Requirement of a Mitogen-Activated Protein Kinase for Appressorium Formation and Penetration of Insect Cuticle by the Entomopathogenic Fungus Beauveria bassiana

Abstract

Beauveria bassiana is an important insect-pathogenic fungus that invades insects by direct penetration of the host cuticle. To delineate the molecular mechanisms involved in fungal infection, a mitogen-activated protein kinase (MAPK) gene, Bbmpk1, which encodes a YERK1 family MAPK was isolated and characterized. Targeted gene disruption of Bbmpk1 resulted in a complete loss of virulence when applied topically to host insects but did not affect growth of the fungus when conidia were injected directly into the hemocoel. Hyphae of the mutant strain growing in the insect hemocoel were unable to penetrate the cuticle growing outwards and consequently failed to sporulate on the cadaver surface. These data suggest that BbMPK1 is essential for penetration of the insect cuticle both from the outside and from the inside-out in order to escape and disperse from the host. Inactivation of BbMPK1 also caused a significant decrease in fungal adhesion to insect cuticles and eliminated their ability to form appressoria. In order to identify downstream genes regulated by BbMPK1, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Thirty-one genes screened from the SSH library were determined to be expressed in the wild-type strain but either significantly reduced or not expressed in the mutant. Ten genes showed high or medium similarity to known protein encoding genes, including proteins involved in cell surface hydrophobicity, lipid metabolism, microtubule dynamics, mitochondrial electron transport, chromatin remodeling, transcription, rRNA processing, small nucleolar RNA accumulation, oxidation of aldehydes, translation, and likely other cellular processes.

The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana

“…As mentioned, both Bbhog1 and Bbmpk1 (Fus3/Kss1 orthologs) mutants are also impaired in virulence, however, Bbmpk1 mutants were impaired when applied topically but were similar in virulence to the wild-type parent when injected into the hemoceol of target insects (although they failed to sporulate normally on the insect cadaver) (Zhang et al, , 2010. All three B. bassiana MAPKs effect conidial yield, but while DBbhog1 cells also displayed decreased spore viability and reduced hydrophobicity similar to DBbslt2 mutants, in the former mutant strain expression levels of the hydrophobin genes (hyd1 and hyd2) was decreased by more than 95% whereas they appeared unaffected in the DBbslt2 strain (decreased hydrophobin expression was also seen in DBbmpk1 mutants).…”

“…While characterization of any sexual development in B. bassiana is lacking to date, inactivation of any of the other two MAPKs of B. bassiana thus far characterized, i.e. Bbmpk1 and Bbhog1, homologous to Fus3/Kss1p and Hog1p family of MAPKs, respectively, also (with respect to Bbslt2) resulted in significant reductions in conidial production, suggesting that in some fungi these pathways may converge on a common downstream conidiation controlling regulatory point (Zhang et al, , 2010. Further examination of crosstalk and coordination between MAPKs is warranted, and although heterotrimeric guanine nucleotide exchange proteins (G-proteins) are known to regulate some MAPK pathways, a link between the heterotrimeric complex and the Fus3/Kss1 and/ or Slt2 pathway in sporulation has not yet been reported.…”

“…Bbslt2 gene containing its upstream and downstream sequences (3012 bp) was amplified with the primers RC1 and RC2 (introducing HindIII site in both the primers, see Supplementary Table 1) using PrimeSTARTM HS DNA polymerase (TaKaRa). After being confirmed by sequencing, the resultant PCR product was digested with HindIII and inserted into the same site of vector pK2ben (Zhang et al, 2010) to form pK2ben-Bbslt2. This plasmid was mobilized into A. tumefaciens AGL-1 for fungal transformation (Fang et al, 2004).…”

“…Typically, the fungal conidia adhere to the target insect cuticle, germinate across the surface, and begin the process of penetrating the cuticle (Holder and Keyhani, 2005;Zhang et al, 2011b). Penetration of the insect cuticle occurs via a combination of mechanical pressure and cuticle-degrading enzymes, principally proteases and chitinases (Clarkson and Charnley, 1996;Zhang et al, 2010). Once inside the insect hemocoel, the penetrating hyphae differentiate into free-floating single cell propagules known as in vivo hyphal bodies that display distinct morphological and biochemical properties Wanchoo et al, 2009).…”

“…Bbhog1 targeted knockout mutants were sensitive to osmotic, temperature, and oxidative stress; displayed defects in conidial germination, appressorium formation and adhesion (to insect cuticles), and were reduced in virulence in insect bioassays . Targeted inactivation of Bbmpk1 reduced the ability of the fungus to penetrate insect cuticles, decreased appressorium formation, and reduced adhesion and virulence (Zhang et al, 2010). Any effects on stress responses, however, remain unknown.…”