Magnaporthe appressoria: wt vs. bip1 deletion mutant. Pyricularia grisea

The identification of genes regulated by the transcription factor Bip1 during appressorial development. The rice blast fungus Magnaporthe grisea develops melanized appressoria with high turgor pressure that inject the fungus into epidermal host cells. A screen for M. grisea non-pathogenic mutants recovered mutant M763 with reduced penetration efficiency and infectious growth. Complementation analysis identified a gene inactivated in M763 that encodes a novel basic leucine zipper (bZIP) transcription factor named Bip1 (B-ZIP Involved in Pathogenesis1). Deletion of Bip1 by targeted replacement generated non-pathogenic mutants that are unable to infect rice or barley. These mutants develop melanized appressoria that are unable to penetrate intact or wounded host leaves. Bip1 mRNA was detected in fungal spores, appressoria and infected leaves but not in mycelia, while a BIP1:GFP fusion was detected only in nuclei of mature appressoria. Genome wide transcriptome analysis identified 44 genes down regulated in Δbip1:hph appressoria. Most of these genes (77%) are specifically expressed in appressoria and encode enzymes involved in secondary metabolism (11), secreted proteins and enzymes (18) and G-Protein Coupled Receptors (GPCRs) (5). Promoters of BIP1 down-regulated genes share a similar GCN4/bZIP DNA binding motif (TGACTC). Gel shift assays showed that the motifs from 6 of the 7 promoters tested bind to recombinant BIP1 in vitro, including MGG_08381.5 from the ACE1 locus. Mutation of BIP1 binding sites in the MGG_08381.5 promoter significantly reduced its appressorium specific expression. Molecular and biochemical studies confirmed that BIP1 controls the expression of a distinct set of M. grisea genes in the appressorium, revealing a novel regulatory network involved in early stages of fungal infection that activates a diverse gene expression program resulting in a “shock and awe” impact on the plant host. Overall design: Two condition experiment, wt P1.2 vs. delta bip1 at 24 h post-inoculation on Teflon. Three biological replicates, four technical replicates per biological replicate consisting of two slides plus two dye-swapped slides.