Biology of actinorhizal symbiosis from genomics to ecology: the 20th International Meeting on Frankia and Actinorhizal Plants

Background

A nitrogen-fixing actinobacterial genus Frankia establishes symbiosis with actinorhizal plants that are comprised of more than 200 species in eight dicotyledonous families (Benson and Silvester 1993; Huss-Danell 1997; Kucho et al. 2010). The symbiosis takes place in nodules formed on roots. Inside the root nodules, Frankia reduces atmospheric dinitrogen to ammonium and supplies it to the host plants. Because of this ability, actinorhizal plants serve as key pioneer species that can colonize nitrogen-poor biotopes such as burned forest, landslide scar, sand dune, volcanic lava flow, etc.

Frankia spp. are classified into several phylogenetic clusters, each of which infects different host plants (Nouioui et al. 2011). Cluster 1 Frankia infects plant species in Casuarinaceae, Myricaceae and the genus Alnus of Betulaceae. Cluster 2 infects plants in four families of the orders Rosales and Cucurbitales. Cluster 3 infects plants in several families of the orders Fagales and Rosales. Cluster 4 includes atypical strains that cannot infect plants or cannot fix nitrogen.

Genome analyses of Frankia and actinorhizal plants have provided crucial insights into the molecular aspects of this plant-microbe symbiosis. In 2007, genome sequences of three Frankia strains belonging to clusters 1 and 3 were first reported (Normand et al. 2007). One of the interesting features of those genomes was that symbiosis-related genes of rhizobia required for establishing root-nodule symbiosis with leguminous plants were apparently not found in the Frankia strains. Therefore, the molecular mechanisms of these two symbioses were thought to be different. On the other hand, genomes of the actinorhizal plants (Alnus glutinosa and Casuarina glauca) contained many of the symbiosis-related genes of leguminous plants (Hocher et al. 2011). Furthermore, homologues of rhizobia symbiosis-related genes were discovered in genomes of a few cluster 2 Frankia strains (Persson et al. 2015; Van et al. 2016, 2019). Based on these results, these two plant-microbe symbioses are now considered to share, at least partially, a common mechanism.

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Since 1978, the International Meeting on Frankia and Actinorhizal Plants has been held almost every two years (Gtari et al. 2019) and researchers discussed a variety of topics such as ecology, physiology, cytology, genetics and omics-biology. The 20th meeting was organized by Japanese researchers and held in May 2021. Participants from all over the world discussed a wide range of topics related to actinorhizal symbiosis from genomics to ecology, and gained new perspectives in this research field. This special issue contains six papers from the research presented there.

Sarkar and Sen (2022) surveyed genes related to metabolic reaction pathways (MPR genes) in 48 Frankia genomes. They predicted expression levels of the MPR genes using GC content in codons and codon adaptation index (CAI), and found that these genes frequently use GC-rich synonymous codons and the codon usage pattern was similar to known highly expressed genes. These results suggested that expression levels of the MPR genes would be high. Besides, they evaluated rate of synonymous and non-synonymous mutations in the MPR-genes, and found that these genes were highly conserved in Frankia genomes and selection pressure acted to preserve the amino acid sequence. Although those genomes derived from Frankia strains with different lifestyles (obligate symbiont, facultative symbiont and non-symbiont), network analysis for specific metabolic pathways suggested that the MPR genes might not have much to do with the lifestyle. Sen et al. (2022) collected soil samples from rhizosphere of Alnus nepalensis and non-rhizosphere at different altitudes of Darjeeling hills in India. They purified total DNA from the soils, amplified 16S rRNA gene by PCR using universal primers, and sequenced the amplicons using a next-generation sequencer. They found that nitrogen-fixing bacteria such as Frankia and cyanobacteria were enriched in rhizosphere soils, suggesting a synergistic role of these bacteria in proper growth of the Alnus plant. Non-rhizosphere soil showed higher bacterial diversity and enriched with Streptomyces, Rubrobacter and Xanthomonas. They also found that altitude might affect the diversity of microbial community in both rhizosphere and non-rhizosphere soil.

Ribeiro-Barros et al. (2022) reviewed salt-stress tolerance of Casuarina in aspects of physiology, genomics, transcriptomics, proteomics, metabolomics and discussed potential contribution of the actinorhizal and arbuscular mycorrhizal symbioses to rehabilitation of soil degradation caused by salinization. Djighaly et al. (2022) examined the antioxidant activity and cell ultrastructure of two Casuarina species grown under salt stress, and found that C. obesa with a higher antioxidant activity had a better salt stress tolerance compared to C. equisetifolia. Besides, they showed that dual inoculation of Frankia and Rhizophagus fasciculatus, an arbuscular mycorrhizal fungus, improved the performance of both plant species under salt stress.

Karthikeyan et al. (2022) examined the antimicrobial activity of Micromonospora isolated from root nodule of Casuarina against a pathogenic bacterium Ralstonia solanacearum. Then, effects of Frankia and Micromonospora on the growth of Casuarina were examined in the greenhouse. The biocontrol activity of Micromonospora against a bacterial wilt was also evaluated in Casuarina plantations in India. Yamanaka et al. (2022) investigated the distribution of Frankia and ectomycorrhizal fungi associated with Alnus sieboldiana in the underlying soil appeared after the landslide at Izu-Ohshima Island, Japan, during a heavy rainstorm caused by Typhoon No. 26 (Yamanaka et al. 2022) in October 2013. Alnus sieboldiana was an initial invader of devastated area after natural disasters. Therefore, these symbiotic associations between alder and the soil microbes are likely to be important for the recovery of vegetation in this area.

