Plant Signaling & Behavior
ISSN: (Print) 1559-2324 (Online) Journal homepage: http://www.tandfonline.com/loi/kpsb20
RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization Enrico Gobbato, Ertao Wang, Gillian Higgins, Syeda Asma Bano, Christine Henry, Michael Schultze & Giles ED Oldroyd To cite this article: Enrico Gobbato, Ertao Wang, Gillian Higgins, Syeda Asma Bano, Christine Henry, Michael Schultze & Giles ED Oldroyd (2013) RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization, Plant Signaling & Behavior, 8:10, e26049, DOI: 10.4161/psb.26049 To link to this article: http://dx.doi.org/10.4161/psb.26049
Published online: 20 Aug 2013.
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Date: 14 September 2015, At: 08:58
Short Communication
Short Communication
Plant Signaling & Behavior 8:10, e26049; October 2013; © 2013 Landes Bioscience
RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization Enrico Gobbato1, Ertao Wang1, Gillian Higgins2, Syeda Asma Bano2, Christine Henry3, Michael Schultze2, and Giles ED Oldroyd1* Department of Cell and Developmental Biology; John Innes Centre; Norwich, UK; 2Department of Biology; University of York; York, UK; 3The Food and Environment Research Agency; Sand Hutton, York, UK
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Keywords: symbiosis, mycorrhiza, arbuscule, Aphanomyces, promoter
The establishment of the symbiotic interaction between plants and arbuscular mycorrhizal (AM) fungi requires a very tight molecular dialog. Most of the known plant genes necessary for this process are also required for nodulation in legume plants and only very recently genes specifically required for AM symbiosis have been described. Among them we identified RAM (Reduced Arbuscular Mycorrhization)1 and RAM2, a GRAS transcription factor and a GPAT respectively, which are critical for the induction of hyphopodia formation in AM fungi. RAM2 function is also required for appressoria formation by the pathogen Phytophtora palmivora. Here we investigated the activity of RAM1 and RAM2 promoters during mycorrhization and the role of RAM1 and RAM2 during infection by the root pathogen Aphanomyces euteiches. pRAM1 is activated without cell type specificity before hyphopodia formation, while pRAM2 is specifically active in arbusculated cells providing evidence for a potential function of cutin momomers in the regulation of arbuscule formation. Furthermore, consistent with what we observed with Phytophtora, RAM2 but not RAM1 is required during Aphanomyces euteiches infection.
During the establishment of the Arbuscular Mycorrhizal (AM) symbiosis between plants and fungi belonging to the Glomeromycota phylum, a suite of molecular signals from both the plant and the fungus play a critical role for the mutual recognition between the two symbionts.1 Plants release strigolactones in root exudates2 and the perception of strigolactones by AM fungi results in increased spore germination, activation of oxidative metabolism and induction of hyphal branching.3 More recently it was shown that AM germinating spores in turn produce lipo-chito oligosaccarides (AM-LCOs) structurally similar to those produced by rhizobial bacteria.4 AM LCOs are perceived by the plant and trigger symbiotic responses including promotion of AM colonization, transcriptional reprogramming and stimulation of lateral root formation.5 When AM fungal hyphae reach the plant root surface, they develop adhesion structures, the hyphopodia, which are structurally simpler than appressoria of pathogenic fungi.6 Critical for the formation of AM hyphopodia is the production of cutin monomers by the plant which can also induce appressoria formation in pathogenic fungi.7 Upon penetration through the epidermal cell layer, which has been shown to be a plant directed process,8 fungal hyphae grow inside the plant root, reaching the inner root cortex within which they develop arbuscules, highly
branched hyphae surrounded by the periarbuscular membrane of plant origin.6 Nutrient exchanges take place at this interface and are mediated by transporters that are specifically expressed within the arbuscule in both the plant and the fungus.9-11 The knowledge we have of the plant signaling pathways that regulate recognition and response to AM fungi has hitherto been derived from the study of nodulation mutants previously identified in model legume plant species.12 Several of these mutants were also proven to be defective for AM symbiosis and the underlying genes are therefore commonly termed Sym (Symbiosis) genes.13 The activation of a common signaling pathway by different microbial symbionts, raises the problem of understanding how plants can achieve specificity in the activation of two genetic programs which are radically different.12 In this respect the identification of genetic components with specific functions in either the nodulation or AM pathway is critical. Within the Nodulation pathway multiple transcription factors were identified acting downstream of the Sym pathway, among which, NSP (Nodulation Signaling Pathway) 1 and NSP2, belong to the GRAS family.14,15 In contrast to the initial assumption that these transcription factors were specifically required for nodulation, recent evidence showed that both NSP1 and NSP2 also contribute to responses to AM fungi5,16
Correspondence to: Giles ED Oldroyd; Email: [email protected] Submitted: 07/29/2013; Accepted: 08/05/2013 Citation: Gobbato E, Wang E, Higgins G, Bano SA, Henry C, Schultze M, Oldroyd GE. RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization. Plant Signaling & Behavior 2013; 8:e26049; http://dx.doi.org/10.4161/psb.26049
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Figure 1. pRAM1 and pRAM2 Activities. A–C Medicago truncatula composite plants expressing pRAM1:GUS and pRAM2:GUS were inoculated with Rhizophagus irregularis (R.i.) purified spores and roots were stained at 6 wpi A. GUS stained pRAM1:GUS roots B. GUS stained pRAM2:GUS roots c. Fuchsin stained pRAM2:GUS roots. In b. and c. an arbusculated cell is indicated with a red arrow. Scale bar = 100 μm D–F pRAM1:GUS root cultures were co-cultivated with R.i., hyphal growth was monitored and co-cultivation was interrupted before physical contact. D. Image of a root portion in co-cultivation with AM fungi. Scale bar = 1 cm E. root portion shown in d. after GUS staining. Scale bar = 1 cm F. Ink stained root in E. No hyphopodia could be observed. Scale bar = 1 mm.
