Skip to main content
Log in

Proteins Associated with Oxidative Burst and Cell Wall Strengthening Accumulate During Citrus-Xanthomonas Non-Host Interaction

  • Original Paper
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript


Citrus proteome changes at 8 and 48 h post inoculation (hpi) were analysed by both 2D gel electrophoresis and nano-LC-MS/MS proteomic approaches during interaction with Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas oryzae pv. oryzae (Xoo) as host and non-host pathogens, respectively. A total of 256 proteins, 72 at 8 hpi and 184 at 48 hpi, differentially accumulated during citrus-Xanthomonas interaction. Of these, 67 and 115 proteins were specific to Xac and Xoo interaction, respectively. In addition, 64 proteins, 10 at 8 hpi and 54 at 48 hpi, variedly accumulated during both the interactions. Proteins related to photosynthesis, carbohydrate metabolism and protein synthesis were in low abundance during both the interactions resulting in reduced rate of photosynthesis. Proteins related to defence response, cell wall (CW) strengthening, lignin deposition and generation of reactive oxygen species (ROS) were in high abundance only during Xoo interaction. Whereas, during Xac interaction, proteins involved in antioxidant metabolism and CW loosening and/or elongation were in high abundance. The precise increase in abundance of these proteins during non-host interaction suggested an important role for CW fortification and ROS accumulation in non-host resistance in plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

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

Similar content being viewed by others



Non-host resistance


Liquid chromatography-tandem mass spectrometry


2D electrophoresis

Xac :

Xanthomonas axonopodis pv. citri

Xac :

Xanthomonas oryzae pv. oryzae


Reactive oxygen species




Superoxide dismutase


Ascorbate peroxidase


Xyloglucan endotransglycosylase/hydrolase


Cell wall


  • An C, Mou Z (2012) Non-host defense response in a novel Arabidopsis-Xanthomonas citri subsp. citri Pathosystem. PLoS One 7:e31130

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Asirvatham VS, Watson BS, Sumner LW (2002) Analytical and biological variances associated with proteomic studies of Medicago truncatula by two-dimensional polyacrylamide gel electrophoresis. Proteomics 2:960–968

    Article  CAS  PubMed  Google Scholar 

  • Austin MJ, Muskett P, Kahn K et al (2002) Regulatory role of SGT1 in early R gene-mediated plant defenses. Science 295:2077–2080

    Article  CAS  PubMed  Google Scholar 

  • Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287

    Article  CAS  PubMed  Google Scholar 

  • Bevan M, Bancroft I, Bent E et al (1998) Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391:485–488

    Article  CAS  PubMed  Google Scholar 

  • Bonfig KB, Schreiber U, Gabler A et al (2006) Infection with virulent and avirulent P. syringae strains differentially affects photosynthesis and sink metabolism in Arabidopsis leaves. Planta 225:1–12

    Article  CAS  PubMed  Google Scholar 

  • Caraux G, Pinloche S (2005) PermutMatrix: a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics 21:1280–1281

    Article  CAS  PubMed  Google Scholar 

  • Daurelio LD, Petrocelli S, Blanco F et al (2011) Transcriptome analysis reveals novel genes involved in nonhost response to bacterial infection in tobacco. J Plant Physiol 168:382–391

    Article  CAS  PubMed  Google Scholar 

  • Daurelio LD, Romero MS, Petrocelli S et al (2013) Characterization of Citrus sinensis transcription factors closely associated with the non-host response to Xanthomonas campestris pv. vesicatoria. J Plant Physiol 170:934–942

    Article  CAS  PubMed  Google Scholar 

  • Garavaglia BS, Thomas L, Zimaro T et al (2010) A plant natriuretic peptide-like molecule of the pathogen Xanthomonas axonopodis pv. citri causes rapid changes in the proteome of its citrus host. BMC Plant Biol 10:e8950

    Article  Google Scholar 

  • Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplast. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

    Article  CAS  PubMed  Google Scholar 

  • Henkel AW, Bieger SC (1994) Quantification of proteins dissolved in an electrophoresis sample buffer. Anal Biochem 223:329–331

    Article  CAS  PubMed  Google Scholar 

  • Huckelhoven R (2007) Cell wall-associated mechanisms of disease resistance and susceptibility. Annu Rev Phytopathol 45:101–127

