Skip to main content

Defense responses in female gametophytes of Saccharina japonica (Phaeophyta) induced by flg22-derived peptides

Abstract

Plants and algae can detect the presence of bacteria via sensing of proteins or peptides of bacterial origin. Flg22, a fragment of bacterial flagellin, is one of these peptides and has been shown to be an effective elicitor in both plants and algae. Here, we investigated the elicitor activity of flg22-derived peptides in the brown alga, Saccharina japonica. By monitoring luminol-dependent fluorescence, we could observe that the release of H2O2 induced by flg22-derived peptides is maximal at 2 h after induction. The elicitor activity was depending on the length of the peptides in the order of flg22 > flg15 > flg14. Cytological observations regarding the presence of reactive oxygen species (ROS) after induction were consistent with quantitative measurements of H2O2 generation using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. Addition of 1 μM each of flg22 and flg15 was sufficient to inhibit growth of female gametophytes. Furthermore, the elicitor activity of C-terminally shortened flg15-derived peptides suggests that flg15 apparently is the smallest peptide with elicitor activity. Amino acid position D43 at the N-terminus of a flagellin was demonstrated to be involved in the elicitor activity. Finally, H2O2 was localized in the plasma membranes of female gametophytes by an NADPH oxidase inhibitor and electron-dense deposits of cerium perhydroxide by transmission electron microscope.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  • Basse CW, Bock K, Boller T (1992) Elicitors and suppressors of the defense response in tomato cells Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase. J Biol Chem 267:10258–10265

    CAS  PubMed  Google Scholar 

  • Bauer Z, Gómez-Gómez L, Boller T, Felix G (2001) Sensitivity of different ecotypes and mutants of Arabidopsis thaliana toward the bacterial elicitor flagellin correlates with the presence of receptor-binding sites. J Biol Chem 276:45669–45676

    CAS  Article  PubMed  Google Scholar 

  • Baureithel K, Felix G, Boller T (1994) Specific, high affinity binding of chitin fragments to tomato cells and membranes. J Biol Chem 269:17931–17938

    CAS  PubMed  Google Scholar 

  • Bestwick CS, Brown IR, Bennett MH, Mansfield JW (1997) Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. Plant Cell 9:209–221

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406

    CAS  Article  PubMed  Google Scholar 

  • Bouarab K, Potin P, Correa J, Kloareg B (1999) Sulfated oligosaccharides mediate the interaction between a marine red alga and its green algal pathogenic endophyte. Plant Cell 11:1635–1650

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Canter HM, Jaworski GHM (1979) The occurrence of a hypersensitive reaction in the planktonic diatom Asterionella formosa Hassall parasitized by the chytrid Rhizophydium planktonicum Canter emend., in culture. New Phytol 82:187–206

    Article  Google Scholar 

  • Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18:465–476

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Cosse A, Leblanc C, Potin P (2007) Dynamic defense of marine macroalgae against pathogens: from early activated to gene-regulated responses. Adv Bot Res 46:221–266

    Article  Google Scholar 

  • Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276

    CAS  Article  PubMed  Google Scholar 

  • Gómez-Gómez L, Felix G, Boller T (1999) A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. Plant J 18:277–284

    Article  PubMed  Google Scholar 

  • Graham TL, Sequeira L, Huang TS (1977) Bacterial lipopolysaccharides as inducers of disease resistance in tobacco. Appl Environ Microb 34:424–432

    CAS  Google Scholar 

  • Jones DA, Takemoto D (2004) Plant innate immunity-direct and indirect recognition of general and specific pathogen-associated molecules. Curr Opin Immunol 16:48–62

    CAS  Article  PubMed  Google Scholar 

  • Koller T, Bent AF (2014) FLS2-BAK1 extracellular domain interaction sites required for defense signaling activation. Plos One 9:e111185

    Article  PubMed  PubMed Central  Google Scholar 

  • Küpper FC, Gaquerel E, Boneberg EM, Morath S, Salaün JP, Potin P (2006) Early events in the perception of lipopolysaccharides in the brown alga Laminaria digitata include an oxidative burst and activation of fatty acid oxidation cascades. J Exp Bot 57:1991–1999