while both NSP1 and NSP2 function in the activation of strigolactone biosynthesis.17 In an effort to identify novel components specific to the AM pathway, we identified two genes, RAM (Reduced Arbuscular Mycorrhization)1 and RAM2.7,18 Plants mutated in either gene retained the capability to support nodulation, but showed dramatically decreased levels of mycorrhization and this was associated with severely reduced numbers of hyphopodia at the root surface. Complementation experiments demonstrated that RAM2 encodes a Glycerol-3-Phosphate Acyl Transferase (GPAT ), involved in the synthesis of cutin monomers.7 RAM1 encodes a GRAS transcription factor which regulates the expression of RAM2,18 consistent with the similarities between ram1 and ram2 symbiotic phenotypes. In addition to the hyphopodia phenotype, ram2 plants also displayed a severe defect in arbuscule formation, which suggests a critical function for RAM2 synthesized cutin monomers during arbuscule development.7 RAM2 is also required to stimulate appressoria formation in the pathogenic oomycete Phytophtora palmivora, suggesting that in order to establish parasitism primordial microbial pathogens
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have hijacked a preexisting symbiotic signaling pathway to manipulate their host metabolism.7 To better understand the mechanism of action of RAM1 and RAM2, we performed a study of RAM1 and RAM2 promoters. We used Agrobacterium rhizogenes transformation of Medicago truncatula to assess root expression of pRAM1 and pRAM2 driving the expression of the GUS reporter gene.19,20 Composite plants were inoculated with purified spores of the AM fungus Rhizophagus irregularis and roots were collected and stained for GUS activity at 6 wpi. As shown in Figure 1A and B, while pRAM1 showed homogeneous activity in all tissues of colonized roots (that was absent in uncolonized roots), the RAM2 promoter was strongly activated in cortical cells harboring arbuscules. To confirm the presence of arbuscular structures within cells displaying pRAM2 activity, we dually stained inoculated root tissues for GUS activity and with Acid Fuchsin to mark fungal structures. As shown in Figure 1B and C overlap confirmed the specific activation of the RAM2 promoter in cortical cells hosting arbuscules.
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Figure 2. Arbuscule development in ram1-2. Wild type (R108) and ram1-2 plants were inoculated by co-cultivation with colonized leek nurse plants. After 6 weeks of co-cultivation root samples were taken and ink stained. While wild-type plants (left panel) showed fully developed arbuscules (Fd arb, red arrow), ram1-2 plants (right panel) predominantly showed small undeveloped arbuscules (Incomp arb, red arrow) consistent with a function of RAM1 in arbuscule development. Scale bar = 50 μm.
Previously we had not been able to observe an arbuscule defect in ram1 mutants. However, this was surprising, since RAM1 regulates RAM2 expression and RAM2 is induced in arbusculated cells and is required for arbuscule development. This suggests that RAM1 may also function during arbuscule development. Under our growth conditions, we observed very little AM colonization in the ram1-1 mutant, limiting the characterization of arbuscule development in this allele. However the ram1-2 mutation is slightly more permissive than ram1-1 and using leek nurse plants, a very strong source of inoculum, we were able to observe a significantly increased number of hyphae reaching the inner cortex in ram1-2, compared with what we had previously seen with ram1-1. In contrast to what we had observed for ram1‑1, we observed a defect in arbuscule formation in ram1-2, that resulted in smaller, undeveloped arbuscules (Fig. 2), similar to what we previously observed for ram2.7 This suggests that both RAM1 and RAM2 have functions at later stages of mycorrhizal colonization, being required for appropriate arbuscule formation. We previously showed that upon Rhizophagus irregularis colonization RAM1 displays a maximum peak in its expression at 30 dpi, but a slight increase in RAM1 transcript levels could already be observed by qPCR at 20 dpi, a time point at which mycorrhizal colonization of the root is limited.18 To monitor in greater detail pRAM1 activation at very early time points, we generated root cultures of M. truncatula pRAM1:GUS. The pRAM1:GUS root cultures were co-cultivated with carrot root cultures colonized by Rhizophagus irregularis as previously described.21 This experimental system permitted us to follow the progression of AM fungal hyphae toward the Medicago roots (as shown in Figure 1D) and allowed us to assess early induction of pRAM1. When stained for GUS activity, pRAM1:GUS roots showed activation of the RAM1 promoter in areas in close proximity to the fungal hyphae before physical contact as shown in Figure 1E. To confirm that co-cultivated roots were sampled before hyphopodia formation, GUS stained roots were also subsequently ink stained and no hyphopodia could be observed