    Article  PubMed  Google Scholar 

  • Isaacson T, Damasceno CM, Saravanan RS et al (2006) Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc 1:769–774

    Article  CAS  PubMed  Google Scholar 

  • Kang L, Li J, Zhao T et al (2003) Interplay of the Arabidopsis nonhost resistance gene NHO1 with bacterial virulence. Proc Natl Acad Sci U S A 100:3519–3524

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kanzaki H, Saitoh H, Ito A et al (2003) Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Mol Plant Pathol 4:383–391

    Article  CAS  PubMed  Google Scholar 

  • Kundu S, Chakraborty D, Pal A (2011) Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo. J Proteomics 74:337–349

    Article  CAS  PubMed  Google Scholar 

  • Li B, Takahashi D, Kawamura Y et al (2012a) Comparison of plasma membrane proteomic changes of Arabidopsis suspension-cultured cells (T87 Line) after cold and ABA treatment in association with freezing tolerance development. Plant Cell Physiol 53:543–554

    Article  CAS  PubMed  Google Scholar 

  • Li W, Xu Y-P, Zhang Z-X et al (2012b) Identification of genes required for nonhost resistance to Xanthomonas oryzae pv. oryzae reveals novel signaling components. PLoS One 7:e42796

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Liu H, Wang Y, Xu J et al (2008) Ethylene signaling is required for the acceleration of cell death induced by the activation of AtMEK5 in Arabidopsis. Cell Res 18:422–432

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Schiff M, Serino G et al (2002) Role of SCF ubiquitin-ligase and the COP9 signalosome in the N genemediated resistance response to tobacco mosaic virus. Plant Cell 14:1483–1496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ma QU (2007) Small GTP-binding proteins and their functions in plants. J Plant Growth Regulation 26:369–388

    Article  CAS  Google Scholar 

  • Matsumura H, Reich S, Ito A et al (2003) Gene expression analysis of plant host-pathogen interactions by SuperSAGE. Proc Natl Acad Sci U S A 100:15718–15723

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Meunier B, Dumas E, Piec I et al (2007) Assessment of hierarchical clustering methodologies for proteomic data mining. J Proteome Res 6:358–366

    Article  CAS  PubMed  Google Scholar 

  • Nakano A, Asada K (1987) Purification of ascorbate peroxidase in spinach chloroplasts: its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol 28:131–140

    CAS  Google Scholar 

  • Nouri M-Z, Komatsu S (2010) Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel-based and LC MS/MS-based proteomics approaches. Proteomics 10:1930–1945

    Article  CAS  PubMed  Google Scholar 

  • Nurnberger T, Lipka V (2005) Non-host resistance in plants: new insights into an old phenomenon. Mol Plant Pathol 6:335–345

    Article  PubMed  Google Scholar 

  • Nurnberger T, Scheel D (2001) Signal transmission in the plant immune response. Trends Plant Sci 6:372–379

    Article  CAS  PubMed  Google Scholar 

  • Oh S-K, Lee S, Chung E et al (2006) Insight into types I and II nonhost resistance using expression patterns of defense-related genes in tobacco. Planta 223:1101–1107

    Article  CAS  PubMed  Google Scholar 

  • Overmyer K, Brosche M, Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death. Trends Plant Sci 8:335–342

    Article  CAS  PubMed  Google Scholar 

  • Rani TS, Podile AR (2014) Extracellular matrix-associated proteome changes during non-host resistance in citrus-Xanthomonas interactions. Physiol Plant 150:565–579

    Article  Google Scholar 

  • Roberts MR, Salinas J, Collinge DB (2002) 14-3-3 proteins and the response to abiotic and biotic stress. Plant Mol Biol 50:1031–1039

    Article  CAS  PubMed  Google Scholar 

  • Schulze-Lefert P, Panstruga R (2011) A molecular evolutionary concept connecting nonhost resistance, pathogen host range, and pathogen speciation. Trends Plant Sci 16:117–125

    Article  CAS  PubMed  Google Scholar 

  • Senthil-Kumar M, Mysore KS (2013) Nonhost resistance against bacterial pathogens: retrospectives and prospects. Annu Rev Phytopathol 51:19.1–19.21

    Article  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O et al (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  CAS  PubMed  Google Scholar 

