How can spores be genetically dissimilar

Some Epulopiscium -like surgeonfish symbionts form mature endospores at night. These spores possess all of the characteristic protective layers seen in B. These are the largest endospores described thus far, with the largest being over times larger than a Bacillus subtilis endospore.

The formation of endospores may help maintain the symbiotic association between these Epulopiscium -like symbionts and their surgeonfish hosts. Since endospore formation coincides with periods in which the host surgeonfish is not actively feeding, the cells do not need to compete for the limited nutrients present in the gut at night.

The protective properties of the endospores also allow them to survive passage to new surgeonfish hosts. The fish may also benefit from this relationship because it is able to maintain stable microbial populations that assist in digestion and may receive a nutritional gain from microbial products released during mother cell death and spore germination.

Endospore formation in some Epulopiscium -like symbionts follows a daily cycle: A Polar septa are formed at the poles of the cell.

B Forespores become engulfed. C Forespores gradually increase in size within the mother cell through the day. D In late afternoon, final preparations for endospore dormancy. E Endospores mature and remain dormant throughout most of the night. F Just before sunrise, the endospores germinate and are released from mother cell to repeat the cycle.

Google Tag Manager. Bacterial Endospores. Sexual reproduction in AMF may, indeed, occur frequently enough to purge deleterious mutations, but may not be frequent enough to be readily observable in experiments, making its experimental observation highly elusive [ 7 ].

The genomic region that defines mating compatibility and sexual reproduction in fungi MAT-locus is not fully conserved among phyla. At least three different transcription factors homeodomain HD , alpha-box, and HMG-box are responsible for sex-determination in different fungal phyla [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. This is supported by its presence in Zygomycetes, Microsporidia, Hemiascomycetes, and Euascomycetes. However, in Ascomycetes and Basidiomycetes, HMG-box transcription factors are replaced by alpha-box and HD transcription factors [ 29 ].

However, the presence of additional genes is possible as in P. Exceptions to the rule also occur as in the homothallic fungus Syzygites megalocarpus [ 31 ] where an individual contains two MAT-loci each containing a SexP and SexM copy. When two individuals of the same species interact, nonself-recognition is initiated by the perception of mating pheromones. The presence of mating pheromones activates at least four different mitogen-activated protein kinase MAPK cascades during the mating process [ 32 ].

Activation of MAT-locus sex-determination transcription factors is also expected [ 33 , 34 ]. This process leads to the formation of the mating tube and the induction of meiosis and sexual sporulation. Despite the likely existence of sexual processes in AMF, the molecular mechanisms of mating in these fungi are poorly understood.

Homologs of genes present in the MAT-locus of closely related Mucoromycotina species were found in R. However, they are not contiguous, and are located in different regions of the genome [ 35 ].

No activation of HMG-box genes was detected when two genetically different R. A study of R. The presence of this locus was confirmed in the Glomerales and Diversiporales, with the exception of Gigasporaceae, where the locus presented no structural conservation [ 19 ].

However, no study has provided any experimental evidence of the involvement of this putative MAT-locus, or any other genes, in the interaction between genetically different AMF isolates of the same species.

Curiously, no studies have ever tried to look for evidence of transcription when isolates of the same AMF species coexist in planta , even though it is known that genetically different isolates of AMF co-colonize roots. In this study, we investigated the molecular mechanisms of nonself-interactions between two genetically different, but closely related, R. We compared gene transcription of two genetically different R. First, we tested if fungal genes were differentially transcribed in the coinoculation treatment compared with both single-inoculation treatments.

Second, we evaluated if there were different fungal transcriptional response in the three host genotypes. Third, we evaluated if the proportion of colonization by each isolate was different among the three host plant genotypes in the coinoculation treatments. Understanding the molecular mechanisms of nonself-interactions is highly relevant in the understanding how AMF interact with each other in plant roots.

Identifying which genes are activated when two AMF meet, and if this occurs in symbiosis with different host plant genetic backgrounds, will help identify the molecular mechanisms of AM fungal coexistence, give clues about AMF nonself-recognition and how it is affected by the genetic background of host plants. We conducted a greenhouse experiment where cassava M. We performed this experiment independently on three different cassava M.

