JE. 1999. Selection and characterization of a proteo-chitinolytic strain of Bacillus thuringiensis, able to grow in shrimp waste media. World J. Microbiol. Biotechnol. 15: 261-268.
  11. Rosas-García NM, Villegas-Mendoza JM. 2008. Bionomics of a novel species of Argyrotaenia (Lepidoptera: Tortricidae) presents in Mexican avocado orchards. Acta Zool. Mex. Nueva Ser. 24: 129-137.
  12. Santi L, Beys da Silva WO, Berger M, Guimaraes JA, Schrank A, Vainstein MH. 2010. Conidial surface proteins of Metarhizium anisopliae: source of activities related with toxic effects, host penetration and pathogenesis. Toxicon 55: 874.
    Pubmed CrossRef
  13. Silva JC, Messias CL. 1986. Virulence of mutants and revertants of Metarhizium anisopliae var. anisopliae toward Rhodnius prolixus. J. Invertebr. Pathol. 48: 368-374.
    CrossRef
  14. St. Leger RJ, Charnely AK, Cooper RM. 1986. Cuticledegrading enzymes of entomopathogenic fungi: synthesis in culture on cuticle. J. Invertebr. Pathol. 48: 85-95.
    CrossRef
  15. St. Leger RJ, Charnely AK, Cooper RM. 1986b. Cuticledegrading enzymes of entomopathogenic fungi: mechanisms of interaction between pathogen enzymes and cuticle. J. Invertebr. Pathol. 47: 295-302.
    CrossRef
  16. St. Leger RJ, Joshi L, Bidochka MJ, Rizzo NW, Roberts DW. 1996. Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles. Appl. Environ. Microbiol. 62: 1257-1264.
    Pubmed
  17. St. Leger RJ, Joshi L, Bidochka MJ, Roberts DW. 1996. Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc. Natl. Acad. Sci. USA 93: 6349-6354.
    Pubmed CrossRef

Related articles in JMB

More Related Articles

Article

Research article

J. Microbiol. Biotechnol. 2014; 24(11): 1495-1502

Published online November 28, 2014 https://doi.org/10.4014/jmb.1404.04044

Copyright © The Korean Society for Microbiology and Biotechnology.

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

1Laboratorio de Biotecnología Ambiental, Centro de Biotecnología Genómica-Instituto Politécnico Nacional, Reynosa 88710, México, 2University of Guanajuato, Life Science Division, Food Department, Irapuato, Guanajuato 36500, México

Received: April 24, 2014; Accepted: July 18, 2014

Abstract

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

References

  1. Bagga S, Hu G, Screen SE, St. Leger RJ. 2004. Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 324: 159-169.
    Pubmed CrossRef
  2. Barboza-Corona JE, Ortiz-Rodriguez T, de la Fuente-Salcido N, Ibarra J, Bideshi DK, Salcedo-Hernández R. 2009. Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis. Antonie Van Leuwenhoek. 96: 31-42.
    Pubmed CrossRef
  3. Bidochka MJ, Khachatourians GG. 1990. Identification of Beauveria bassiana extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanopus sanguinipes. J. Invertebr. Pathol. 56: 362-370.
    CrossRef
  4. Cha mplin FR, Cheung PYK, Pekrul S, Smith RJ, Burton RL, Grula EA. 1981. Virulence of Beauveria bassiana mutants for the pecan weevil. J. Econom. Entomol. 74: 617-621.
  5. Dhar P, Kaur G. 2010. Production of cuticle-degrading proteases by Beauveria bassiana and their induction in different media. Afr. J. Biochem. Res. 4: 65-72.
  6. Fang W, Feng J, Fan Y, Zhang Y, Bidochka MJ, St. Leger RJ, Pei Y. 2009. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana. J. Invertebr. Pathol. 102: 155-159.
    Pubmed CrossRef
  7. Freimoser FM, Screen S, Bagga S, Hu G, St. Leger RJ. 2003. Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology. 149: 239-247.
    Pubmed CrossRef
  8. Gillespie JP, Bateman R, Charnley K. 1998. Role of cuticledegrading proteases in the virulence of Metarhizium spp. for the desert locust, Schistocerca gregaria. J. Invertebr. Pathol. 71: 128-137.
    Pubmed CrossRef
  9. Raeder U, Broda P. 1985. Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbiol. 1: 17-20.
    CrossRef
  10. Rojas-Aveli zapa LI, Cruz-Camarillo R, Guerrero MI, RodriguezVazquez R, Ibarra JE. 1999. Selection and characterization of a proteo-chitinolytic strain of Bacillus thuringiensis, able to grow in shrimp waste media. World J. Microbiol. Biotechnol. 15: 261-268.
  11. Rosas-García NM, Villegas-Mendoza JM. 2008. Bionomics of a novel species of Argyrotaenia (Lepidoptera: Tortricidae) presents in Mexican avocado orchards. Acta Zool. Mex. Nueva Ser. 24: 129-137.
  12. Santi L, Beys da Silva WO, Berger M, Guimaraes JA, Schrank A, Vainstein MH. 2010. Conidial surface proteins of Metarhizium anisopliae: source of activities related with toxic effects, host penetration and pathogenesis. Toxicon 55: 874.
    Pubmed CrossRef
  13. Silva JC, Messias CL. 1986. Virulence of mutants and revertants of Metarhizium anisopliae var. anisopliae toward Rhodnius prolixus. J. Invertebr. Pathol. 48: 368-374.
    CrossRef
  14. St. Leger RJ, Charnely AK, Cooper RM. 1986. Cuticledegrading enzymes of entomopathogenic fungi: synthesis in culture on cuticle. J. Invertebr. Pathol. 48: 85-95.
    CrossRef
  15. St. Leger RJ, Charnely AK, Cooper RM. 1986b. Cuticledegrading enzymes of entomopathogenic fungi: mechanisms of interaction between pathogen enzymes and cuticle. J. Invertebr. Pathol. 47: 295-302.
    CrossRef
  16. St. Leger RJ, Joshi L, Bidochka MJ, Rizzo NW, Roberts DW. 1996. Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles. Appl. Environ. Microbiol. 62: 1257-1264.
    Pubmed
  17. St. Leger RJ, Joshi L, Bidochka MJ, Roberts DW. 1996. Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc. Natl. Acad. Sci. USA 93: 6349-6354.
    Pubmed CrossRef