  • Takahashi D, Kawamura Y, Uemura M (2013) Changes of detergent-resistant plasma membrane proteins in oat and rye during cold acclimation: association with differential freezing tolerance. J Proteome Res

  • Takahashi D, Kawamura Y, Yamashita T et al (2012) Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants. J Proteome Res 11:1654–1665

    Article  CAS  PubMed  Google Scholar 

  • Talla S, Riazunnisa K, Padmavathi L et al (2011) Ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosynthesis and protect against photoinhibition. J Biosci 36:163–173

    Article  CAS  PubMed  Google Scholar 

  • Tao Y, Xie Z, Chen W et al (2003) Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae. Plant Cell 15:317–330

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Thordal-Christensen H, Zhang Z, Wei Y et al (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11:1187–1194

    Article  CAS  Google Scholar 

  • Uma B, Rani TS, Podile AR (2011) Warriors at the gate that never sleep: non-host resistance in plants. J Plant Physiol 168:2141–2152

    Article  CAS  PubMed  Google Scholar 

  • Vallet C, Chabbert B, Czaninski Y et al (1996) Histochemistry of lignin deposition during sclerenchyma differentiation in alfalfa stems. Annu Bot 78:625–632

    Article  CAS  Google Scholar 

  • Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective roles of exogenous polyamines. Plant Sci 151:59–66

    Article  CAS  Google Scholar 

  • Wang X, Li X, Li Y (2007) A modified commassie brilliant blue staining method at nanogram sensitivity compatible with proteomic analysis. Biotechnol Lett 29:1599–1603

    Article  CAS  PubMed  Google Scholar 

  • Zamany A, Liu J-J, Ekramoddoullah AKM (2012) Comparative proteomic profiles of Pinus monticola needles during early compatible and incompatible interactions with Cronartium ribicola. Planta 236:1725–1746

    Article  CAS  PubMed  Google Scholar 

  • Zimaro T, Gottig N, Garavaglia BS et al (2011) Unraveling plant responses to bacterial pathogens through proteomics. J Biomed Biotechnol 2011:354801

    Article  PubMed Central  PubMed  Google Scholar 

Download references


We thank UGC-CAS programme and Proteomics Facility (DBT-CREBB) of School of Life Sciences and DST-FIST facility of Plant Sciences Department at University of Hyderabad. TSR acknowledges the research fellowship from DBT-CREBB and DST-INSPIRE, India. The research performed in Iwate University was in part supported by Grants-in-Aid (#22120003 and #24370018to MU and #24-7373 to DT) from Japan Society for the Promotion of Science (JSPS).

Conflict of interests

The authors declare that they have no competing interests.

Author’s contributions

TSR carried 2DE, biochemical analysis and statistical analysis. TSR and DT performed nano-LC-MS/MS. ARP designed the experiments, and TSR, DT, MU and ARP wrote the final draft of the manuscript. All authors read and approved the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Appa Rao Podile.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. S1

Representation of functional categories of differentially regulated proteins: Proteins responded to pathogen challenge (Xac or Xoo) were assigned to functional categories based on biological process, shown as percent of total response in each category at 8 hpi (right panel) and at 48 hpi (left panel) are shown (GIF 140 kb)

High resolution image (TIFF 331 kb)

Supplementary Fig. S2

Venn diagram analysis of the expression patterns of differentially expressed proteins during regulated proteins: Proteins differentially expressed during Xac and Xoo interaction a) at 8 hpi, b) at 48 hpi, and c) overlapping proteins at 8 and 48 hpi are shown (GIF 83 kb)

High resolution image (TIFF 95 kb)

Supplementary Fig. S3

Measurement of photosynthetic rate: Pattern of photosynthetic oxygen evolution in citrus during mock, Xac and Xoo challenge at 8 and 48 hpi were measured. Error bars indicate standard deviation of the mean from three independent experiments. hpi-hours post inoculation (GIF 97 kb)

High resolution image (TIFF 259 kb)

Supplementary Tables S1-S5

(XLSX 903 kb)


(PPTX 289 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rani, T.S., Takahashi, D., Uemura, M. et al. Proteins Associated with Oxidative Burst and Cell Wall Strengthening Accumulate During Citrus-Xanthomonas Non-Host Interaction. Plant Mol Biol Rep 33, 1349–1360 (2015).

Download citation

  • Published:

  • Issue Date:

  • DOI: