Expression of phenazine biosynthetic genes during the arbuscular mycorrhizal symbiosis of Glomus intraradices.

To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr) of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010). Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD) is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

INTRODUCTION

The arbuscular mycorrhiza (AM) is a complex and intimate association of organisms formed by fungi of the Glomeromycota phylum and different taxonomic groups of plants, including 80% of the terrestrial flora (Schüssler et al., 2001; Smith and Read, 1997). After the consistent discovery of endocellular bacteria within mycorrhizal fungi (Macdonald M.R. and Chandler R.M. 1981; Bianciotto ), AM has been considered to be the result of a tripartite symbiosis (Bonfante, 2003). The absence of these bacteria from fungal spore, results in important changes in fungal presymbiotic growth and sporal morphology, suggesting that they are important for mycellium germination and possibly ecological fitness (Lumini ); however, their main role during the establishment and maintenance of the AM symbiosis remains unknown. Studies attempting to elucidate their physiological role have been hindered by a lack of protocols allowing their cultivation in vitro (Bianciotto ; Bonfante and Anca, 2009), a difficulty that could be related to the impossibility of reproducing essential conditions of the fungal cytoplasmic milieu necessary for their survival (Jargeat ).

Some understanding of the role played by non-AM bacterial endosymbionts has emerged from studies of plant pathogenic fungi belonging to the genus Rhizopus, in which the antimitotic polyketide rhizoxin - responsible for causing seedling blight in rice - is biosynthesized by bacteria of the genus Burkholderia (Partida-Martínez and Hertweck, 2005). Rhizopus microsporus does not form sporangia and spores in the absence of its endosymbionts, indicating that Burkholderia produce metabolic factors that are essential for the fungal life cycle (Partida-Martínez ). In addition to confirming the potential for a large genetic diversity intrinsic to Burkholderia and perhaps other families of endosymbiotic bacteria (Komatsu ), the recent elucidation of a Burkholderia rhizoxinica genome revealed an evolutionary tendency not only towards specialized uptake of fungal metabolites, but also to a bacterial-dependent provision of putative phytotoxins and virus-related factors that could promote nutritional uptake from decaying plants (Lackner ). The phenotypic diversity of endosymbionts is also reflected by their capacity for producing a large variety metabolites involved in antagonistic interactions such as phenazines, a large family of heterocyclic nitrogen antibiotical compunds produced by different bacteria such as Burkholderia, Streptomyces and Pseudomonas ssp., but not animals or plants (Thomashow and Weller, 1988; Komatsu ; Delaney ; Blankenfeldt ; Laursen and Nielsen, 2004; Fitzpatrick, 2009).

In contrast to non-AM endocellular bacteria, the genetic diversity, metabolic potential, and physiological contribution of AM endosymbionts remains largely unexplored. Endocellular bacteria have been reported in several Glomeromycota species that include Glomus versiforme, Acaulospora laevis, and Gigaspora margarita (MacDonald and Chandler, 1981; Bianciotto, 2000; Naumann, 2010), as well as the ectomycorrhizal basidiomycete Laccaria bicolor for which a complete genome is available (Bertaux ; Martin ). In several Gigaspora species, rod-shaped Gram negative endosymbiotic bacteria were demonstrated to be present in spores, germ tubes, and hyphae, but not arbuscules (Bianciotto ). A genomic library of G. margarita had a partial representation of the genome of its bacterial endosymbionts (van Buuren ), and although subsequent studies suggested that some sequences could have resulted from contamination with foreign bacterial DNA (Jargeat ), a gene encoding a putative phosphate transporter, and a gene involved in cell colonization by entero-invasive pathogenic bacteria were unequivocally confirmed as being derived from genetic material contained in the spores G. margarita (Ruiz-Lozano and Bonfante, 1999; Anca ), demonstrating the presence of bacterial DNA within the fungus.