The cassava cultivars were obtained from the cassava genebank resource at the International Center for Tropical Agriculture and two R. This experiment was run in parallel with another experiment designed to answer a separate question regarding how cassava genotypes affected cassava and R.

The phenotypic data on single-inoculations and the mock-inoculated samples was already published in the previous publication. The RNA-seq data on the single-inoculation samples and the mock-inoculated treatments were already made publicly available under the accession number PRJNA The fungal colonization, plant response, and transcript data of the coinoculation treatments have not previously been published.

The source code describing the steps of the bioinformatic analysis and the differential gene transcription analysis can be found in Supplementary File 1. We performed a greenhouse experiment by inoculating micropropagated cassava plants with spores produced in in vitro cultures Supplementary Note 2. The methods concerning the fungal colonization and plant growth responses can be found in Supplementary Note 3. The RNA extraction, library preparation, sequencing, and the bioinformatic analysis were performed exactly according to a previously published dual RNA-seq analysis experiment [ 37 ] Supplementary Note 4.

In order to test for contamination by other fungi in the RNA sequencing data set, we estimated the presence of different fungal taxa in the raw sequencing reads with the Mash screen algorithm [ 38 ]. We first downloaded a single genome assembly for every fungal species present in the RefSeq NCBI database, resulting in different genome assemblies. We used the Mash sketch algorithm to create a combined reference file containing the different genomes.

We screened each sample raw sequencing reads against the reference file by using the Mash screen algorithm with the winner-take-all strategy. To illustrate these results, we produced a heatmap in R.

We used the DEseq2 pipeline for detecting differential gene transcription among treatments [ 39 ]. We used the raw read count data tables from feature Counts as input. We constructed the DEseq data set by including the read count tables per sample and the different conditions to be evaluated. We then performed the default DESeq differential transcription analysis based on a negative-binomial distribution and producing Wald statistics.

The DEseq pipeline performed an internal normalization method that corrects for library size and RNA composition bias. Previously, genetically different plant genotypes have shown to strongly affect fungal gene transcription [ 37 ]. Therefore, we independently analyzed the data for each plant host genotype, to avoid the effect of plant genotype variability on fungal gene transcription.

For illustration purposes, we showed the normalized counts of all treatments together and not the pairwise comparisons. We produced a single variant calling VCF file for each sample and we analyzed the inoculated samples single-inoculations and coinoculations for each cultivar independently.

We then filtered and kept: 1 the positions where SNPs were detected in at least one sample. We then measured the identity and frequency of the reference and alternative allele of each position by using the fields genotype GT and depth DP of the VCF file.

The allele frequency estimation and displays were performed in R and with the ggplot2 package. The alignment figures were generated by the Expasy Boxshade v3. We reconstructed the phylogenetic relationship among sequences by first identifying the most fitted substitution model of the sequences with MEGA X software [ 44 ].

We then performed maximum likelihood phylogenies on gap-free sites, and we used bootstraps of resamplings. We blasted the upregulated genes against the NCBI nonredundant protein sequence database to find gene identity [ 47 ]. In addition, we identified homologs on fungal model species by doing a reciprocal blasting to genes observed in model fungal organisms as Saccharomyces cerevisiae , Schizosaccharomyces pombe , Aspergillus nidulans , Ustilago maydis , and Mucor circinelloides. We used published available R.

We used the console NCBI app blast functions to identify homologous genes between the different genomes assemblies and used Easyfig [ 48 ] for the synteny visualization. We obtained an average of 42 million reads per sample, from which Six million reads did not map to the cassava genome and 3.

We did not observe any statistically significant difference in the number of total reads, reads uniquely mapped to cassava, reads uniquely mapped to R. However, we found that the total number of reads differed between the coinoculation treatment and both single-inoculation treatments in plant genotype CM We also observed that the number of genes that were sequenced differed between the coinoculation and DAOM isolate on plant genotype BRA Supplementary File 2.

In addition, a detailed analysis of data quality and its robustness were previously shown for the mock and single-inoculation treatments in Mateus et al. We observed a negligible number of fungal transcripts in the mock-inoculated plants and a large number of fungal transcripts in the fungal inoculated plants in all three cultivars and in all fungal inoculated treatments.

