Cycad Coralloid Roots Contain Bacterial Communities Including Cyanobacteria andCaulobacterspp. That Encode Niche-Specific Biosynthetic Gene Clusters

Abstract

Cycads are the only early seed plants that have evolved a specialized root to host endophytic bacteria that fix nitrogen. To provide evolutionary and functional insights into this million-year old symbiosis, we investigate endophytic bacterial sub-communities isolated from coralloid roots of species from Dioon (Zamiaceae) sampled from their natural habitats. We employed a sub-community co-culture experimental strategy to reveal both predominant and rare bacteria, which were characterized using phylogenomics and detailed metabolic annotation. Diazotrophic plant endophytes, including Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium, and Nostoc species, dominated the epiphyte-free sub-communities. Draft genomes of six cyanobacteria species were obtained after shotgun metagenomics of selected sub-communities. These data were used for whole-genome inferences that suggest two Dioon-specific monophyletic groups, and a level of specialization characteristic of co-evolved symbiotic relationships. Furthermore, the genomes of these cyanobacteria were found to encode unique biosynthetic gene clusters, predicted to direct the synthesis of specialized metabolites, mainly involving peptides. After combining genome mining with detection of pigment emissions using multiphoton excitation fluorescence microscopy, we also show that Caulobacter species co-exist with cyanobacteria, and may interact with them by means of a novel indigoidine-like specialized metabolite. We provide an unprecedented view of the composition of the cycad coralloid root, including phylogenetic and functional patterns mediated by specialized metabolites that may be important for the evolution of ancient symbiotic adaptations.

Unlocking a high bacterial diversity in the coralloid root microbiome from the cycad genus Dioon

“…Endophytes associated with cycad coralloid roots have been studied for more than a century mostly by methods that underestimated or did not aim to measure the non-cyanobacterial bacterial diversity present inside the root. Our study supports very early studies [4,8,9], and two more recent studies, the 16S rRNA-based Zheng et al [17] and the metagenomic-based Gutiérrez-García et al [18], that report a diverse bacterial microbiome beyond cyanobacteria.…”

“…Zheng et al [17] found significant differences in the abundance of some bacterial families compared to regular cycad roots, with a dominance of cyanobacteria in the coralloid roots [17]. Likewise, Gutiérrez-García et al [18] sampled the bacterial coralloid microbiome of Dioon edule using complete genomes and shotgun metagenomes from axenic cultures and liquid co-cultures that aim to describe the functional diversity of the endophytic microbiome [19], and also found several bacterial groups in addition to cyanobacteria endophytes. There appears to be a broad spectrum of non-cyanobacteria inside the coralloid root, which we are only beginning to explore.…”

“…In plants, comparison between soil and rhizosphere microbiomes often show differences in alpha [24] and beta diversity [24,25] and slight differences in taxonomic composition and community structure [26]. The endosphere typically has the lowest bacterial diversity [25], sometimes enriched with specific bacterial groups [27] and functionally specialized microbial communities [18]. In cycads, only two studies mention the influence of bacterial soil diversity in the bacterial coralloid endosphere.…”

Specialized bacteriome uncovered in the coralloid roots of the epiphytic gymnosperm, Zamia pseudoparasitica

“…The cyanobacterial sequences from Panamanian Zamia belong to a single clade based on rb-cL-rbcX. Our study is in line with findings in the Mexican cycad genus Dioon where cyanobionts were also monophyletic using 16S rRNA (Gutiérrez-García et al, 2018;Suárez-Moo et al, 2019).…”

“…To investigate microbial diversity within coralloid roots, researchers have used a broad set of techniques, including DNA restriction patterns (Lindblad, Haselkorn, Bergman, & Nierzwicki-Bauer, 1989;Lotti, Giovannetti, Margheri, Ventura, & Materassi, 1996), protein fingerprinting (Zimmerman & Rosen, 1992), PCR fingerprinting (Costa, Paulsrud, & Lindblad 1999;Costa, Romero, & Lindblad 2004;Zheng, Song, Bao, Bergman, & Rasmussen, 2002), and Sanger sequencing (Cuddy, Neilan, & Gehringer, 2012;Gehringer et al, 2010;Gutiérrez-García et al, 2018;Thajuddin et al, 2010;Yamada, Ohkubo, Miyashita, & Setoguchi, 2012). Only two studies have so far addressed the diversity of endosymbionts using metagenomic approaches (e.g., amplicon sequencing of the 16S for bacteria).…”