Here we analyze a sample of cDNA clones generated from mRNA present in Glomus intraradices BE3 prior to the establishment of the AM symbiosis, identifying numerous transcripts with homology to eukaryotic or prokaryotic genes. We show that a gene with homology to the bacterial pleiotopic regulator Phenazine Biosynthesis Regulator (Pbr) is expressed within fungal cells at presymbiotic stages, and present in genomic DNA samples extracted from bacterial isolates cultivated from Glomus intraradices BE3 spores. We also show that a homolog of the bacterial phenazine biosynthetic gene phzD - also present in the genome of bacterial isolates from Glomus intraradices BE3 spores - is expressed at the time of the establishment of the fungal-plant symbiosis. Our results indicate that phenazine biosynthetic genes are active in G. intraradices BE3, opening possibilities for studying its function and regulatory mechanisms during the AM symbiosis.

MATERIALS AND METHODS

Mycorrhizal material

Spores of Glomus intraradices strains BE2 and BE3 were obtained from the in vitro collection of Departamento de Biotecnología y Bioingeniería CINVESTAV Zacatenco, and recovered following chelation with sodium citrate as described in Doner and Bécard (1991). Spores of Gigaspora margarita BE2 were isolated from potted plant trap cultures with Sorghum sp. and Lolium sp., following the method of tween-sucrose flotation (Gerdemann and Nicholson, 1963).

cDNA library construction and sequencing

Spores of G. intraradices BE3 were germinated in the presence of Ri T-DNA transformed roots of carrot. No physical interaction was allowed by insertion of a cellophane membrane between the fungus and plant tissues. Germinated spores were recovered with insulin syringes (Beckton-Dickinson) under a stereo-microscope and immediately frozen in liquid nitrogen. Total RNA was obtained using the RNAeasy mini kit (Qiagen) and treated with RNase-free DNase I according to manufacturer's instructions (Invitrogen). Total RNA was quantified by NanoDrop® ND-1000 (Spectrophotometer Termo Scientific), and PCR-tested for integrity using primers r18S and f18S (sequence available in Supplementary Materials).

Total RNA concentration was adjusted to 450 ng and used for cDNA synthesis using a CDS III/3' PCR primer [5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30-(A,G,C) -(A,G,C,T)-3'] and the SMART kit (Clonetech). Double-stranded cDNA was purified and cloned into pCR 2.1 TOPO (Invitrogen). A random sample of 960 cDNA clones was sequenced using M13 forward and reverse primers and the Terminador Bigdye kit (ABI). Resulting sequences were assembled and filtered using parameters of minimum overlap 25, maximum gap 2, minimum overlap identity 80%, and maximum ambiguity 4 (Drummond et al., 2010. Geneious v 5.0); sequences representing unique genes, were compared to entries of the NCBI database using the non-redundant BLASTX algorithm (Worley ).

Fungal spore cultivation

Spores of G. intraradices BE3 were recovered from petri dishes divided by 10 mM sodium acetate. Spore disinfection was performed with 0.05% Tween 20, 2% chloramine T twice for 5 minutes, followed by 3 rinses in sterile distilled water. Samples were subsequently stored in a 100 ppm of gentamicin, 200 ppm streptomycin solution at 4°C (Bécard and Piché, 1992). For spore bursting and bacterial isolation, approximately 500 spores/plate were inoculated in minimal mineral media (M) medium (Fortin ), in the absence of sucrose and following a pH gradient from 3 to 10 with increments of 0.5 units. Three replicates of each plate were incubated at 25°C until spore bursting and bacterial growth occurred. Successfully isolated bacterial colonies were transferred into two different culture media: a rich Bacto Nutrient Agar culture medium (Difco), and a poor M culture medium containing sucrose and potato dextrose agar (Bioxon), and subsequently cultivated in Bacto Nutrient broth culture for DNA extraction following Harwood and Cutting (1990). FD1 and RD1 primers were used for 16S rDNA PCR amplification (Weisburg ) and PCR products were cloned into the pCR 2.1 TOPO vector and sequenced by Sanger. Sequence comparisons were conducted following conventional BLAST algorithms (Worley ). Closely related sequences were analyzed using Geneious 5.0 under a Tamura-Nei genetic distance model, (Drummond ; Geneious v 5.0). New DNA sequences were deposited in the National Center for Biotechnology Information database (NCBI).