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(Fig. 1F). No GUS staining was observed in clonal root cultures grown in the absence of the AM fungus (data not shown). This very early activation of pRAM1 is consistent with the role of RAM1 in promoting hyphopodia formation. RAM2 is necessary also for appropriate root colonization by the pathogen Phytophtora palmivora. To assess if this reflected a broader role in pathogen colonization we tested the capability of another root pathogen, Aphanomyces euteiches to colonize ram1 and ram2 roots. ram2 but not ram1 plants showed reduced susceptibility to A. euteiches, measured as reduced percentage of root length colonized by the pathogen (Fig. 3): ram2 plants showed ~3% of their root length colonized, equal to one quarter of the colonization levels of wild type roots. Our work confirms the importance of RAM2 for normal colonization by root oomycete pathogens and this further supports the hypothesis that the function of RAM2 has been coopted by pathogenic oomycetes to facilitate their own colonization. It will be interesting to test whether root pathogens belonging to the Ascomycota or Basidiomycota also require RAM2 during their colonization as could be hypothesized given the capability of cutin to induce appressoria formation in their foliar pathogen counterparts.22,23 We have shown that RAM1 and RAM2 are required for the formation of hyphopodia at the root surface and for appropriate arbuscule formation in the root cortex. These two stages represent the periods of intracellular colonization by mycorrhizal fungi and we propose that the regulation of RAM2 to activate cutin production may be necessary for all stages of mycorrhizal intracellular colonization. The expression of RAM1 is consistent with a role at both the root surface and the root cortex, although we do not see expression restricted to colonized cells. In contrast the induction of RAM2 appears to be associated with cortical cells containing arbuscules. This is consistent with a role for RAM2 in the root cortex, but does not explain its function at the root surface. Longer GUS staining in uninoculated roots revealed apparent low level RAM2 expression throughout
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Figure 3. Aphanomyces euteiches colonization levels. Wild type (A17), ram1 and ram2 plants were inoculated with Aphanomyces euteiches. Roots were collected and ink stained at 14 dpi and the percentage of colonized root length measured. Error bars represent standard errors (n = 15–18).
the root (data not shown). This low level constitutive expression may explain RAM2 function at the root surface. Material and Methods A region of 2,451 Kb from the RAM1 start codon to the end of the next predicted gene in the genome was selected as the RAM1 promoter. This region was amplified using Gateway ® compatible primers (pRAM1 – f GGGGACAAGT TTGTACAAAA AAGCAGGCTT CCCTT TTCTTGTCCT TTTACCACC pRAM1 – r GGGGACCACT TTGTACAAGA AAGCTGGG TCTTTTTTCC CACCCTTTTT TATTTG), cloned into a pDONR 207 vector by BP reaction and finally transferred by LR reaction into the pBGWFS724 vector (resulting in pBGWFS7pRAM1). A region of 2.3 kb of the RAM2 promoter was used, which is sufficient to drive RAM2 expression in complementation experiments.7 This region was amplified using Gateway compatible primers (pRAM2-gus-f: GGGACAAGTT TGTAC AAAAAAGCAGG CTATTGCCGG TGGGATAAAC AC pRAM2-gus-r: GGGACCACTT TGTACAAGAA AG CTGGGTGTGA AGTTGTTGTG TTTA TATACAC) and inserted into pDONOR207 by Gateway technology. Also this construct was recombined by LR reaction with pBGWFS7 (resulting in pBGWFS7-pRAM2). PBGWFS7-pRAM1 and pBGWFS7-pRAM2 were transformed into roots of M. truncatula wild-type using A. rhizogenes 20 and assessed for promoter activity with G. intraradices at 6 weeks post inoculation. The mycorrhizal colonized roots were GUS stained.19 pBGWFS7pRAM2 transformed roots were subsequently co-stained for with acid fuchsin.25 The stained roots were imaged with a Nikon Eclipse 800 microscope with a filter set for TRITC.
