Molecular Dialogues between Early Divergent Fungi and Bacteria in an Antagonism versus a Mutualism

Abstract

Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.IMPORTANCE Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

Keywords: Mycetohabitans; Rhizopus microsporus; cell wall remodeling; innate immunity; reactive oxygen species.

FIG 1

Positions of the nonhost Rm ATCC 11559 and the previously cured host Rm ATCC 52813 relative to Mycetohabitans sp. B13 endobacteria isolated from the host ATCC 52813 at the time of harvest for the RNA-seq experiment before contact and after physical contact. (A) The nonhost interacting with Mycetohabitans precontact. (B) The host interacting with Mycetohabitnas precontact. (C) The nonhost interacting with Mycetohabitans after contact. (D) The host interacting with Mycetohabitans after contact. White arrowheads indicate sites of bacterial inoculation, and black arrowheads point to sites of fungal inoculation.

FIG 2

ROS output quantification in the nonhost Rm ATCC 11559 and the previously cured host Rm ATCC 52813 cocultivated with and without Mycetohabitans sp. B13 endobacteria isolated from the host ATCC 52813. (A) The nonhost interacting with Mycetohabitans. (B) The nonhost grown without Mycetohabitans. (C) The host interacting with Mycetohabitans. (D) The host grown without Mycetohabitans. In panels A to D, culture plates were stained with yellow, water-soluble NBT, which is reduced by superoxide radicals to blue, water-insoluble formazan, indicating ROS accumulation; white arrowheads show sites of Mycetohabitans or mock inoculation; black arrowheads point to sites of Rm inoculation. (E) Mean RGB color intensity ratios between portions of fungal colonies proximal and distal to the site of Mycetohabitans or mock inoculation. Means are the values from 9 or 10 replicate culture plates. Error bars indicate 1 standard error of the mean. P values from Student's t test are shown next to comparisons.

FIG 3

Hypotheses describing molecular dialogues between early divergent fungi and bacteria in a mutualism versus an antagonism before and after physical contact. The presented hypotheses were formulated on the basis of changes in gene expression patterns described in this study and by Lastovetsky et al. (24), shown in orange. Bacterial cells are represented as purple ovals, and fungal mycelia are depicted in green (host) and brown (nonhost).