RT-PCR

Total RNA was extracted from growing hyphae using TRIzol (Invitrogen). Reactions were performed using One-Step RT-PCR kit (QIAGEN) and the annealing temperature was standardized for each pair of primers used (Supplementary Table 2). In all cases reverse-transcription reactions were performed with 3 biological replicates representing independent events, using 100 ng of total RNA and 50 pmoles/μl of each primer. PCR conditions were 1 minute at 94°C (denaturation), 30 seconds at 55-60°C (alignment), 1 min at 72°C (extension) for 28 cycles, followed by a final extension at 72°C for 5 minutes.

RESULTS

Transcripts with homology to eukaryotic or prokaryotic genes are expressed at pre-symbiotic stages of the G. intraradices BE3 life cycle

To conduct a transcriptional analysis of AM spore germination and early hyphal growth, we constructed a cDNA library using mRNA from an in vitro culture of G. intraradices BE3 at asymbiotic to presymbiotic stages, in the absence of physical interaction with plant tissues (D.G. León-Martínez, J-Ph. Vielle-Calzada, and V. Olalde-Portugal, unpublished results). An initial screen of the 104 col/μg titer colony collection resulted in the identification of 458 distinct open reading frames on the basis of 960 sequenced clones (Supplementary Table 1). A comparison of assembled coding sequences to publically available databases revealed that the corresponding genes encode for a large variety of proteins covering a wide spectrum of predicted molecular functions in eukaryotes. Whereas 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, 9% showed high homology to previously characterized bacterial genes, and 41% did not show homology with reported genomic sequences. cDNA sequences with homology to bacterial genes encode proteins involved in housekeeping, secondary metabolism or signal transduction pathways, putative transcription factors, or proteins conferring resistance to antibiotics such as tetracycline (Table 1).

Table 1

Identification of sequences with homology to prokaryotic genes and expressed during pre-symbiotic stages of Glomus intradices BE3.

Length (pb)	PROTEIN	ORGANISM	Accession number	E-value
635	PBR	Burkholderia cenocepacia	ACJ54935	1.15e-11
647	hypothetical protein	Clostridium nexile	ZP_03289628	2.71e-31
813	hypothetical 16.9K protein	Salmonella typhimurium	JQ1541	8.72e-40
269	hypothetical protein	Azorhizobium caulinodans	YP_001526574	5.88e+00
437	hypothetical protein	Bacteroides uniformis	ZP_02071990	9.91e+00
439	helix-hairpin-helix DNA-binding motif-containinG protein	Methylobacterium sp.	YP_001772034	2.77e-02
230	oxidoreductase aldo/keto reductase family1	Vibrio coralliilyticus	ZP_05886239	2.84e-02
235	NADH pyrophosphatase	Vibrio splendidus	ZP_00991846	2.64e+00
288	GGDEF domain protein	Desulfovibrio magneticus	YP_002953232	7.77e+00
421	hypothetical protein	Salmonella enterica subsp. arizonae serovar 62:z4,z23	YP_001571636	2.03e+00
538	Tetracycline resistance protein	Bacillus cereus	ZP_04234556	8.92e-01
528	putative nitrogen metabolite repression regulator	Rhizobium leguminosarum bv. viciae	YP_766861	5.57e-08
262	arginine biosynthesis bifunctional protein ArgJ	Clostridium papyrosolvens	ZP_05498017	6.74e-04
335	hypothetical protein BDI	Parabacteroides distasonis	YP0130449_03	6.89e-01
220	oxidoreductase aldo/keto reductase family	Vibrio coralliilyticus	ZP_05886239	5.38e-09
292	aldo/keto reductase	Azorhizobium caulinodans	YP_001527069	5.36e-01
642	sulfotransferase	Methanocaldococcus vulcanius	ZP_05303437	1.87e+00
220	hypothetical protein	Bacteroides fragilis	YP_212901	5.58e-07
314	mobilization (Mob)/recombination (Pre) protein	Listeria monocytogenes	AAA93293	7.79e+00
694	hypothetical protein CYB_1972	Synechococcus sp.	YP_478184	2.28e-05
302	high-affinity zinc transporter periplasmic component	Actinobacillus succinogenes	YP_001345069	1.40e-01

