AM fungi constitute an important group of fungi for sustainable agriculture benefits; however, the genome sequences and gene repertoires of most of the AM species are not yet explored. The information on genetic structure of these fungi could provide important information about molecular mechanisms underlying the host-specific interaction with different species of crop plants and associated agriculture benefits (Prasad et al., 2019). For majority of fungal classes and species, the information regarding their genetic structure and function has commonly been acquired by comparative studies with the genomes of model species belonging to Ascomycota and Basidiomycota. However, it is difficult to achieve understanding about the genetic architecture of AM fungi by similar comparisons as Ascomycota and Basidiomycota are only distantly related with Glomeromycota and extensive divergence between them over the long evolutionary period has occurred (Sanders and Croll 2010). With such a background, the exceptional identifications regarding the lack of many genes constituting the basic machineries for eukaryotic metabolic pathways in Glomeromycota, expansion of kinome and reduction of CAZymes are being cautiously probed.
The investigation reported here provided first draft of the genome sequence and genome annotation of R. proliferus, which is one of the important species of AM fungi known to provide benefits to multiple crops. The estimated size of genome is ~ 110 Mbps, which is the smallest of all the reported AM fungi till date. Like the previously reported AM fungi, conservation with respect to fewer carbohydrate active enzymes and higher number of protein kinases was predicted in R. proliferus in comparison to EM and Ascomycetes fungi. High proportion of protein classes representing “establishment of localization” and “signal transduction” proteins were seen in GO classification in R. proliferus. Genes coding for “establishment of localization” could be crucially involved in the development of plant-microbial interactions in a symbiotic association.
The remarkable enlargement of protein kinase gene family and especially of tyrosine kinase-like (TKL) genes in is supported by the previous reports in AM fungi (Tisserant et al., 2013; Lin et al., 2014; Salvioli et al., 2016; Tang et al., 2016). Protein kinases influence most cellular activities, especially cell signaling, by protein phosphorylation. The expansion of kinase gene family has been suggested to be crucial in signal transduction processes that are involved in establishment of symbiotic interaction between AM fungi and plant. Interestingly, TKL-containing proteins have been observed to over-express in germinating spores and intraradical mycelium in R. irregularis (Tisserant et al., 2013). Conservation of alpha protein kinases, which is an ancient class of protein (Drennan and Ryazanov 2004), in R. proliferus and other AM fungi unlike the other fungal groups, may either indicate inefficiency of AM fungi to expel the genetic load through sexual reproduction or a strong conservation of the molecular mechanisms supportive of lifestyle of AM fungi.
The reduced presence of Glycoside hydrolases found in R. proliferus #24 in comparison to other fungal division was in conformity with the other species of AM fungi. Expansins (EXPN) and Polysaccharide Lyases (PL) were absent in R. proliferus genome similar to the previous reports in AM species. Expansins (EXPN) functions in cell wall loosening and help the accommodation process of the fungus inside the cortical cells (Cosgrove et al., 2002). Expansins of fungal origin are supposed to function in the loosening of interfacial material loose (Balestrini et al., 2005). Polysaccharides lyases (PL) play a role in degradation of pectin layers of wood (Kristiina and Miia 2018). These observations in AM fungi, unlike the EM and the pathogenic fungi, has been proposed as “functional tradeoffs” in an obligate symbiont for achieving a stealth entry and colonization into root while evading plant immune response (Tisserant et al., 2012). In contrast to the previous reports in AM fungi (Tisserant et al., 2013; Tang et al., 2016; Kobayashi et al., 2018; Sun et al., 2018; Morin et al., 2019; Venice et al., 2020), presence of proteins belonging to AA4 family in R. proliferus was striking. AA4 codes for Vanilly-alcohol oxidase (VAO), which are intracellular FAD-dependent enzymes that act on activated aromatic alcohols like 4-hydroxybenzyl alcohols. AA4 is directly not involved in lignocellulolysis but in the metabolism of lignin-derived compounds. Another noteworthy observation for CAZymes in R.poliferus is higher abundance of carbohydrate esterases (CE) in comparison to the other reported AM fungi. CE is a special class of enzyme identified in microorganisms, which de-acetylate hemicellulose and pectin units of plant polysaccharides (Sista and Qin 2018). Deacetylation leads to breaking of glycosidic linkages and help in degradation of plant cell wall, which further enables entry of microorganisms in plants. The abundance of CEs and presence of unique lytic enzyme in R. proliferus may indicate towards higher saprotrophic activity in comparison to R. irregularis.