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For the generation of pBGWFS7-pRAM1 root cultures, transformed roots were excised and cultured on M plates25 containing augmentin (400 mg/ml). Transgenic roots were subsequently co-cultivated with mycorrhized transgenic carrot roots21 for a period variable between 2 and 4 weeks until fungal hyphae had grown in proximity of the M. truncatula transformed root. M. truncatula roots were then GUS stained and subsequently ink stained.25 For co-cultivation of M. truncatula with leek, six-week old leek plants colonized with R. irregularis were used. Colonized leek plants were produced by planting seedlings in 9 parts of a mixture of autoclaved sand and Terragreen (equal volumes) and 1 part (by volume) of leek root pieces colonized by R. irregularis. The plants were grown for 6 weeks and then individual colonized leek plants were used as nurse plants. The leeks were potted in the center of a P6 (11 cm diameter) round pot surrounded by 4–5 Medicago plants. Root length colonization was tested 6 weeks after the start of cocultivation by ink staining as described previously.18 For inoculation with Aphanomyces euteiches Drechs (ATCC201684) seeds of M. truncatula were pre-germinated on moist filter paper for 2 d in the dark. Seedlings were subsequently transferred into pots containing a 1:2 mixture of sterilized expanded clay and vermiculite. Plants were grown under constant conditions in a greenhouse (243 mE μmol m-2 s -1 for 12 h; 24°C, 65% humidity) and fertilized with phostrogen (10 ml/9 L) 3 d after transplanting and 7 d after inoculation. To generate the A. euteiches inoculum, 10 disks of 1 cm 2 fungal mycelium grown on corn meal agar were cut out and cultured in 20 ml maltose peptone broth (MPB) for 9 d at room temperature. Zoospore production was induced by washing the mycelium twice in autoclaved lake water. Zoospores were counted and diluted to appropriate concentrations. After 10 d, the plantlets were inoculated by application of zoospore suspensions at the stem basis. Control plants were mock inoculated with autoclaved lake water. At 14 d after inoculation control and inoculated roots were harvested, incubated in 10% (w/v) KOH for 4 d at room temperature, and then stained with ink and acetic acid.25 The percentage of root length colonized by oospores was determined by utilizing the grid line intersection method.26 Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We thank Frank Colditz for providing a culture of and protocols for growth of A. euteiches. This work was supported by the BBSRC, as grants BB/E003850/1 and BB/E001408/1; the European Research Council, as SYMBIOSIS; and the Higher Education Commission, Pakistan.
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References 1. Oldroyd GE. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 2013; 11:252-63; PMID:23493145 and http://dx.doi.org/10.1038/ nrmicro2990. 2. Xie X, Yoneyama K, Yoneyama K. The strigolactone story. Annu Rev Phytopathol 2010; 48:93-117; PMID:20687831 and http://dx.doi.org/10.1146/ annurev-phyto-073009-114453. 3. Parniske M. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat Rev Microbiol 2008; 6:763-75; PMID:18794914 and http://dx.doi. org/10.1038/nrmicro1987. 4. Gough C, Cullimore J. Lipo-chitooligosaccharide signaling in endosymbiotic plant-microbe interactions. Mol Plant Microbe Interact 2011; 24:867-78; PMID:21469937 and http://dx.doi.org/10.1094/ MPMI-01-11-0019. 5. Maillet F, Poinsot V, André O, Puech-Pagès V, Haouy A, Gueunier M, Cromer L, Giraudet D, Formey D, Niebel A, et al. Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 2011; 469:58-63; PMID:21209659 and http://dx.doi. org/10.1038/nature09622. 6. Harrison MJ. Signaling in the arbuscular mycorrhizal symbiosis. Annu Rev Microbiol 2005; 59:1942; PMID:16153162 and http://dx.doi.org/10.1146/ annurev.micro.58.030603.123749. 7. Wang E, Schornack S, Marsh JF, Gobbato E, Schwessinger B, Eastmond P, Schultze M, Kamoun S, Oldroyd GE. A common signaling process that promotes mycorrhizal and oomycete colonization of plants. Curr Biol 2012; 22:2242-6; PMID:23122843 and http://dx.doi.org/10.1016/j.cub.2012.09.043. 8. Bonfante P, Genre A. Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis. Nat Commun 2010; 1:48; PMID:20975705; http://dx.doi.org/10.1038/ncomms1046 9. Pumplin N, Zhang X, Noar RD, Harrison MJ. Polar localization of a symbiosis-specific phosphate transporter is mediated by a transient reorientation of secretion. Proc Natl Acad Sci USA 2012; 109:E66572; PMID:22355114.
