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

Abstract

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.

Use of Ecologically-and Evolutionary Relevant Transcriptomic Data to Infer Functions of Fungal Pathogen Gene Orthologs Important for Limiting Fungal Stresses Caused by Interacting Host Plants and Bacteria

“…Shows in red genes and processes dominating during maintenance if expression is normalized for growth using the His2b gene [10] in plant pathogens highlights especially maintenance needed to counteract plant ROS defenses and ROS generated as NO by non-self-recognition fungal innate immunity. CPA=MoCPA1 [19] and FgCPA1 (this study), ArgLy=MoArgLy [20] and FgArgLy (this study), CYP(NO,ERG)=FgCYP(NO,ERG) [17] and MoCYP(NO,ERG) (this study), ATG8=MoATG8 [21] and FgATG8 [12], PARP=FgPrp (this study) and MoPrp (this study), ADE4=MoADE4 [15] and FgADE4 (this study), NOD=FgNOD1 and FgNOD2 [17] and MgNOD and (this study), finally catalases Catalase=FgCAT1-5 (this study) and MoCAT1&3 (this study). For more info information on these genes see Table 2.…”

“…Arginine is also heavily used as the main source together with oxygen for production of nitric oxide (NO) that is a ROS produced in fungal innate immunity. It is triggered both during the transition between biotrophy to necrotrophy and when the fungus is exposed to bacterial MAMPs (microbial associated molecular patterns) [17]. Together these, through evolutions, very conserved genes fit into a conceptual model for how these genes are likely to be differentially expressed during different phases of the plant colonization by the pathogen.…”

“…However, it is in addition possible to normalize for growth using the His2B expression as indicative for DNA-synthesis to focus on the relative gene expression needed for only cell maintenance [10]. Using a large number of transcriptomes from previous papers [10,17], we set out to test the hypothesis that that the stress weighted network (Fig. 1B) should be most active just before penetration and in the transition between biotrophy to necrotrophy when plant defenses are activated.…”

“…For F. graminearum and M. oryzae we used 64 respective 47 transcriptomes sampled at different times post infection [10]. For F. graminearum we in addition also tested the activity of the genes of interest under non-growing conditions short time (<4h) after challenging with purified bacterial MAMPs in a dataset of 113 transcriptomes [17,22]. Exposure to bacterial MAMPs triggers NO production and should mainly be reflected as an increased response of the stress weighted network (Fig.…”

“…Exposure to bacterial MAMPs triggers NO production and should mainly be reflected as an increased response of the stress weighted network (Fig. 1B) with increasing expression of the cytochrome p450 gene (CYP(NO,ERG)) that is the gene mainly responsible for the intrinsic NO production with accompanying ROS stress [17].…”