    Article  PubMed  Google Scholar 

  • Küpper FC, Kloareg B, Guern J, Potin P (2001) Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata. Plant Physiol 125:278–291

    Article  PubMed  PubMed Central  Google Scholar 

  • Medzhitov R, Janeway CA (2002) Decoding the patterns of self and nonself by the innate immune system. Science 296:298–300

    CAS  Article  PubMed  Google Scholar 

  • Meindl T, Boller T, Felix G (2000) The bacterial elicitor flagellin activates its receptor in tomato cells according to the address–message concept. Plant Cell 12:1783–1794

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Meindl T, Boller T, Felix G (1998) The plant wound hormone systemin binds with the N-terminal part to its receptor but needs the C-terminal part to activate it. Plant Cell 10:1–11

    Article  Google Scholar 

  • Naito K, Taguchi F, Suzuki T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2008) Amino acid sequence of bacterial microbe-associated molecular pattern flg22 is required for virulence. Mol Plant Microbe Interact 21:1165–1174

    CAS  Article  PubMed  Google Scholar 

  • Nürnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 198:249–266

    Article  PubMed  Google Scholar 

  • Nürnberger T, Nennstiel D, Jabs T, Sacks WR, Hahlbrock K, Scheel D (1994) High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses. Cell 78:449–460

    Article  PubMed  Google Scholar 

  • Paul C, Mausz MA, Pohnert G (2013) A co-culturing/metabolomics approach to investigate chemically mediated interactions of planktonic organisms reveals influence of bacteria on diatom metabolism. Metabolomics 9:349–359

    CAS  Article  Google Scholar 

  • Potin P, Bourarab K, Salaun JP, Pohnert G, Kloareg B (2002) Biotic interactions of marine algae. Curr Opin Plant Biol 5:308–317

    CAS  Article  PubMed  Google Scholar 

  • Sharp JK, McNeil M, Albersheim P (1984) The primary structure of one elicitor-active and seven elicitor-inactive hexa(beta-D-glucopyranosyl)-D-glucitols isolated from the mycelial walls of Phytophthora megasperma f. sp. glycinea. J Biol Chem 259:11321–11336

    CAS  PubMed  Google Scholar 

  • Song X, She X (2010) The generation and the role of hydrogen peroxide in plant. J Lianyungang Teachers College 4:99–103 (In Chinese with English abstract)

    Google Scholar 

  • Sun W, Dunning FM, Pfund C, Weingarten R, Bent AF (2006) Within-species flagellin polymorphism in Xanthomonas campestris pv campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2–dependent defenses. Plant Cell 18:764–779

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Sun YD, Li L, Macho AP, Han ZF, Hu ZH, Zipfel C, Zhou JM, Chai J (2013) Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science 342:624–628

    CAS  Article  PubMed  Google Scholar 

  • Takai R, Isogai A, Takayama S, Che FS (2008) Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice. Mol Plant Microbe Interact 21:1635–1642

    CAS  Article  PubMed  Google Scholar 

  • Wang SS, Wei XJ, Lu BJ, Wang GG (2012) Preliminary study on flg22-induced defense responses in sporophytes of Saccharina japonica (Phaeophyta). J Fish China 36:1834–1841 (In Chinese with English abstract)

    CAS  Article  Google Scholar 

  • Wang SS, Zhao FY, Wei XJ, Lu BJ, Duan DL, Wang GG (2013) Preliminary study on flg22-induced defense response in female gametophytes in Saccharina japonica (Phaeophyta). J Appl Phycol 25:1215–1223

    CAS  Article  Google Scholar 

  • Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:85–88

    CAS  Article  PubMed  Google Scholar 

  • Weinberger F (2007) Pathogen-induced defense and innate immunity in macroalgae. Biol Bull 213:290–302

    CAS  Article  PubMed  Google Scholar 

  • Weinberger F, Friedlander M, Hoppe HG (1999) Oligoagars elicit a physiological response in Gracilaria conferta (Rhodophyta). J Phycol 35:747–755

    CAS  Article  Google Scholar 

  • Weinberger F, Friedlander M (2000) Response of Gracilaria conferta (Rhodophyta) to oligoagars results in defense against agar-degrading epiphytes. J Phycol 36:1079–1086