To validate the presence of some of these transcripts in G. intraradices BE3 and determine their temporal pattern of expression, we conducted reverse-transcriptase PCR (RT-PCR) in a random group of genes at different developmental phases during the establishment of the AM symbiosis (Figure 1 and Supplementary Table 2). These phases include fungal spores at the time of germination (asymbiotic phase), germinated spores during early hyphal growth (pre-symbiotic phase), initial penetration of fungal hyphae into plant roots prior to the appearance of arbuscular structures (symbiotic phase), and fully established AM symbiosis showing external hyphae with second generation spores and well developed arbuscular structures within plant roots (extradical phase). Most genes were expressed either before or at the time of spore germination, confirming that all of them are transcribed during the presymbiotic phase of hyphal growth. Whereas most transcripts could also be detected during the extradarical phase of the life cycle, only 2 out of 8 were expressed at symbiosis, suggesting that an important shift in gene expression distinguishes the pre-symbiotic and symbiotic phases. These results indicate that transcripts identified in our presymbiotic cDNA library are expressed in germinating spores of G. intraradices BE3.

Figure 1

RT-PCR of genes expressed during the G. intraradices BE3 life cycle. cDNA amplification was performed during the asymbiotic (a), presymbiotic (p), symbiotic (s), and extraradical (e) phase of the fungal life cycle (see text for details), and (nc) negative control. (1) Hypothetical Protein homologue of Cryptococcus neoformans XP570866.1; (2) Hypothetical Protein homologue of Schizosaccharomyces pombe NP594031.1; (3) Hypothetical Protein homologue of Caenorhabditis briggsae CAE71635.1; (4) Hypothetical Protein homologue of Coprinopsis cinerea EAU86123.); (5) Hypothetical Protein homologue of Botryotinia fuckeliana XP001557861.1; (6) Hypothetical Protein homologue of Coprinopsis cinerea EAU88851.1; (7) Hypothetical protein with no significant homology; (8) Hypothetical Protein homologue of Yarrowia lipolytica XP502676.1.

Expression of GintPbr1a, a gene encoding a protein homolog of Phenazine Biosynthesis Regulator (Pbr)

We identified a cDNA clone with high homology to Phenazine Biosynthesis Regulator (Pbr), a transcription regulator of the Burkholderia cenocepacia complex required for the expression of phzD and phzF, two genes encoding proteins with significant homology to Pseudomonas choloroaphis PhzD and PhzF and involved in phenazine biosynthesis. We named this sequence GintPbr1a (for Glomus intraradices Pbr protein 1a; Franken P. 2002). The cDNA includes a transcript sequence corresponding to a 57-amino acid residue containing a predicted helix-residue-helix motif that is commonly found on prokaryotic transcriptional regulators of Burkholderia cenocepacia (Figure 2A). At the amino acid level, both sequences only differ at Pbr amino acid positions 27, in which glycine (G) is replaced by alanine (A), and positions 29 to 32, in which an arginine-valine-valine (R-V-V) motif is replaced by threonine-isoleucine-glycine-isoleucine (T-I-G-I). The strong conservation of the amino acid sequence between Pbr and GintPbr1a suggested that both proteins could play equivalent biochemical roles, despite being present in bacteria with highly divergent biological habits.