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10. Doidy J, Grace E, Kühn C, Simon-Plas F, Casieri L, Wipf D. Sugar transporters in plants and in their interactions with fungi. Trends Plant Sci 2012; 17:413-22; PMID:22513109 and http://dx.doi. org/10.1016/j.tplants.2012.03.009. 11. Rausch C, Daram P, Brunner S, Jansa J, Laloi M, Leggewie G, Amrhein N, Bucher M. A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 2001; 414:462-70; PMID:11719809 and http://dx.doi.org/10.1038/35106601. 12. Charpentier M, Oldroyd G. How close are we to nitrogen-fixing cereals? Curr Opin Plant Biol 2010; 13:556-64; PMID:20817544 and http://dx.doi. org/10.1016/j.pbi.2010.08.003. 13. Kistner C, Parniske M. Evolution of signal transduction in intracellular symbiosis. Trends Plant Sci 2002; 7:511-8; PMID:12417152 and http://dx.doi. org/10.1016/S1360-1385(02)02356-7. 14. Kaló P, Gleason C, Edwards A, Marsh J, Mitra RM, Hirsch S, Jakab J, Sims S, Long SR, Rogers J, et al. Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 2005; 308:1786-9; PMID:15961668 and http://dx.doi.org/10.1126/science.1110951. 15. Smit P, Raedts J, Portyanko V, Debellé F, Gough C, Bisseling T, Geurts R. NSP1 of the GRAS protein family is essential for rhizobial Nod factorinduced transcription. Science 2005; 308:1789-91; PMID:15961669 and http://dx.doi.org/10.1126/ science.1111025. 16. Delaux PM, Bécard G, Combier JP. NSP1 is a component of the Myc signaling pathway. New Phytol 2013; 199:59-65; PMID:23663036 and http://dx.doi. org/10.1111/nph.12340. 17. Liu W, Kohlen W, Lillo A, Op den Camp R, Ivanov S, Hartog M, Limpens E, Jamil M, Smaczniak C, Kaufmann K, et al. Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-type transcription factors NSP1 and NSP2. Plant Cell 2011; 23:3853-65; PMID:22039214 and http://dx.doi.org/10.1105/tpc.111.089771. 18. Gobbato E, Marsh JF, Vernié T, Wang E, Maillet F, Kim J, Miller JB, Sun J, Bano SA, Ratet P, et al. A GRAS-type transcription factor with a specific function in mycorrhizal signaling. Curr Biol 2012; 22:2236-41; PMID:23122845 and http://dx.doi. org/10.1016/j.cub.2012.09.044.
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19. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 1987; 6:3901-7; PMID:3327686. 20. Boisson-Dernier A, Chabaud M, Garcia F, Bécard G, Rosenberg C, Barker DG. Agrobacterium rhizogenestransformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant Microbe Interact 2001; 14:695-700; PMID:11386364 and http://dx.doi. org/10.1094/MPMI.2001.14.6.695. 21. Kosuta S, Hazledine S, Sun J, Miwa H, Morris RJ, Downie JA, Oldroyd GE. Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes. Proc Natl Acad Sci USA 2008; 105:98238; PMID:18606999 and http://dx.doi.org/10.1073/ pnas.0803499105. 22. Mendoza-Mendoza A, Berndt P, Djamei A, Weise C, Linne U, Marahiel M, Vranes M, Kämper J, Kahmann R. Physical-chemical plant-derived signals induce differentiation in Ustilago maydis. Mol Microbiol 2009; 71:895-911; PMID:19170880 and http://dx.doi.org/10.1111/j.1365-2958.2008.06567.x. 23. Skamnioti P, Gurr SJ. Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence. Plant Cell 2007; 19:2674-89; PMID:17704215 and http:// dx.doi.org/10.1105/tpc.107.051219. 24. Karimi M, Inzé D, Depicker A. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 2002; 7:193-5; PMID:11992820 and http://dx.doi.org/10.1016/S1360-1385(02)02251-3. 25. Chabaud M, Harrison M, De Carvalho-Niebel F, Becard G, Barker DG. Medicago truncatula Handbook: Inoculation and growth of mycorrhizal fungi. 2006. 26. Giovannetti M, Mosse B. Evaluation of Techniques for Measuring Vesicular Arbuscular Mycorrhizal Infection in Roots. New Phytol 1980; 84:489500; http://dx.doi.org/10.1111/j.1469-8137.1980. tb04556.x.
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ISSN: (Print) 1559-2324 (Online) Journal homepage: http://www.tandfonline.com/loi/kpsb20
RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization Enrico Gobbato, Ertao Wang, Gillian Higgins, Syeda Asma Bano, Christine Henry, Michael Schultze & Giles ED Oldroyd To cite this article: Enrico Gobbato, Ertao Wang, Gillian Higgins, Syeda Asma Bano, Christine Henry, Michael Schultze & Giles ED Oldroyd (2013) RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization, Plant Signaling & Behavior, 8:10, e26049, DOI: 10.4161/psb.26049 To link to this article: http://dx.doi.org/10.4161/psb.26049
Published online: 20 Aug 2013.
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Article views: 1648
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kpsb20 Download by: [111.175.192.103]
Date: 14 September 2015, At: 08:58
Short Communication
Short Communication
Plant Signaling & Behavior 8:10, e26049; October 2013; © 2013 Landes Bioscience
RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization Enrico Gobbato1, Ertao Wang1, Gillian Higgins2, Syeda Asma Bano2, Christine Henry3, Michael Schultze2, and Giles ED Oldroyd1* Department of Cell and Developmental Biology; John Innes Centre; Norwich, UK; 2Department of Biology; University of York; York, UK; 3The Food and Environment Research Agency; Sand Hutton, York, UK
1
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Keywords: symbiosis, mycorrhiza, arbuscule, Aphanomyces, promoter
The establishment of the symbiotic interaction between plants and arbuscular mycorrhizal (AM) fungi requires a very tight molecular dialog. Most of the known plant genes necessary for this process are also required for nodulation in legume plants and only very recently genes specifically required for AM symbiosis have been described. Among them we identified RAM (Reduced Arbuscular Mycorrhization)1 and RAM2, a GRAS transcription factor and a GPAT respectively, which are critical for the induction of hyphopodia formation in AM fungi. RAM2 function is also required for appressoria formation by the pathogen Phytophtora palmivora. Here we investigated the activity of RAM1 and RAM2 promoters during mycorrhization and the role of RAM1 and RAM2 during infection by the root pathogen Aphanomyces euteiches. pRAM1 is activated without cell type specificity before hyphopodia formation, while pRAM2 is specifically active in arbusculated cells providing evidence for a potential function of cutin momomers in the regulation of arbuscule formation. Furthermore, consistent with what we observed with Phytophtora, RAM2 but not RAM1 is required during Aphanomyces euteiches infection.