Topics presented in the 20th International Meeting on Frankia and Actinorhizal Plants

The 20th International Meeting on Frankia and Actinorhizal Plants was originally planned to be held in Kagoshima, Japan in May 2020. However, due to the COVID-19 pandemic, this meeting was rescheduled and held online on May 29–31, 2021. Fifty-seven scientists attended the meeting while the number of registrations in the originally planned face-to-face meeting had been only 35. This suggests that people who had given up on traveling to Japan was able to join this online event. On the other hand, the time difference was a big problem as the participants were getting together from 15 countries on every continent except Antarctica. There were 19 oral and 8 poster presentations. The oral sessions were conducted using Zoom. The poster sessions were conducted using Zoom and file sharing function of Microsoft OneDrive. Participants viewed PDF files of the posters in a shared folder in OneDrive and had discussions in the Zoom breakout rooms assigned to each poster.

Due to rapid advance in DNA sequencing technology, “Omics” approach was a major topic in this meeting. Majority of researchers used this approach and gained new insights into the actinorhizal symbiosis. Transcriptome analysis of Alnus glutinosa (Hocher et al. 2011) revealed that a lipid transfer protein AgLTP24 was upregulated in roots during establishment of symbiosis, and Gasser et al. (2021) reported this protein was localized at deformed root hairs and Frankia vesicles. Several metagenome analyses of cluster-2 Frankia strains had been reported (Persson et al. 2015; Van et al. 2016, 2019). Berckx et al. (2021a) and Berckx et al. (2021b) presented new metagenome analyses using nodules collected from Philippines, Taiwan and New Zealand and discussed evolution of the cluster-2 strains. Herrera-Belaroussi et al. (2021) talked about genome analyses of spore-positive (Sp+) Aluns-infective strains that sporulate inside nodules and have never been isolated in pure culture (Herrera-Belaroussi et al. 2020; Pozzi et al. 2020). Genomes of the Sp+ strains were significantly reduced in size and lacked more than 1000 genes compared to spore-negative (Sp-) strains (Herrera-Belaroussi et al. 2021). Hahlin et al. (2021) reviewed research history of nitrogen fixation, hydrogenase and hydrogen evolution, and reported genome analyses of local source of Frankia strains.

Kagiya et al. (2021) analyzed nifD-K intergenic spacer sequences of Frankia in Alnus hirsta nodules and its rhizosphere soils collected from Uryu experimental forest, Hokkaido, Japan. Significantly different Frankia operational taxonomic units were detected between the two ecological niches, suggesting that host plants filter infecting partners. Jabberi et al. (2021) evaluated effect of static magnetic field (SMF) on bacterial community in Tunisian phosphogypsum soils by amplicon sequencing of 16S rRNA gene. They found that exposure to SMF increased several nitrogen-fixing taxa such as Frankia. Sarkar et al. (2021) applied the reverse ecology approach to genomes of Frankia and co-inhabiting bacteria in nodules. The results suggested that co-inhabiting bacteria did not compete with Frankia but played a supportive role in symbiosis process.

An important topic of this meeting was genetic transformation of Frankia. Two laboratories had previously reported successful transformation of Frankia spp. by filter mating with an Escherichia coli strain (Pesce et al. 2019) or by electroporation (Gifford et al. 2019). Pesce et al. (2021) presented an application of this mating method to genome editing using CRISPR/CAS9 system (Doudna and Charpentier 2014). On the other hand, however, Kucho (2021) reported that all attempts at the mating transformation made in his laboratory had failed. It was also discussed that cultivation of the Frankia transformants had not been successful outside of the two laboratories. Therefore, transformation of Frankia is not easily reproducible by universal researchers at present.

Rehan et al. (2021) reported Frankia strains accumulated cadmium (Cd) and tolerated high concentration of Cd. Worth et al. (2021) reported that co-inoculation of Alnus glutinosa with an alder-infecting Frankia strain and with an atypical Frankia strain enhanced nodulation under copper stress.

Studies about ecology of actinorhizal symbiosis were also reported. Katayama and Tateno reported that even in late autumn Alnus firma continued photosynthesis without resorbing nitrogen from leaves and provided photosynthetic products to Frankia to support nitrogen fixation (Tateno 2003). They also reported that litter of Fallopia japonica enhanced growth of Alnus inokumae in volcanic soil by supplying phosphate (Katayama et al. 2021). Tobita et al. (2021) investigated N₂-fixing capacity of three Alnus species that were naturally regenerated at the massive landslide area on Mt. Ontake, Japan, using N stable isotope ratio analysis. They found that contribution of N₂ fixation to N absorption varied not only with elevation but also with vegetation recovery status. Krishnamoorthi et al. (2021) reported nodulation and blister bark disease incidence of Casuarina junghuhniana and C. cunninghamiana in Tamil Nadu state in southern India. Rajendran (2021) reported exopolysaccharide production by Casuarina equisetifolia and its beneficial role for growth of the plants.

Three researchers presented analysis of chemical compounds from actinorhizal plants. Jin et al. (2021) purified compounds from root nodules of Casuarina equisetifolia and found that tyramine was a dominant constituent. Banerjee et al. (2021) reported phytochemical profiling of Elaeagnus wine and isolated compounds with anti-cancer activity. Nisad et al. (2021) reviewed pharmacological and therapeutic potential of myricetin, which is a flavonol purified from bayberry Myrica nagi plant.

Finally, we thank all the participants and presenters for their corporation to make this unusual meeting successful. We sincerely hope the next meeting will be face-to-face and be able to have more intimate communication with each other.

Acknowledgments

We thank Dr. Rikiya Endoh for his dedication to the editorial work as a coordinating editor.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.