    CAS  Article  Google Scholar 

  • Weinberger F, Leonardi P, Miravalles A, Correa JA, Lion U, Kloareg B, Potin P (2005) Dissection of two distinct defense related responses to agar oligosaccharides in Gracilaria chilensis (Rhodophyta) and Gracilaria conferta (Rhodophyta). J Phycol 41:863–873

    CAS  Article  Google Scholar 

  • Zipfel C (2009) Early molecular events in PAMP-triggered immunity. Curr Opin Plant Biol 12:414–420

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

This study was sponsored by the National Natural Science Foundation of China (No. 41576158) granted to G.G. Wang; Science and Technology Development Project of Qingdao, China, 12-1-4-1-(4)-jch; the Shandong Agriculture Breeding Engineering Biological Resources Innovation of Research Project; and Student Research Training Program (SRTP) of Ocean University of China (No. 201210423048).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Dongmei Zhan or Gaoge Wang.

Additional information

Bojun Lu and Dandan Li contributed equally to this work and should be considered joined first authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1
figure 9

Release of H2O2 in female gametophytes of S. japonica by different concentrations of flg14 and flg22D43A after 2 h of incubation. Values are means ± SD (n=6) (JPG 1.14 mb)

Fig. S2
figure 10

Fluorescence images of ROS production in female gametophytes of S. japonica induced by flg15-Δ1 after 2 h of incubation A: Control experiment treated with sterilized seawater; B: Control experiment treated with 0.1 % BSA+ sterilized seawater; C-G: The green fluorescent spots indicated ROS production at concentrations of 200, 400, 600, 800 and 1000 μM. Bars: 10 μm (JPG 1.63 mb)

Fig. S3
figure 11

Fluorescence images of ROS production in female gametophytes of S. japonica induced by flg15-Δ2 after 2 h of incubation A: Control experiment treated with sterilized seawater; B: Control experiment treated with 0.1 % BSA+ sterilized seawater; C-G: The green fluorescent spots indicated ROS production at concentrations of 200, 400, 600, 800 and 1000 μM. Bars: 10 μm (JPG 1.51 mb)

Fig. S4
figure 12

Fluorescence images of ROS production in female gametophytes of S. japonica induced by flg15-Δ3 after 2 h of incubation A: Control experiment treated with sterilized seawater; B: Control experiment treated with 0.1 % BSA+ sterilized seawater; C-G: The green fluorescent spots indicated ROS production at concentrations of 200, 400, 600, 800 and 1000 μM. Bars: 10 μm (JPG 1.33 mb)

Fig. S5
figure 13

Fluorescence images of ROS production in female gametophytes of S. japonica induced by flg15-Δ4 after 2 h of incubation A: Control experiment treated with sterilized seawater; B: Control experiment treated with 0.1 % BSA+ sterilized seawater; C-G: The green fluorescent spots indicated ROS production at concentrations of 200, 400, 600, 800 and 1000 μM. Bars: 10 μm (JPG 1.43 mb)

Fig. S6
figure 14

Fluorescence images of ROS production in female gametophytes of S. japonica induced by flg15-derived-peptide after 2 h of incubation A: Control experiment treated with sterilized seawater; B: Control experiment treated with 0.1 % BSA+ sterilized seawater; C-F: The green fluorescent spots indicated ROS production induced by flg15-Δ5、flg15-Δ6、flg15-Δ7 and flg15-Δ8 in female gametophytes at concentrations of 1000 μM. Bars: 10 μm (JPG 1.33 mb)

High resolution image (TIF 954 kb)

High resolution image (TIF 4.34 mb)

High resolution image (TIF 3.47 mb)

High resolution image (TIF 2.86 mb)

High resolution image (TIF 3.14 mb)

High resolution image (TIF 3.64 mb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lu, B., Li, D., Zhang, R. et al. Defense responses in female gametophytes of Saccharina japonica (Phaeophyta) induced by flg22-derived peptides. J Appl Phycol 28, 1793–1801 (2016). https://doi.org/10.1007/s10811-015-0721-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-015-0721-3

Keywords

  • Defense response
  • flg22 derivative peptides
  • Saccharina japonica
  • Reactive oxygen species