Sfp-Type 4′-Phosphopantetheinyl Transferase Is Indispensable for Fungal Pathogenicity

Abstract

In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (a-aminoadipate reductase [AAR]) and secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTasedeficient mutants (Dppt1). Dppt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of secondary metabolites produced by wild-type and Dppt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Dppt1 strains. Rarely formed appressoria of Dppt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Dppt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Daar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.

Infection Structure-Specific Expression of -1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of -Glucan-Triggered Immunity in Maize

“…Interestingly, several appressoria of class I RNAi strains that did not explode ( Figure 4C, white arrow; Figure 4F), but they developed hyphae reminiscent of biotrophic primary hyphae ( Figure 4C, black arrow). Hyphae developing from appressoria of class I RNAi strains on the plant surface had a diameter of 7.8 6 3.2 µm, which is in good agreement with and statistically not different (P > 0.05) from diameters of 8.1 6 3.2 µm measured for biotrophic primary hyphae formed in planta (Politis and Wheeler, 1973;Horbach et al, 2009). As tight adhesion of appressoria is required for directed growth of the penetration peg into the plant cell wall, failure in penetration and development of primary hyphae on the cuticle is likely due to compromised adhesion.…”

“…Appressoria of RNAi strains of C. graminicola, in contrast with vegetative hyphae, did not hypermelanize, and leakage of melanin from the appressorial cell wall reported here suggests that an intact b-1,3-glucan polymer network is essential for incorporation and linking of melanin to the cell wall. However, insufficient melanization of the appressorial wall of RNAi strains is unlikely to cause lack of rigidity, as melanin-deficient mutants of C. graminicola (Horbach et al, 2009), C. lagenarium (Kubo et al, 1991), and M. oryzae (Chida and Sisler, 1987) did not show increased elasticity.…”

“…Infection structure differentiation of the wild-type and RNAi strains was induced on polyester, glass, or onion epidermis as described (Horbach et al, 2009), with 0.15 M KCl added as an osmolyte.…”

“…Maize leaf segments and onion epidermal layers were inoculated with 10-mL droplets containing 10 4 conidia in 0.01% (v/v) Tween 20. Wound inoculation was performed as described (Horbach et al, 2009). Leaf segments were incubated in sealed Petri dishes (diameter of 9 cm) in a BOD 400 incubator (Uni Equip) in darkness at 25°C.…”

Hexose Transporters of a Hemibiotrophic Plant Pathogen

“…The ascomycete C. graminicola is a hemibiotrophic fungus and, therefore, switches from an initial biotrophic growth phase during initial host colonization to a necrotrophic phase that leads to the destruction of the host (1,36). This developmental switch is paralleled by a transition from apoplastic growth to the feeding on cytoplasmic contents after breaching the host's plasma membranes and, thus, is also attended by a change in the available carbon sources and their concentrations.…”