The Golgin Protein RUD3 Regulates Fusarium graminearum Growth and Virulence

Abstract

Golgins are coiled-coil proteins that play prominent roles in maintaining the structure and function of the Golgi complex. However, the role of golgin proteins in phytopathogenic fungi remains poorly understood. In this study, we functionally characterized Fusarium graminearum golgin protein RUD3, a homolog of ScRUD3/GMAP-210 in Saccharomyces cerevisiae and mammalian cells. Cellular localization observation revealed that RUD3 is located in the cis-Golgi. Deletion of RUD3 caused defects in vegetative growth, ascospore discharge, deoxynivalenol (DON) production, and virulence. Moreover, the Δrud3 mutant showed reduced expression of tri genes and impairment of the formation of toxisomes, both of which play essential roles in DON biosynthesis. We further used GFP-tagged SNARE protein SEC22 (SEC22-GFP) as a tool to study the transport between endoplasmic reticulum (ER) and Golgi, and observed that SEC22-GFP was retained in the cis-Golgi in the Δrud3 mutant. RUD3 contains the coiled coil (CC), GRAB-associated 2 (GA2), GRIP-related Arf binding (GRAB), and GRAB-associated 1 (GA1) domains; which except for GA1, are indispensable for normal localization and function of RUD3, whereas only CC is essential for normal RUD3–RUD3 interaction. Together, these results demonstrate how the golgin protein RUD3 mediates retrograde trafficking in the ER-to-Golgi pathway and is necessary for growth, ascospore discharge, DON biosynthesis, and pathogenicity in F. graminearum. IMPORTANCE Fusarium head blight (FHB) caused by the fungal pathogen Fusarium graminearum is an economically important disease of wheat and other small grain cereal crops worldwide, with limited effective control strategies are available. A better understanding of the regulation mechanisms of F. graminearum development, DON biosynthesis and pathogenicity is therefore important for development of effective control management of this disease. Golgins are attached via their extreme carboxy terminus to the Golgi membrane and involved in vesicle trafficking and organelle maintenance in eukaryotic cells. In this study, we systematically characterized a highly conserved Golgin protein RUD3, and found that it is required for vegetative growth, ascospore discharge, DON production, and pathogenicity in F. graminearum. Our findings provide a comprehensive characterization of the Golgin family protein RUD3 in plant pathogenic fungus, which could help to identify a new potential target for effective control of this devastating disease.

The Endoplasmic Reticulum Cargo Receptor FgErv14 Regulates DON Production, Growth and Virulence in Fusarium graminearum

“…During this process, Erv14, as an ER cargo receptor, regulates cargo protein loading during ER exit [ 56 ]. FgRud3 is an early-Golgi localization protein required for toxisome generation and virulence in F. graminearum [ 27 ]. Whether FgErv14 affects the localization of FgRud3 was determined by monitoring FgRud3-GFP signals in PH-1 and the Δ Fgerv14 mutant strains.…”

“…In the former, FgRud3-GFP colocalized with mCherry-FgSed5 ( Figure 8 A), the early-Golgi marker, whereas in the mutant lacking FgERV14 , it was instead diffusely distributed throughout the cytosol ( Figure 8 A), implying that the normal localization of FgRud3 depends on FgErv14. We then used late secretory pathway markers (GFP-FgSnc1-PEM: mutant V42A and M45A) to investigate whether FgErv14 is involved in the late secretory pathway, which mediates trafficking from late Golgi to PM [ 27 ]. In WT PH-1 hyphae, GFP-FgSnc1-PEM accumulated on the PM, whereas in hyphae of the Δ Fgerv14 mutant, although the signal was mostly localized to the PM, multiple green dots were also detected in the cytoplasm ( Figure 8 B) colocalized with the late Golgi marker FgKex2-tdTomato, indicating that GFP-FgSnc1-PEM was partly trapped in the late Golgi.…”

“…Vesicular transport from the ER to the Golgi in the early secretory pathway is necessary for growth and development in eukaryotes, including phytopathogenic fungi [ 27 , 34 , 36 , 37 , 59 ]. Erv14, an ER cargo receptor, circulates between the ER and Golgi apparatus and participates in the direct selection of COPII cargo proteins [ 38 , 43 , 45 , 46 , 47 ].…”

“…Each fungal strain expressing fluorescence-tagged proteins from plasmids and conidia stained with calcofluor white (CFW) were examined via fluorescence microscopy using confocal microscopy (LSM 880 NLO system; Zeiss, Oberkochen, Germany) or an upright fluorescence microscope (Eclipse Ni-U; Nikon) [ 27 ]. At least 10 different microscopic fields were observed for each sample with one or two channels (Zeiss confocal microscopy: 488 nm for GFP and 561 nm for mCherry/tdTomato.…”

“…TRI6 and TRI10 encode transcriptional regulators to control DON biosynthesis by regulating the TRI gene cluster expression [ 23 , 24 ]. In addition to coding products of these TRI genes, the proteins involved in vesicle transport have been reported as essential regulators of DON biosynthesis, which play critical roles in the pathogenesis of phytopathogenic fungi [ 25 , 26 , 27 , 28 , 29 , 30 ]. However, the regulatory mechanisms of these proteins are different.…”