Figure 2

Expression of a phenazine biosynthetic pathway in endocellular bacteria of Glomus intraradices BE3. (A) Predicted amino acid alignment of GintPbr1a of G. intraradices BE3 and Pbr of Burkholderia cenocepacia. (B) RT-PCR expression of GintPbr1a and BGintphzD in G. intraradices BE3; cDNA amplification was performed during the asymbiotic (1), presymbiotic (2) and, symbiotic (3), phase of the fungal life cycle (see text for details). (C) Genomic PCR amplification of BGintPbr1 (for bacterial GintPbr1a) and BGintphzD (for bacterial GintphzD) in a bacteria isolated from spores of G. intraradices BE3. Whereas lane 1 represents the isolate BG1 from G. intraradices BE3, lanes 2 to 5 represent isolates BG3, BG4, BG7 and BG10 from G.margarita BE2, respectively.

To confirm the expression of GintPbr1a within cells of G. intraradices BE3 and determine its temporal pattern of activity, we conducted RT-PCR at previously described developmental phases during the establishment of the AM symbiosis. GintPbr1a was abundantly expressed at the presymbiotic phase and weakly at the extraradical phase, but not in the asymbiotic or symbiotic phase (Figure 2B), suggesting that its expression is mainly restricted to initial stages of hyphal growth, before the establishment of the AM symbiosis. These results validated the expression of the Burkholderia Pbr homolog in G. intraradices BE3, and indicated that its transcriptional activity occurs mainly during the presymbiotic phase of the fungal life cycle.

Bacterial isolates from in vitro cultivated G. intraradices spores express genes involved in phenazine biosynthesis

The molecular nature and the pattern of expression of GintPbr1a suggested that the corresponding gene could have a bacterial origin within the AM fungus considering that the cDNA library was generated using polyadenylated primers that can potentially target prokaryotic transcripts present within spores and hyphae. To determine if the genome of a potential bacterial symbiont could contain a GintPbr1a gene, we isolated bacterial cultures originating from sterilized spore inocula of G. intraradices BE3, and of Gigaspora margarita BE2 under in vitro culture and variable pH conditions. Whereas one bacterial isolate could be recovered from G. intraradices BE3 (pH range from 4 to 7), four were recovered from G. margarita BE2 (pH range from 4 to 9). After the extraction of sufficient DNA from these isolates, and to attempt their taxonomic identification, we amplified and sequenced PCR products of approximately 1.5 kb corresponding to the conserved genomic DNA 16S ribosomal subunit (Table 2; Weisburg ). Comparison to publicly available genomic databases revealed that the isolate recovered from G. intraradices BE3 corresponds to a previously described "uncultured bacteria" that we named BG1, whereas isolates from G. margarita BE2 correspond to presumed Brevibacillus sp. (BG10), Paenibacillus sp. (BG4), and two uncultured bacterium (BG3 and BG7), respectively.

Table 2

Identification of bacteria derived from spores of G. intraradices BE3 and G. margarita BE2.

Name	Lenth (pb)	Phylogenetic relationship and GenBank accession number	Isolated from	Similarity %	E-value	Identity %
BG10	1523	Brevibacillus centrosporus(AB112719.1)	G. margarita	97	0.0	99
BG4	1575	Paenibacillus graminis(AB428571.1)	G. margarita	98	0.0	98
BG3	1523	Uncultured bacterium(EU236261.1)	G. margarita	98	0.0	99
BG7	1524	Uncultured bacterium(EU560794.1)	G. margarita	98	0.0	96
BG1	1434	Uncultured bacterium GU223217.1	G. intraradices	100	0.0	99

To find if some of these bacteria could include a GintPbr1a copy in their genome, we attempted the amplification of a 246 bp fragment using genomic DNA from all 5 previously cultivated bacterial isolates. A PCR product of the correct size was only amplified from BG1, the bacterial isolate from G. intraradices BE3. (Figure 2C). PCR products derived from BG4 and BG10 were also amplified, but their molecular size did not correspond to the predicted GintPbr1a fragment, indicating that a different genomic version of a Pbr-like gene could be present in the genome of some G. margarita BE2 endosymbionts. We also conducted genomic PCR amplification with primers specific to phzD, a gene that is under transcriptional control of Pbr in Burkholderia. As expected, a PCR product was amplified in BG1 (Figure 2C), confirming that key regulatory enzymes of the phenzine biosynthetic pathway are encoded in the genome of bacteria isolated from in vitro cultures of G. intraradices BE3 spores. A PCR product was also derived from BG7 and BG10, confirming that isolates from G. margarita BE2 are also likely to express the same pathway. Finally, we conducted RT-PCR to determine if G. intraradices BE3 could express a homologue of phzD, a gene encoding a protein D isochorismatase phenazine biosynthetic enzyme involved in the pathway of pyocianin (Komatsu et al., 2003). As shown in Figure 2B, a gene encoding a phzD ortholog is expressed at the symbiotic phase, confirming that a phenazine transcriptional regulatory pathway is active in G. intraradices BE3.