During the establishment of the Arbuscular Mycorrhizal (AM) symbiosis between plants and fungi belonging to the Glomeromycota phylum, a suite of molecular signals from both the plant and the fungus play a critical role for the mutual recognition between the two symbionts.1 Plants release strigolactones in root exudates2 and the perception of strigolactones by AM fungi results in increased spore germination, activation of oxidative metabolism and induction of hyphal branching.3 More recently it was shown that AM germinating spores in turn produce lipo-chito oligosaccarides (AM-LCOs) structurally similar to those produced by rhizobial bacteria.4 AM LCOs are perceived by the plant and trigger symbiotic responses including promotion of AM colonization, transcriptional reprogramming and stimulation of lateral root formation.5 When AM fungal hyphae reach the plant root surface, they develop adhesion structures, the hyphopodia, which are structurally simpler than appressoria of pathogenic fungi.6 Critical for the formation of AM hyphopodia is the production of cutin monomers by the plant which can also induce appressoria formation in pathogenic fungi.7 Upon penetration through the epidermal cell layer, which has been shown to be a plant directed process,8 fungal hyphae grow inside the plant root, reaching the inner root cortex within which they develop arbuscules, highly
branched hyphae surrounded by the periarbuscular membrane of plant origin.6 Nutrient exchanges take place at this interface and are mediated by transporters that are specifically expressed within the arbuscule in both the plant and the fungus.9-11 The knowledge we have of the plant signaling pathways that regulate recognition and response to AM fungi has hitherto been derived from the study of nodulation mutants previously identified in model legume plant species.12 Several of these mutants were also proven to be defective for AM symbiosis and the underlying genes are therefore commonly termed Sym (Symbiosis) genes.13 The activation of a common signaling pathway by different microbial symbionts, raises the problem of understanding how plants can achieve specificity in the activation of two genetic programs which are radically different.12 In this respect the identification of genetic components with specific functions in either the nodulation or AM pathway is critical. Within the Nodulation pathway multiple transcription factors were identified acting downstream of the Sym pathway, among which, NSP (Nodulation Signaling Pathway) 1 and NSP2, belong to the GRAS family.14,15 In contrast to the initial assumption that these transcription factors were specifically required for nodulation, recent evidence showed that both NSP1 and NSP2 also contribute to responses to AM fungi5,16
Correspondence to: Giles ED Oldroyd; Email: [email protected] Submitted: 07/29/2013; Accepted: 08/05/2013 Citation: Gobbato E, Wang E, Higgins G, Bano SA, Henry C, Schultze M, Oldroyd GE. RAM1 and RAM2 function and expression during Arbuscular Mycorrhizal Symbiosis and Aphanomyces euteiches colonization. Plant Signaling & Behavior 2013; 8:e26049; http://dx.doi.org/10.4161/psb.26049
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Figure 1. pRAM1 and pRAM2 Activities. A–C Medicago truncatula composite plants expressing pRAM1:GUS and pRAM2:GUS were inoculated with Rhizophagus irregularis (R.i.) purified spores and roots were stained at 6 wpi A. GUS stained pRAM1:GUS roots B. GUS stained pRAM2:GUS roots c. Fuchsin stained pRAM2:GUS roots. In b. and c. an arbusculated cell is indicated with a red arrow. Scale bar = 100 μm D–F pRAM1:GUS root cultures were co-cultivated with R.i., hyphal growth was monitored and co-cultivation was interrupted before physical contact. D. Image of a root portion in co-cultivation with AM fungi. Scale bar = 1 cm E. root portion shown in d. after GUS staining. Scale bar = 1 cm F. Ink stained root in E. No hyphopodia could be observed. Scale bar = 1 mm.