This showed that R. We also observed that the coinoculation treatment resulted in distinct patterns of fungal gene transcription from either of the single inoculations Supplementary Fig. We analyzed the raw reads for the presence of fungal sequences that could have contaminated the plants in the greenhouse. We identified only R. Furthermore, we did not find any fungal sequences in the mock-inoculated samples. Taken together, these results mean that the data from inoculated plants only contained transcripts of R.

We observed that the mock-inoculated samples did not display any fungal colonization. We also observed no statistically significant differences in the colonization rates between the coinoculation and the single-inoculation treatments on any plant genotype Supplementary Fig. We did not observe any significant differences in plant growth in any host plant genotype between the two single-inoculation and the coinoculation treatments Supplementary File 5.

We did not observe any significant differences in growth between the coinoculation and the single-inoculation treatments in plant genotype CM Supplementary Fig. We observed 79 genes that displayed a significantly different level of transcription in the coinoculation treatment compared with the two single-inoculation treatments when associated with the host plant genotype COL Fig.

Most of the upregulated genes represented proteins of unknown function. Normalized counts of transcripts that displayed significantly higher gene transcription in the coinoculation treatment with isolates B1 and DAOM compared with the two single-inoculation treatments B1 or DAOM The three upregulated HMG-box genes are shown in bold type. Previous studies in several fungi have shown that genes containing an HMG-box domain are involved in fungal mating in two different ways: 1 they represent part of the MAT-locus [ 49 ] or 2 they are involved in the pheromone response during the recognition and mating process i.

Prf1 in U. We reconstructed the phylogenetic relationships among the different HMG-box genes involved in sexual reproduction in fungi, including the three specifically upregulated HMG-box genes. We found that the three upregulated HMG-box genes were grouped in an independent clade within the HMG-box genes involved in fungal sexual reproduction Supplementary Fig. We found partial similarity between the R. These results suggest that R. Out of the remaining 77 genes specifically upregulated in the coinoculation treatment compared with single-inoculation treatments, in host genotype COL, 15 of these were genes with a known function in different stages of mating in other fungal species Fig.

These genes were homologs of genes involved in: 1 pheromone perception; 2 encoded in MAT-loci HMG-box and RNA helicases ; 3 involved in MAPK pathways STE20 and Mkk2 ; 4 cell survival to pheromones; 5 formation of mating tubes; 6 meiosis; 7 sexual sporulation; and 8 mating regulators see Supplementary Note 6 for a detailed description of the 15 upregulated genes.

The graphs show normalized counts of transcripts that displayed significantly higher gene transcription in the coinoculation treatment with isolates B1 and DAOM together compared with the two single-inoculation treatments B1 or DAOM The number of genes reflects the genes that were found in common between 1 isolate DAOM vs.

Together, these results suggest that different stages of a putative mating response were elicited when two genetically different R.

A previous study identified a putative MAT-locus in R. However, no evidence has ever been presented demonstrating that HD transcription factors are only transcribed in the presence of two genetically different R. We did not find any reads mapping to HD2 Supplementary Fig. We identified different upregulated genes in the coinoculation treatment compared with both single-inoculation treatments among the three plant genotypes COL 79 genes, CM 26 genes, and BRA 13 genes; Fig.

We observed that compared with host genotype COL, fewer genes were specifically upregulated in the coinoculation treatments in host genotypes CM and BRA Each number reflects the genes that were found in common between 1 isolate DAOM vs.

We observed that 26 genes were specifically upregulated in the coinoculation treatment with host genotype CM Supplementary File 8. Of these, 18 genes were differentially transcribed in the coinoculation treatment in both host genotypes CM and COL These genes included several that are known to be involved in mating and sexual reproduction in other fungi: the GBC The interaction between the two isolates in symbiosis with host genotype BRA resulted in the upregulation of 13 genes that were differentially transcribed between the coinoculation treatment compared with the two single-inoculations Supplementary File 9.

In summary, all the genes related to a putative fungal mating responses identified in host genotypes CM and BRA were detected in host genotype COL The results from the three host genotypes taken together represent the first demonstration of genes involved in several steps of a putative mating response in AMF Fig.

We identified homologs of genes involved in 1 pheromone reception, 2 survival to pheromones, 3 different pheromone response MAPK cascades, 4 encoded in MAT-locus, 5 mating regulation, 6 meiosis, 7 formation of the mating tube, and 8 sexual sporulation.

