Nanoscale enrichment of the cytosolic enzyme trichodiene synthase near reorganized endoplasmic reticulum in Fusarium graminearum.

Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms.

CbCTB2, an O-methyltransferase is essential for biosynthesis of the phytotoxin cercosporin and infection of sugar beet by Cercospora beticola.

BACKGROUND: Cercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beets (Beta vulgaris) worldwide. Cercosporin, a light-inducible toxin, is essential for necrosis of the leaf tissue and development of the typical leaf spots on sugar beet leaves. RESULTS: In this study we show that the O-methyltransferase gene CTB2 is essential for cercosporin production and pathogenicity in two C. beticola isolates. We established a transformation system for C. beticola protoplasts, disrupted CTB2, and transformed the Δctb2 strains as well as a wild type strain with the DsRed reporter gene. The Δctb2 strains had lost their pigmentation and toxin measurements demonstrated that the Δctb2 strains were defective in cercosporin production. Infection of sugar beets with the wild type and Δctb2 DsRed strains showed that the deletion strain was severely impaired in plant infection. Histological analysis revealed that the CTB2-deficient isolate cannot enter the leaf tissue through stomata like the wild type. CONCLUSIONS: Taken together, these observations indicate that cercosporin has a dual function in sugar beet infection: in addition to the well-known role in tissue necrosis, the toxin is required for the early phase of sugar beet infection.

Fusarium graminearum forms mycotoxin producing infection structures on wheat.

BACKGROUND: The mycotoxin producing fungal pathogen Fusarium graminearum is the causal agent of Fusarium head blight (FHB) of small grain cereals in fields worldwide. Although F. graminearum is highly investigated by means of molecular genetics, detailed studies about hyphal development during initial infection stages are rare. In addition, the role of mycotoxins during initial infection stages of FHB is still unknown. Therefore, we investigated the infection strategy of the fungus on different floral organs of wheat (Triticum aestivum L.) under real time conditions by constitutive expression of the dsRed reporter gene in a TRI5prom::GFP mutant. Additionally, trichothecene induction during infection was visualised with a green fluorescent protein (GFP) coupled TRI5 promoter. A tissue specific infection pattern and TRI5 induction were tested by using different floral organs of wheat. Through combination of bioimaging and electron microscopy infection structures were identified and characterised. In addition, the role of trichothecene production for initial infection was elucidated by a ΔTRI5-GFP reporter strain. RESULTS: The present investigation demonstrates the formation of foot structures and compound appressoria by F. graminearum. All infection structures developed from epiphytic runner hyphae. Compound appressoria including lobate appressoria and infection cushions were observed on inoculated caryopses, paleas, lemmas, and glumes of susceptible and resistant wheat cultivars. A specific trichothecene induction in infection structures was demonstrated by different imaging techniques. Interestingly, a ΔTRI5-GFP mutant formed the same infection structures and exhibited a similar symptom development compared to the wild type and the TRI5prom::GFP mutant. CONCLUSIONS: The different specialised infection structures of F. graminearum on wheat florets, as described in this study, indicate that the penetration strategy of this fungus is far more complex than postulated to date. We show that trichothecene biosynthesis is specifically induced in infection structures, but is neither necessary for their development nor for formation of primary symptoms on wheat.