The methylome of the model arbuscular mycorrhizal fungus, Rhizophagus irregularis, shares characteristics with early diverging fungi and Dikarya.

Early-diverging fungi (EDF) are distinct from Dikarya and other eukaryotes, exhibiting high N6-methyldeoxyadenine (6mA) contents, rather than 5-methylcytosine (5mC). As plants transitioned to land the EDF sub-phylum, arbuscular mycorrhizal fungi (AMF; Glomeromycotina) evolved a symbiotic lifestyle with 80% of plant species worldwide. Here we show that these fungi exhibit 5mC and 6mA methylation characteristics that jointly set them apart from other fungi. The model AMF, R. irregularis, evolved very high levels of 5mC and greatly reduced levels of 6mA. However, unlike the Dikarya, 6mA in AMF occurs at symmetrical ApT motifs in genes and is associated with their transcription. 6mA is heterogeneously distributed among nuclei in these coenocytic fungi suggesting functional differences among nuclei. While far fewer genes are regulated by 6mA in the AMF genome than in EDF, most strikingly, 6mA methylation has been specifically retained in genes implicated in components of phosphate regulation; the quintessential hallmark defining this globally important symbiosis.

Generation of unequal nuclear genotype proportions in Rhizophagus irregularis progeny causes allelic imbalance in gene transcription.

Arbuscular mycorrhizal fungi (AMF) form mutualisms with most plant species. The model AMF Rhizophagus irregularis is common in many ecosystems and naturally forms homokaryons and dikaryons. Quantitative variation in allele frequencies in clonally dikaryon offspring suggests they disproportionately inherit two distinct nuclear genotypes from their parent. This is interesting, because such progeny strongly and differentially affect plant growth. Neither the frequency and magnitude of this occurrence nor its effect on gene transcription are known. Using reduced representation genome sequencing, transcriptomics, and quantitative analysis tools, we show that progeny of homokaryons and dikaryons are qualitatively genetically identical to the parent. However, dikaryon progeny differ quantitatively due to unequal inheritance of nuclear genotypes. Allele frequencies of actively transcribed biallelic genes resembled the frequencies of the two nuclear genotypes. More biallelic genes showed transcription of both alleles than monoallelic transcription, but biallelic transcription was less likely with greater allelic divergence. Monoallelic transcription levels of biallelic genes were reduced compared with biallelic gene transcription, a finding consistent with genomic conflict. Given that genetic variation in R. irregularis is associated with plant growth, our results establish quantitative genetic variation as a future consideration when selecting AMF lines to improve plant production.

Genetically Different Isolates of the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis Induce Differential Responses to Stress in Cassava.

Water scarcity negatively impacts global crop yields and climate change is expected to greatly increase the severity of future droughts. The use of arbuscular mycorrhizal fungi (AMF) can potentially mitigate the effects of water stress in plants. Cassava is a crop that feeds approximately 800 million people daily. Genetically different isolates of the AMF R. irregularis as well as their clonal progeny have both been shown to greatly alter cassava growth in field conditions. Given that cassava experiences seasonal drought in many of the regions in which it is cultivated, we evaluated whether intraspecific variation in R. irregularis differentially alters physiological responses of cassava to water stress. In a first experiment, conducted in field conditions in Western Kenya, cassava was inoculated with two genetically different R. irregularis isolates and their clonal progeny. All cassava plants exhibited physiological signs of stress during the dry period, but the largest differences occurred among plants inoculated with clonal progeny of each of the two parental fungal isolates. Because drought had not been experimentally manipulated in the field, we conducted a second experiment in the greenhouse where cassava was inoculated with two genetically different R. irregularis isolates and subjected to drought, followed by re-watering, to allow recovery. Physiological stress responses of cassava to drought differed significantly between plants inoculated with the two different fungi. However, plants that experienced higher drought stress also recovered at a faster rate following re-watering. We conclude that intraspecific genetic variability in AMF significantly influences cassava physiological responses during water stress. This highlights the potential of using naturally existing variation in AMF to improve cassava tolerance undergoing water stress. However, the fact that clonal progeny of an AMF isolate can differentially affect how cassava copes with natural drought stress in field conditions, highlights the necessity to understand additional factors, beyond genetic variation, which can account for such large differences in cassava responses to drought.