We did not observe upregulation of homeodomains proposed on a putative R. We did not find upregulation of genes encoding the mating pheromones or those involved in plasmogamy, karyogamy, and recombination. Genes in red were observed in the three host plant genotypes in the coinoculation treatment specifically.

Few studies have evaluated genome-wide gene transcription during the mating response in Mucoromycota species. In order to understand if the list and number of activated genes obtained in this study was representative of the genes expected to be identified in coinoculation studies compared with single-inoculations, we compared the results found in this study to an experiment that compared gene transcription in a coinoculation treatment to a single-inoculation treatment in the Mucoromycotina species Rhizopus microsporus [ 54 ].

In that experiment, the authors identified a set of genes transcribed during confirmed mating in R. We evaluated the homology of the coinoculation-specific differentially transcribed genes in R. Some of these genes were common to both organisms but a number of genes were either activated in R.

We analyzed SNPs in the R. We identified positions with no missing data across the samples, where isolate DAOM displayed the reference allele previously samples had been mapped to isolate DAOM and where isolate B1 displayed an alternative allele. In total, we kept positions from transcripts found in plant genotype COL, positions in plant genotype BRA, and positions in plant genotype CM Supplementary File We observed for almost all the positions, independently of the host plant genotype, that isolate DAOM displayed the reference allele, isolate B1 displayed an alternative allele and the coinoculation samples displayed a combination of both reference and alternative alleles see Supplementary Fig.

We then combined all the positions together to make allele frequency distributions of each sample. We observed that the majority of positions in isolate DAOM displayed an allele frequency of 1, meaning that the majority of positions displayed the reference allele. In contrast, the majority of positions in isolate B1 displayed an allele frequency of 0, meaning that the majority of positions displayed the alternative allele.

Finally, we observed that the coinoculation samples displayed intermediate levels of allele frequency confirming that both isolates indeed coexisted in the coinoculation treatment Fig. In contrast, in plant genotype CM, isolate B1 predominantly colonized the plant roots in the ratio in one sample and in the second sample of isolate DAOM to B1, respectively Fig.

Thus, we observed a more even coexistence in host genotype COL, where the greatest number of specifically upregulated genes where detected. We plotted the allele frequency distribution of the reference and alternative allele in the single-inoculation and the coinoculation samples. In contrast, the coinoculation samples display different proportions of each isolate represented by the peak of the distribution.

We used an experimental approach to identify AM fungal genes that were exclusively transcribed in planta when two genetically different R. We did not observe any concordant plant growth response to the coinoculation treatments.

However, we observed several fungal genes that were differentially transcribed in the coinoculation treatments compared with the single-inoculations. To further confirm the efficiency of spore surface sterilization and the presence of bacteria on spore wall, the disinfected spores were viewed under scanning electron microscope SEM. The bacterial colonies growing around the spores were purified and subcultured. Cluster analysis were performed using dice coefficient model with unweighted pair grouping with mathematic average UPGMA in BioNumerics 7.

All SAB isolated from each spore type at different disinfection time interval were tested for Gram staining and spore association characters. Gram staining was performed using Gram staining kit and the bacterial suspensions were examined under upright microscope Olympus CX41 for cell morphology. Qualitative chitinolytic activity was determined on plates containing colloidal chitin as described in Liu et al.

Quantitative estimation of chitinase production was assayed as described in Rojas-Avelizapa et al. Protease activity was determined by growing the bacteria in skim milk agar plates. Nearly complete 16S rDNA were aligned and the closest identities were obtained by searching against the type strain genes in EzTaxon. The evolutionary distance between the isolates were calculated according to Jukes and Cantor model [ 31 ] and bootstrapped with replications using MEGA 6 software [ 32 ].

Percentage data were subjected to arcsine square root transformation before analysis of variance ANOVA was performed. Morphologically differentiated spores were identified at the species level by molecular identification. A total of SAB were isolated from three different types of spore at different disinfection time interval. From F. From R. There was no bacterial growth in completely surface sterilized spores at 30 min disinfection. Arrow marks indicate bacterial cells of various shapes adhering to the spore wall; filamentous bacterial cells a, i , coccus-shaped bacterial cells c.

