Imaging Gene Expression Dynamics in Pseudomonas fluorescens In5 during Interactions with the Fungus Fusarium graminearum PH-1

Hennessy, Rosanna C.; Stougaard, Peter; Olsson, Stefan

doi:10.21769/bioprotoc.3264

Cited by 1 publication

(1 citation statement)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recordings was made through the bottom of the plate and a lid was kept on to avoid drying of the plate. The plate reader was set up to produce one big image for the whole plate that could be exported as a TEXT image and imported into the freeware ImageJ to produce a TIFF Z-stack that can be measured for the fluorescence development over time in any defined area (Hennessy et al ., 2017, 2019). The fluorescence increase from the hyphae of each agar plug could then be analyzed and compared.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary conserved multifunctional nitric oxide synthesis proteins responding to bacterial MAMPs are located at the endoplasmic reticulum

Zheng

Ipcho

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Nitric oxide is known to be produced by most Eukaryotic organisms although the NO production mechanism is only known for animals (Canovas et al., 2016). Mammals have a set of different nitric oxide synthases (NOSs) with basically similar mechanism for producing NO under different circumstances (Velayutham and Zweier, 2013; Yu et al., 2014). NO-production is characteristically induced as part of innate immunity reactions to bacterial Microbial Associated Molecular Patterns (MAMPs). We knew from our previous work that the plant pathogen Fusarium graminearum quickly upregulate genes characteristic for an innate immune response so we set out to test if it also produces NO as part of this response and find the responsible genes if it does. We found that F. graminearum produces NO in response to bacterial MAMPs and that the NO production system must be similar in fungi as in mammals where NO is activated by a homo-dimerization of nitric oxide synthase proteins (NOSs) containing an N-terminal cytochrome P450 domain (CYP) and a C-terminal NADPH dependent cytochrome P450 reductase (NCP) domain. The electrons are transferred from the C-terminal NCP domains to the CYP domain in the paired protein to produce NO. We could not find a candidate NOS in the fungus. Thus, we tested the hypothesis that an NCP-protein similar to the NCP part of a NOS is brought together with a CYP-protein to produce NO in another way than the dimerization of a classic NOS. We found that in F. graminearum NO is produced by an FgNCP and an FgCYP located to the endoplasmic reticulum membrane where both proteins are predicted to be N-terminally attached by a transmembrane or embedded hydrophobic alpha helix. Deletion of any of these proteins lowered pathogenicity to wheat and stopped NO-production. Knockout of the FgNCP also completely blocked deoxynivalenol synthesis needed for pathogenicity indicating that the FgNCP also delivers electrons to another CYP (TRI4) located at the ER. The FgCYP we found to be involved in NO-productions is the same or similar to proteins involved in Eukaryote sterol synthesis (CYP51) reducing lanosterol on the way to the final main sterols different for different Eukaryotes. Lanosterol enriched membranes are known to be inhibited in endocytosis and we found that the deletion of the FgCYP producing NO also completely stopped endocytosis. We further tested consequences of these indications of more than one function and suggest the CYP-protein is most likely an FgCYPNO,ERG involved in both NO and ergosterol synthesis and the NCP is involved in NO, trichothecene and ergosterol synthesis, an FgNCPNO,TRI,ERG. The two proteins shown here to be responsible for NO production in F. graminearum are both highly conserved in Eukaryotes from amoeba to human and homologues are likely candidates for the production of NO in many other eukaryotes including mammals. The multiple functions of these proteins can be part of the explanation for the links between chronic inflammation, sterols and blood pressure in human.

show abstract

Section: Methodsmentioning

confidence: 99%