Performing Department
(N/A)
Non Technical Summary
Rhizopus microsporus is emerging as a model for studying the basic biology of the early-diverging fungal subphylum Mucoromycotina, an understudied group containing significant post-harvest spoilage agents, phytopathogens, and opportunistic human pathogens. Unlike most other eukaryotic lineages that rely on 5-methylcytosine (5mC) DNA modifications, early-diverging fungi such as the Mucoromycotina employ N6-methyladenine (6mA) as the predominant epigenetic modification. Bacteria also use 6mA as their predominant epigenetic mark and Mucoromycotina fungi including R. microsporus frequently harbor highly coevolved bacterial endosymbionts. The absence of cognate adenine methyltransferases in plants and animals and the shared epigenetic program between these fungi and their endosymbionts inspired my hypotheses that 1) adenine methylation is a novel biocontrol target for early-diverging fungal pathogens and 2) 6mA is integral to the symbiosis of R. microsporus and its bacterial endosymbiont, Mycetohabitans. To address my objectives, I will first develop an in vitro screening strategy of small-molecule libraries for inhibition of the adenine methyltransferase complex in R. microsporus to identify candidate biocontrol agents. Second, I will compare the genomes and 6mA profiles of ~30 R. microsporus isolates that naturally harbor Mycetohabitans and ~30 that do not in order to identify candidate symbiosis factors regulated by 6mA. Adenine methyltransferase deletion mutants in Mycetohabitans will further assess the role of 6mA in symbiosis. By defining the role of a unique eukaryotic epigenetic modification in an agriculturally relevant fungal-bacterial symbiosis and through assessing adenine methylation as a novel biocontrol target, I strive to inform management strategies and the basic biology of the Mucoromycotina fungi.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The goals of this project are 1) to evaluate N6-methyladenine (6mA) as a novel biocontrol target for early-diverging fungal pathogens and food-spoilage agents of the subphylum Mucoromycotina using Rhizopus microsporus as a model organism and 2) investigate the role of 6mA in the symbiosis of R. microsporus and its bacterial endosymbiont Mycetohabitans. The motivation for this investigation stems from the distinct epigenomes of early-diverging fungi and the high prevalence of bacterial endosymbionts found within fungi of this group. Unlike most eukaryotic lineages that are enriched in 5-methylcytosine (5mC) DNA modifications, early-diverging fungi such as the Mucoromycotina employ N6-methyladenine (6mA) as the predominant epigenetic modification, similar to bacterial genomes. The paucity of 6mA modifications in the genomes of plants and animals coupled with its significance in early-diverging fungal pathogens and food-spoilage agents make it a promising target for biocontrol strategies. In addition, the shared use of 6mA modifications between fungi in the Mucoromycotina and their bacterial endosymbionts may underlie the molecular basis of these symbioses. To address these hypotheses, I will screen a small-molecule collection for inhibition of the R. microsporus adenine methylation machinery to identify compounds with the potential for controlling R. microsporus as a plant pathogen and post-harvest food spoilage agent. I will also generate genomes and genome-wide methylation data for ~60 R. microsporus strains that both do and do not harbor Mycetohabitans endosymbionts. By comparing the 6mA profiles of the genomes of fungi with and without endosymbionts, I aim to identify candidate symbiosis factors that are regulated by 6mA. I will also generate adenine methyltransferase mutants of Mycetohabitansand assess their ability to reestablish a functional symbiosis with their original host fungus to further test the hypothesis that 6mA is significant to symbiosis maintenance.
Project Methods
Assess small-molecule collection for inhibition of the Rhizopus microsporus methylation complex with a high-througput in vitro methylation reactionsPrepare DNA from ~60 R. microsporus strains for PacBio sequencing in collaboration with the JGI followed by population-level analyses of the adenine methylation landscape of these genomesGenerate adenine methyltransferase mutants of Mycetohabitans, the bacterial endosymbiont of R. microsporus followed by determining if these mutants can reform a functional symbiosis with their original fungal host.