Arrowhead indicates the formation of mucilaginous product replacing outer hyaline layer b, j, k. To understand the genetic relationship between the cultivated bacteria from all three different spores, BOX-PCR fingerprinting method was approached. The molecular typing of BOX-PCR generated well resolved banding pattern with 1—11 amplification products and the band size varied from bp to over bp Fig 2. SAB isolated from all three types of spores at different time interval disinfection were analyzed.

Bacteria associated with each spore type were analyzed separately to understand the genetic similarity of SAB within single AMF species. The low similarity between the clusters in each spore type suggests that genetically diverse bacterial communities were associated with each AMF species. The percentage of Gram positive bacteria isolated after 10 min and 20 min disinfected spores were significantly higher compared to SAB isolated from 0 min disinfected spores S6 Fig. The percentage of SAB which showed protease activity were significantly higher in 20 min disinfected spores suggesting that protease activity might help bacteria to strictly associate with AMF spores.

Cellulase activity followed the same trend with protease activity where significantly higher percentage of SAB showed cellulase activity isolated from 20 min disinfected spores Fig 3C.

The percentage of SAB which showed cellulase activity were higher at 10 and 20 min disinfected spores suggesting that cellulase enzyme activity play a major role in AMF and bacteria association.

To further understand the spore association characters of SAB, bacteria isolated from different disinfection time interval were grouped based on their association characters. The remaining seven bacteria which did not show any of the association characters were isolated from 0 min disinfected spores. SAB isolated from 10 min disinfected spores showed a minimum of one or more characters for association whereas SAB isolated from 20 min disinfected spores showed a minimum of two or more characters for association suggesting that bacteria having multiple association characters can have close association with AMF spores.

Fifty-one SAB which showed at least three spore association characters were sequenced and the closest neighbors were identified Fig 5. Nine different species from 0 min, three different species from 10 and three different species from 20 min disinfected spores were isolated Table 1.

SAB isolated from three types of spores at different disinfection time intervals possessing a minimum of three association characters were sequenced. The numbers at the nodes indicate the bootstrap support levels based on a neighbor joining analysis of resampled data sets. The numbers in the parenthesis are the nucleotide sequence accession numbers in the GenBank. The molecular identification of these SAB were chosen based on their ability to possess at least three spore association characters.

Twenty one SAB were able to solubilize tricalcium phosphate. Only four SAB were able to produce siderophore. All of which are Pseudomonas species. Interaction between AMF and bacteria is of considerable interest due to their beneficial effect on plant growth and yield [ 34 , 35 ].

AMF spore germination, viability, root colonization and spore density have been influenced by spore associated bacteria [ 36 ]. Spore associated bacteria enhance spore germination by removing the spore outer hyaline layer and inhibiting the toxic compounds that affects spore germination [ 11 ]. In the present study, SAB associated with the spore walls of F.

The number of bacteria associated with AMF spores also varied from species to species. The highest number of SAB were obtained from 0 min disinfected spores. When disinfection time increased, the number of associated bacteria reduced showing that only strictly associated bacteria remained after 20 min of disinfection. SEM observation of spores before and after treating them with disinfection solution revealed that there were no damage on the spore wall even after 30 min of disinfection.

Our observation on the spore wall revealed the occurrence of various shapes of bacterial cells at 0, 10 min and 20 min disinfected spores. The complete surface sterilization of AMF spores after 30 min disinfection shows that the bacterial attachment on the spore wall was limited mostly to the spore outer hyaline layer [ 37 ]. Cocci-shaped bacterial cells were clearly visible on the spore walls of F.

Ames et al. We also observed that the outer hyaline layer was replaced by mucilaginous products and the reason is that the spore outer hyaline layer was sloughing off or decaying in mature spores [ 11 ]. Amplification of BOX-PCR elements provide a strain level fingerprints to find out phylogenetic relatedness among the different isolates [ 40 ].

In the present study, we found that diverse bacterial communities were associated with AMF spores and the bacterial communities belonging to the same cluster were able to associate with different AMF species.