while both NSP1 and NSP2 function in the activation of strigolactone biosynthesis.17 In an effort to identify novel components specific to the AM pathway, we identified two genes, RAM (Reduced Arbuscular Mycorrhization)1 and RAM2.7,18 Plants mutated in either gene retained the capability to support nodulation, but showed dramatically decreased levels of mycorrhization and this was associated with severely reduced numbers of hyphopodia at the root surface. Complementation experiments demonstrated that RAM2 encodes a Glycerol-3-Phosphate Acyl Transferase (GPAT ), involved in the synthesis of cutin monomers.7 RAM1 encodes a GRAS transcription factor which regulates the expression of RAM2,18 consistent with the similarities between ram1 and ram2 symbiotic phenotypes. In addition to the hyphopodia phenotype, ram2 plants also displayed a severe defect in arbuscule formation, which suggests a critical function for RAM2 synthesized cutin monomers during arbuscule development.7 RAM2 is also required to stimulate appressoria formation in the pathogenic oomycete Phytophtora palmivora, suggesting that in order to establish parasitism primordial microbial pathogens
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have hijacked a preexisting symbiotic signaling pathway to manipulate their host metabolism.7 To better understand the mechanism of action of RAM1 and RAM2, we performed a study of RAM1 and RAM2 promoters. We used Agrobacterium rhizogenes transformation of Medicago truncatula to assess root expression of pRAM1 and pRAM2 driving the expression of the GUS reporter gene.19,20 Composite plants were inoculated with purified spores of the AM fungus Rhizophagus irregularis and roots were collected and stained for GUS activity at 6 wpi. As shown in Figure 1A and B, while pRAM1 showed homogeneous activity in all tissues of colonized roots (that was absent in uncolonized roots), the RAM2 promoter was strongly activated in cortical cells harboring arbuscules. To confirm the presence of arbuscular structures within cells displaying pRAM2 activity, we dually stained inoculated root tissues for GUS activity and with Acid Fuchsin to mark fungal structures. As shown in Figure 1B and C overlap confirmed the specific activation of the RAM2 promoter in cortical cells hosting arbuscules.
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Figure 2. Arbuscule development in ram1-2. Wild type (R108) and ram1-2 plants were inoculated by co-cultivation with colonized leek nurse plants. After 6 weeks of co-cultivation root samples were taken and ink stained. While wild-type plants (left panel) showed fully developed arbuscules (Fd arb, red arrow), ram1-2 plants (right panel) predominantly showed small undeveloped arbuscules (Incomp arb, red arrow) consistent with a function of RAM1 in arbuscule development. Scale bar = 50 μm.
Previously we had not been able to observe an arbuscule defect in ram1 mutants. However, this was surprising, since RAM1 regulates RAM2 expression and RAM2 is induced in arbusculated cells and is required for arbuscule development. This suggests that RAM1 may also function during arbuscule development. Under our growth conditions, we observed very little AM colonization in the ram1-1 mutant, limiting the characterization of arbuscule development in this allele. However the ram1-2 mutation is slightly more permissive than ram1-1 and using leek nurse plants, a very strong source of inoculum, we were able to observe a significantly increased number of hyphae reaching the inner cortex in ram1-2, compared with what we had previously seen with ram1-1. In contrast to what we had observed for ram1‑1, we observed a defect in arbuscule formation in ram1-2, that resulted in smaller, undeveloped arbuscules (Fig. 2), similar to what we previously observed for ram2.7 This suggests that both RAM1 and RAM2 have functions at later stages of mycorrhizal colonization, being required for appropriate arbuscule formation. We previously showed that upon Rhizophagus irregularis colonization RAM1 displays a maximum peak in its expression at 30 dpi, but a slight increase in RAM1 transcript levels could already be observed by qPCR at 20 dpi, a time point at which mycorrhizal colonization of the root is limited.18 To monitor in greater detail pRAM1 activation at very early time points, we generated root cultures of M. truncatula pRAM1:GUS. The pRAM1:GUS root cultures were co-cultivated with carrot root cultures colonized by Rhizophagus irregularis as previously described.21 This experimental system permitted us to follow the progression of AM fungal hyphae toward the Medicago roots (as shown in Figure 1D) and allowed us to assess early induction of pRAM1. When stained for GUS activity, pRAM1:GUS roots showed activation of the RAM1 promoter in areas in close proximity to the fungal hyphae before physical contact as shown in Figure 1E. To confirm that co-cultivated roots were sampled before hyphopodia formation, GUS stained roots were also subsequently ink stained and no hyphopodia could be observed
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(Fig. 1F). No GUS staining was observed in clonal root cultures grown in the absence of the AM fungus (data not shown). This very early activation of pRAM1 is consistent with the role of RAM1 in promoting hyphopodia formation. RAM2 is necessary also for appropriate root colonization by the pathogen Phytophtora palmivora. To assess if this reflected a broader role in pathogen colonization we tested the capability of another root pathogen, Aphanomyces euteiches to colonize ram1 and ram2 roots. ram2 but not ram1 plants showed reduced susceptibility to A. euteiches, measured as reduced percentage of root length colonized by the pathogen (Fig. 3): ram2 plants showed ~3% of their root length colonized, equal to one quarter of the colonization levels of wild type roots. Our work confirms the importance of RAM2 for normal colonization by root oomycete pathogens and this further supports the hypothesis that the function of RAM2 has been coopted by pathogenic oomycetes to facilitate their own colonization. It will be interesting to test whether root pathogens belonging to the Ascomycota or Basidiomycota also require RAM2 during their colonization as could be hypothesized given the capability of cutin to induce appressoria formation in their foliar pathogen counterparts.22,23 We have shown that RAM1 and RAM2 are required for the formation of hyphopodia at the root surface and for appropriate arbuscule formation in the root cortex. These two stages represent the periods of intracellular colonization by mycorrhizal fungi and we propose that the regulation of RAM2 to activate cutin production may be necessary for all stages of mycorrhizal intracellular colonization. The expression of RAM1 is consistent with a role at both the root surface and the root cortex, although we do not see expression restricted to colonized cells. In contrast the induction of RAM2 appears to be associated with cortical cells containing arbuscules. This is consistent with a role for RAM2 in the root cortex, but does not explain its function at the root surface. Longer GUS staining in uninoculated roots revealed apparent low level RAM2 expression throughout
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Figure 3. Aphanomyces euteiches colonization levels. Wild type (A17), ram1 and ram2 plants were inoculated with Aphanomyces euteiches. Roots were collected and ink stained at 14 dpi and the percentage of colonized root length measured. Error bars represent standard errors (n = 15–18).