DISCUSSION

To explore the molecular mechanisms that prevail during the establishment of the AM symbiosis involving the genus Glomus, we initiated a systematic global expression analysis of early phases of the G. intraradices life cycle. The large amount of expressed genes found at presymbiotic stages confirmed that fungal cells are active during early hyphal growth, and contain a wide diversity of transcripts with homology to eukaryotic and prokaryotic genes. While some of the identified transcripts with homology to prokaryotic genes (such as a sulfotransferase, a pyrophosphastase, and several reductases) could reflect global housekeeping functions related to the general metabolism, other expressed genes such as a G protein with a DNA binding domain, a high affinity Zn transporter, or a nitrogen metabolism negative regulator, could suggest that a cross-talk between trancriptionally active bacterial cells and fungal cells occurs early during the fungal life cycle. While a large group of hypothetical and unknown proteins requires further annotation and functional elucidation, additional prokaryotic expressed genes include those related to the biosynthesis of essential components such as amino acids, and those involved in the protection against potential pathogens or the production of antibiotics.

Phenazines are secondary metabolites of bacterial origin that have been implicated in the control of plant pathogens, contributing to the ecological fitness and pathogenicity of the producing strains. While the evolution and distribution of phenazine genes has revealed that they are mainly found in soil-dwelling or plant associated bacterial species (Mavrodi et al., 2010), their presence in the genome of AM fungal species had not been reported. The establishment of a pH gradient-based protocol allowed the isolation of bacterial colonies from G. intraradices BE3 spores. Although endocellular bacteria have not been reported in G. intraradices, this type of endosymbionts have been reported in other Glomus species (Naumann ). The identification of a numerous transcripts with homology to eukaryotic genes, associated to the recovery of bacterial colonies from spores, suggests that G. intraradices BE3 indeed could contain bacterial cells within hyphae. While their taxonomic identification remains elusive, successful cultivation and DNA extraction confirmed that both Pbr and phzD homologs are present in genomic DNA extracted from G. intraradices spores and its associated bacterial isolates.

Our overall results indicate that a phenazine biosynthetic pathway is active during the G. intraradices BE3 life cycle.

They also suggest that this pathway is not exclusive of G. intraradices, but is also active in G. margarita. As in previous studies of the biochemical mechanisms that regulate phenazine biosynthesis in Burholderia and Pseudomonas (Laursen ; Mavrodi ; Parsons ), GintPbr1a is transcriptionally active at developmental stages that precede the expression of GintphzD, a result in agreement with the role of GintPbr1a as a pleiotropic transcriptional regulator necessary for the activation of phenazine biosynthetic enzymes (Ramos ), presuming that GintPbr1a transcripts precede the translation of the corresponding protein at subsequent developmental stages. Interestingly, initial expression of GintphzD occurs at the onset of the establishment of the fungal-plant symbiotic interaction, suggesting that phenazine production could play a role a later stages of the AM symbiosis establishment; however, a detailed molecular and biochemical analysis will be require to confirm the presence of endocellular bacteria in hyphal cells, as well as the presence of phenazines within roots before elucidating the physiological role of these molecules during AM symbiosis. Taken together, our results open new possibilities for using G. intraradices BE3 as a model system to study the molecular and biochemical mechanisms that allow the successful establishment of the tripartite AM symbiosis.