For instance, members of Bacillales order that were grouped in the same cluster were associated with all three AMF species. The same trend of association was observed by Agnolucci et al. In our study, we found that both Gram positive and Gram negative bacteria adhered with the spore wall, however, only Gram positive bacteria were able to attach or adhere more strictly with the spore walls. Other studies also reported that Gram positive bacteria were more active in bulk soil containing mycorrhiza with low-nutrient content [ 41 ] and closely associate with the external mycelium of AMF [ 5 ].

These results and our findings confirmed the proposal by Artursson et al. Only limited number of studies have attempted to investigate the mechanism of AMF and bacteria interaction and their associative characters. According to previous studies, it is difficult to conclude which bacteria can attach on the AMF spore walls and how closely they are associated.

Lecomte et al. So far, the identified hyphal exudates such as formate, acetate, glucose and oligosaccharides can be utilized by bacteria as carbon sources [ 44 , 45 ], and other unidentified trace substances might also influence bacterial growth [ 46 ].

Bacteria having hydrolytic enzyme activities such as chitinase and cellulase were associated with AMF spores [ 14 ]. Hydrolytic enzyme activity may help bacteria to associate with AMF spore wall and hyphae [ 11 , 47 ]. Our results revealed that bacteria closely associated with F.

Chitinolytic activity of bacteria also enhances AMF and plant root recognition [ 48 ] by stimulating the spores to produce more signaling molecules. In a recent study, Genre et al. Chitinolytic activity of the spore associated bacteria have previously been reported. However, other possible nutrient sources present in the spore walls are not clearly understood. In our results, we found that higher percentage of SAB isolated from disinfected spores had protease activity suggesting that protease activity might play a vital role in AMF and bacteria association by enabling the bacteria to utilize the protein as a nutrient source.

Likewise, high percentage of cellulolytic SAB have been found on the spore walls which is in line with Albertsen et al. EPS production has been reported to be involved in bacterial association with AMF spore and hyphae [ 34 , 35 , 51 , 52 ]. Bianciotto et al. In our study, we found that SAB produce exopolysaccharide, a binding material which help the bacteria aggregate and attach to the spore walls.

Molecular identification of SAB revealed that bacteria belonging to different genera were associated with each AMF spores. Bacteria isolated from 20 min disinfected spores, Bacillus spp. In our study, we found that bacterial genera Bacillus and Lysinibacillus were associated with all three types of spores. The occurrence of Bacillales, Sphingomonadales, Burkholderiales, Pseudomonadales, Actinomycetales and Flavobacteriales is consistent with previous studies [ 7 , 16 , 53 — 55 ] suggesting that order Actinomycetales and Burkholderiales were particularly abundant in the AMF spores.

Bacillus species are often reported to have close association with AMF spores. Several studies have reported that Bacillus species were associated with AMF mycelium [ 5 ], decontaminated spores walls [ 6 ] and within mycorrhizal roots [ 55 ].

The members of order Bacillales have been reported to potentially improve spore germination, hyphal growth and mycorrhizal colonization [ 22 , 56 ]. Pseudomonas species, a widespread soil bacteria, were often found to associate with AMF spores. Xavier and Germida [ 6 ] reported that Pseudomonas species such as P.

Pseudomonas sp. The most common spore associated bacteria Variovorax paradoxus [ 5 , 6 ] has been retrieved in our study as well.

However, we also retrieved bacterial cultures of Bacillus anthracis from Racocetra alborosea and Funneliformis mosseae spores, and yet their roles in association with AMF spores are not clear. Recently, Battini et al. Fungi and bacteria are present everywhere and have been found to live side by side and interact. The range of mechanisms for both symbiosis and antagonism takes place during AMF and bacterial interaction.

Our results demonstrate that bacteria which produce hydrolytic enzymes and exopolysaccharides are closely associated with AMF spore walls. Diverse bacterial communities were associated with each AMF spores. Bacteria belonging to the same species were associated with different AMF species. Bacteria belonging to orders Bacillales, Sphingomonadales, Burkholderiales, Pseudomonadales and Actinomycetales are found to be closely associated with AMF.

The results of our study provide insights into the bacterial communities associated with spores of F. Further molecular studies may reveal the exact mechanism for AMF and bacteria association which will bring us new insights in the field of fungal-bacterial interaction. Data curation: GS RK. Funding acquisition: TS. Investigation: GS KK.

Methodology: TS GS.