the root (data not shown). This low level constitutive expression may explain RAM2 function at the root surface. Material and Methods A region of 2,451 Kb from the RAM1 start codon to the end of the next predicted gene in the genome was selected as the RAM1 promoter. This region was amplified using Gateway ® compatible primers (pRAM1 – f GGGGACAAGT TTGTACAAAA AAGCAGGCTT CCCTT TTCTTGTCCT TTTACCACC pRAM1 – r GGGGACCACT TTGTACAAGA AAGCTGGG TCTTTTTTCC CACCCTTTTT TATTTG), cloned into a pDONR 207 vector by BP reaction and finally transferred by LR reaction into the pBGWFS724 vector (resulting in pBGWFS7pRAM1). A region of 2.3 kb of the RAM2 promoter was used, which is sufficient to drive RAM2 expression in complementation experiments.7 This region was amplified using Gateway compatible primers (pRAM2-gus-f: GGGACAAGTT TGTAC AAAAAAGCAGG CTATTGCCGG TGGGATAAAC AC pRAM2-gus-r: GGGACCACTT TGTACAAGAA AG CTGGGTGTGA AGTTGTTGTG TTTA TATACAC) and inserted into pDONOR207 by Gateway technology. Also this construct was recombined by LR reaction with pBGWFS7 (resulting in pBGWFS7-pRAM2). PBGWFS7-pRAM1 and pBGWFS7-pRAM2 were transformed into roots of M. truncatula wild-type using A. rhizogenes 20 and assessed for promoter activity with G. intraradices at 6 weeks post inoculation. The mycorrhizal colonized roots were GUS stained.19 pBGWFS7pRAM2 transformed roots were subsequently co-stained for with acid fuchsin.25 The stained roots were imaged with a Nikon Eclipse 800 microscope with a filter set for TRITC.
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For the generation of pBGWFS7-pRAM1 root cultures, transformed roots were excised and cultured on M plates25 containing augmentin (400 mg/ml). Transgenic roots were subsequently co-cultivated with mycorrhized transgenic carrot roots21 for a period variable between 2 and 4 weeks until fungal hyphae had grown in proximity of the M. truncatula transformed root. M. truncatula roots were then GUS stained and subsequently ink stained.25 For co-cultivation of M. truncatula with leek, six-week old leek plants colonized with R. irregularis were used. Colonized leek plants were produced by planting seedlings in 9 parts of a mixture of autoclaved sand and Terragreen (equal volumes) and 1 part (by volume) of leek root pieces colonized by R. irregularis. The plants were grown for 6 weeks and then individual colonized leek plants were used as nurse plants. The leeks were potted in the center of a P6 (11 cm diameter) round pot surrounded by 4–5 Medicago plants. Root length colonization was tested 6 weeks after the start of cocultivation by ink staining as described previously.18 For inoculation with Aphanomyces euteiches Drechs (ATCC201684) seeds of M. truncatula were pre-germinated on moist filter paper for 2 d in the dark. Seedlings were subsequently transferred into pots containing a 1:2 mixture of sterilized expanded clay and vermiculite. Plants were grown under constant conditions in a greenhouse (243 mE μmol m-2 s -1 for 12 h; 24°C, 65% humidity) and fertilized with phostrogen (10 ml/9 L) 3 d after transplanting and 7 d after inoculation. To generate the A. euteiches inoculum, 10 disks of 1 cm 2 fungal mycelium grown on corn meal agar were cut out and cultured in 20 ml maltose peptone broth (MPB) for 9 d at room temperature. Zoospore production was induced by washing the mycelium twice in autoclaved lake water. Zoospores were counted and diluted to appropriate concentrations. After 10 d, the plantlets were inoculated by application of zoospore suspensions at the stem basis. Control plants were mock inoculated with autoclaved lake water. At 14 d after inoculation control and inoculated roots were harvested, incubated in 10% (w/v) KOH for 4 d at room temperature, and then stained with ink and acetic acid.25 The percentage of root length colonized by oospores was determined by utilizing the grid line intersection method.26 Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We thank Frank Colditz for providing a culture of and protocols for growth of A. euteiches. This work was supported by the BBSRC, as grants BB/E003850/1 and BB/E001408/1; the European Research Council, as SYMBIOSIS; and the Higher Education Commission, Pakistan.
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