Intraspecific adaptive response of Periconia macrospinosa, a ubiquitous root-associated fungus, to persistent salinity stress provides evidence for its adaptive variability and high-osmolarity glycerol pathway mediated adaptive response

Abstract

Root associated fungi are critical to environmental success of their plant hosts. Whether through their interactions with hosts, nutrient recycling, decomposition, or even contribution to soil structure, soil-dwelling fungi play a pivotal role in environmental preservation and function. As climate change continues to affect and change patterns in climate, researchers are increasingly concerned about the impacts our global environments may endure as a result. In a terrestrial ecosystem, mean annual precipitation (MAP) is an important control of biological productivity. As water leaves a system, via evapotranspiration, mass flow, surface flow, or other means, salts remain and concentrate in soils causing salinity stress for organisms that remain. This increasing salt concentration is a major stressor for organisms and requires specialized osmoadaptive responses. Plant communities as well as those of soil-dwelling fungi and bacteria change along precipitation gradients, indicating their responses to water availability. However, it is less certain if conspecific organisms also differ across similar gradients, especially in their abilities to tolerate salt stress. To investigate the inter- and intraspecific adaptive responses of root-associated ascomycetes to salinity, we devised an experiment wherein conspecific isolates representing five ascomycete species were subjected to increasing concentrations of salinity in an effort to quantify and compare the effective dose of salt (NaCl) necessary to limit colony growth by 50% (ED₅₀). For each of the five species, we selected three conspecifics originating from drier “arid” environments and compared those to three conspecifics originating from wetter “mesic” environments. We hypothesized that (1) ascomycete species differ in their growth response to salinity across species; (2) conspecific strains from drier sites have greater salt tolerance than those from more mesic sites. Each isolate was tested three times in triplicate and exposed to four levels of NaCl concentrations in a quad-plate. We measured colony growth and used regression analyses to estimate the isolates’ ED₅₀. In support of our first hypothesis, we observed that species differed in their growth response to salinity according to ED₅₀. However, we observed no consistent, strong evidence to support our second hypothesis. Still, we observed non-significant differences in ED₅₀ within three species observed, partial support in Periconia macrospinosa isolates and the most significant difference across sites within Fusarium cf. equiseti isolates. While isolate results within F. cf. equiseti do support our second hypothesis, Periconia macrospinosa appear to demonstrate an inverse response. This coupled with the non-significant differences between ED₅₀ results across site conditions of the other three species – our second hypothesis was rejected. To further investigate the underlying mechanisms and differences in adaptive response among conspecific isolates, we designed an experiment in which two isolates, DS 1091 and DS 0982, of Periconia macrospinosa that differed most in their salt tolerance as inferred from NaCl ED₅₀ estimated in the experiment described above were subjected to prolonged salinity stress followed by proteomic analysis. The selection of Periconia macrospinosa isolates over Fusarium equiseti isolates was based on the species’ critical importance globally in ecosystems experiencing low nutrient and MAP inputs as well as the availability of a recently annotated genome. We hypothesized that (3) the highest and lowest performing strains within a species will differ in their proteomic profiles; (4) colony expansion under saline stress (ED₅₀) will correlate with cell wall related protein abundances under salt stress (signaling cell wall modifications); (5) similarly to the cell wall associated proteins, colony growth (ED₅₀) will correlate with plasma membrane related protein abundances under salt stress (signaling cell membrane alterations); (6) the mitogen-activated protein kinase (MAPK) signaling pathway will be one strategy for tolerating prolonged salinity amongst the conspecifics (suggesting “compatible-solute” strategy); and, (7) conspecifics associated with the highest ED₅₀ will demonstrate greater abundance of proteins involved in salt-tolerant strategies such as cell wall, plasma membrane, and MAPK-related proteins than their counterparts originating from more mesic sites. Our third hypothesis was supported as the two isolates demonstrated unique proteomic profiles both with and without salinity induced responses. Our fourth and fifth hypotheses, which proposed correlation between isolate colony growth and the abundance profiles of proteins involved in cell walls and cell membranes respectively, were also supported by our proteomic data. The fourth hypothesis was reinforced by our proteomic analysis of our high-ED₅₀ isolate, DS 0982, through the upregulated chitin synthase coupled with downregulation of chitinase demonstrating an investment in cell wall maintenance. The fifth hypothesis was supported through the evident DS 0982 responses to salinity where indicators of sphingolipid biosynthetic processing and ergosterol biosynthesis were observed. Specifically, support for the cell membrane related protein increase correlated with the colony growth was through ergosterol biosynthesis contributors CDP-diacylglycerol synthase, sterol C-14 reductase-like protein, and sterol 24-C methyltransferase. The sixth hypothesis, which focused on the positive correlation between colony growth and MAPK signaling interactions was also supported. The MAPK high-osmolarity glycerol HOG pathway regulates and initiates osmoadaptive responses within the cell, the most critical of which being the production of the compatible-solute – glycerol. This hypothesis was strongly supported due to observations of abundance differences in response to salinity of several key HOG pathway MAPK proteins, most notably Gdp1 the downstream initiator of glycerol production. Finally, our seventh hypothesis was supported by the observed inverse responses between our two conspecific isolates. As per our hypothesis, the isolate with greatest ED₅₀, DS 0982, did indeed have the greatest abundance of proteins involved with osmoadaptive strategy across the board from those involved in cell wall modifications such as chitin synthase, to proteins involved in plasma membrane amendments such as including such observations as C-14 reductase-like protein, and even the critical MAPK compatible osmolyte production initiator protein Gdp1. Understanding soil-inhabiting and root-associated fungi is critical to ecosystem health. Whether through providing nutrient cycling and ecosystem services, or direct interactions with host plants, fungal impacts cannot be ignored. Through the effects of climate change and corresponding changes in precipitation, soil salinization is a major threat to plant and animal health as well as the overall function of our natural world. This research aims to fill the gap in our knowledge on the impact of salinity toward root-associated fungi and their limits in salt-tolerance both within and across species. Further, this study aids in identifying varying functional adaptations soil-dwelling fungi depend on for survival in response to salt stress in soil matrices.