A widespread notion of the absence of sexual recombination in AM fungi was challenged by the contrasting observations made in the whole genome analysis of R. irregularis (Lin et al., 2014). An exploration of the sexual potential of R. irregularis identified a putative AM fungi mating-type locus with prominent similarities to the mating-type locus of Basidiomycota (Ropars et al., 2016). In addition, 76 HMG (high mobility group) box containing genes were identified in R. irregularis (Riley et al., 2014). Also, in G. rosea #48 meiosis-related genes were found (Tang et al., 2016). In agreement with these findings, #89 HMG (high mobility group) box containing genes and #47 meiosis-related genes were identified in R. proliferus. Such a conservation of meiosis-related genes re-emphasized existence of a yet unknown sexual reproduction mechanism in Glomeromycotan fungi and particularly in R. proliferus.
In context of presence of CUG in R. proliferus a total of 234/248 ultra-conserved CEGs were predicted in R. proliferus using CEGMA analysis. The absence of the fatty acid synthesis, type I multienzyme complex (FAS-I) was in agreement with previously reported species of AM fungi. Homology search based prediction in R. proliferus revealed all components of the bacterial type FAS (type II FAS) genes only. The FAS-I complex is responsible for the cytosolic fatty acid synthesis, which produces the bulk of long-chain fatty acids in other fungi (Leibundgut et al., 2008). This gene has been reported missing in the gene repertoires of AM fungi (Tisserant et al., 2013; Wewer et al., 2014; Tang et al., 2016; Sun et al., 2018; Kobayashi et al., 2018; Morin et al., 2019; Venice et al., 2020), which has motivated extensive exploration to understand how AM fungi may generate lipid reserves. Interestingly, in AM-colonized cells of plant roots intensive stimulation of genes involved in lipid metabolism occur, perhaps to provision the increased demand for lipids for the periarbuscular membrane. Based on these findings it has been suggested that AM fungi may receive fatty acids synthesized by plant cells. In this regard, recent studies have demonstrated that AM fungal lipids are, at least partially, derived from the plant host (Bravo et al., 2017; Jiang et al., 2017; Luginbuehl et al., 2017; Rich et al., 2017; Keymer et al., 2018).
Thiamine is a cofactor for enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and therefore it is an essential constituent of all cells. The biosynthetic pathway for thiamine has been reported missing in AM fungi. In congruence with the previous reports, thiamine biosynthetic pathway genes were not predicted in R. proliferus.
Proteins, uridine permease, uracil permease and dihydroorotate dehydrogenase support uracil metabolism, transport and maintain the intracellular level of uracil. Tight control of the intracellular uracil has been suggested important to reduce the rate of uracil incorporation into DNA (Sun et al., 2013). Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11), which is the fourth enzyme of the pyrimidine de novo biosynthesis pathway, was the only gene from the pathway that was present in both the R. irregularis and R.proliferus genomes. Genes for glutamate metabolism and glutathione metabolism were predicted in R. proliferus, which indicated for its potential for the metabolism of nucleic acids and proteins (Yelamanchi et al., 2016) and detoxification of xenobiotics and the oxidative stress response (Shen et al., 2015) respectively, similar to other AM fungi. Transporters and channels for potassium transport from the soil to the host by the AM fungi are still not completely deciphered in AM fungi. Seven sequences from an EST library of R. irregularis were annotated as K+ transport systems (Casieri et al., 2013), which coded for SKC-type channels and KT/KUP/HAK transporter. Noticeably, no Trk and TOK members were identified in either the EST library or the sequenced nuclear genome (http://genome.jgi.doe.gov/Gloin1/Gloin1.home.html). In congruence with the previous reports, no homologue gene for the yeast TOK1 was identified in R. proliferus. However, a Trk-type K + transport system in R. proliferus was predicted. Probable ferric reductase transmembrane component 8, which is expected to function in the assimilation of iron, was identified only in R. proliferus by the conserved domain analysis and comparison with the proteins coded by yeast. In our comparative analysis, the absence of several CUG in R. proliferus was mostly in confirmation with the previously reported AM fungi, which suggested high conservation in genetic features among all species belonging to Glomeromycotina.