Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis.

Compartmentalization of metabolic pathways to particular organelles is a hallmark of eukaryotic cells. Knowledge of the development of organelles and attendant pathways under different metabolic states has been advanced by live cell imaging and organelle specific analysis. Nevertheless, relatively few studies have addressed the cellular localization of pathways for synthesis of fungal secondary metabolites, despite their importance as bioactive compounds with significance to medicine and agriculture. When triggered to produce sesquiterpene (trichothecene) mycotoxins, the endoplasmic reticulum (ER) of the phytopathogenic fungus Fusarium graminearum is reorganized both in vitro and in planta. Trichothecene biosynthetic enzymes accumulate in organized smooth ER with pronounced expansion at perinuclear- and peripheral positions. Fluorescence tagged trichothecene biosynthetic proteins co-localize with the modified ER as confirmed by co-fluorescence and co-purification with known ER proteins. We hypothesize that changes to the fungal ER represent a conserved process in specialized eukaryotic cells such as in mammalian hepatocytes and B-cells.

The adenylyl cyclase plays a regulatory role in the morphogenetic switch from vegetative to pathogenic lifestyle of Fusarium graminearum on wheat.

Cyclic 3',5'-adenosine monophosphate (cAMP) is a nucleotide derived from adenosine triphosphate that acts as a second messenger throughout all kingdoms. Intracellular cAMP levels are synthesized by a membrane-bound protein, the adenylyl cyclase. In order to analyze the function of this gene and the importance of cAMP in the life cycle of the cereal pathogen Fusarium graminearum, the adenylyl cyclase gene (FGSG_01234) was deleted by gene replacement (ΔFgac1). The ΔFgac1 mutant displayed a drastically reduced growth on agar medium which could be rescued by a cAMP analogon. Furthermore, the ΔFgac1 mutant was unable to produce perithecia on detached wheat nodes. However, artificial conditions like carrot agar allowed perithecia development. Pathogenicity towards wheat was drastically reduced in ΔFgac1 compared to the wild type. Point-inoculated spikelets showed only small lesions but no typical head blight disease symptoms. Fluorescence microscopy using dsRed-expressing strains revealed that the ΔFgac1 strain was unable to develop any complex infection structures like lobate appressoria and infection cushions. Instead, hyphal anastomosis occurs frequently. Scanning electron microscopy demonstrated the lack of fungal penetration. Hence, the formation of compound appressoria seems to be essential for infection of wheat. Hyphae on flower leaves produced huge amounts of new conidia, thereby circumventing the infection cycle. This abundant sporulation on wheat epidermis was not observed in wild type. Intriguingly, the Fgac1 deletion mutant was able to infect maize cobs as wild type, indicating that cAMP signaling is not important for maize infection. The ΔFgac1 mutant was unable to produce the mycotoxin deoxynivalenol both in vitro and during wheat infection. In this study, we show that cAMP signaling controls important cellular processes such as development of infection structures, pathogenicity, secondary metabolite production and sexual reproduction. For the first time, we show that cAMP regulates the switch from vegetative to pathogenic lifestyle of F. graminearum on wheat.

Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors.

Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H(2)O(2) treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses.