Marroquin-Guzman M, Wilson RA.

PLoS Pathog. 2015 Apr 22;11(4):e1004851. doi: 10.1371/journal.ppat.1004851. eCollection 2015.

http://www.ncbi.nlm.nih.gov/pubmed/25901357

Abstract

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

Fig 1. ASD4 is involved in nitrogen assimilation.

(A) Δasd4 mutant strains were grown for 10 days on minimal media (MM) containing 1% (w/v) glucose (GMM) and 10 mM of the indicated sole nitrogen sources. L-isomers were used throughout this study. (B) After 10 days, Δasd4 growth was not further attenuated on GMM containing low concentrations of ammonium as the sole nitrogen. (C) Strains were grown for 10 days on MM containing 10 mM L-glutamine as the sole carbon and nitrogen source. WT = wild type Guy11 isolate, Δasd4 ASD4GFP = Δasd4 complementation strain expressing Asd4 fused to GFP. Bars are added as a visual aid to the plate images in (B) and (C) to demarcate colony size. doi:10.1371/journal.ppat.1004851.g001