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A proteomic approach analysing the Arabidopsis thaliana response to virulent and avirulent Pseudomonas syringae strains

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Abstract

The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.

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Acknowledgments

This work was carried out with financial support from the Spanish ‘‘Ministerio de Educación y Ciencia’’, Project BIO-2006-14790. Besma Sghaier-Hammami is supported by a Grant from the Spanish “La Agencia Española para la Cooperación Internacional”. We gratefully acknowledge support from Dr. Consuelo Gómez (Proteomics Service, University of Córdoba) and Dr. Lola Gutiérrez Blázquez (Proteomics Service, University of Madrid) for mass spectrometry facilities and technical assistance. Thanks to Professor Diana Puntas for his help with the English text. Especially thanks to Salvador Martínez de Bartolomé Izquierdo (ProteoRed, National Center for Biotechnology, Madrid Spain) and David Ovelleiro (PRIDE Group Proteomics Services Team, Hinxton, Cambridge, UK) for their helps in the submission of the data in PRIDE repository.

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  1. Department of Biochemistry and Molecular Biology, Agricultural and Plant Biochemistry and Proteomics Research Group, University of Cordoba, Campus de Rabanales, Ed. Severo Ochoa (C6), 14071, Cordoba, Spain

    Besma Sghaier-Hammami, Inmaculada Redondo-López, Ana M. Maldonado-Alconada, Sira Echevarría-Zomeño & Jesús V. Jorrín-Novo

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  1. Besma Sghaier-Hammami
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  2. Inmaculada Redondo-López
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Correspondence to Besma Sghaier-Hammami.

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Communicated by M. Stobiecki.

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Supplementary Fig. 1: Symptom development on leaves of non-inoculated plants (Col-0), mock-inoculated plants with a 10-mM MgCl2 solution (M) and plants inoculated by virulent (V) and avirulent (A) pathogens during 4, 8 and 24 h post inoculation. No symptoms are observed on non-inoculated and infiltrated plants. Leaves inoculated with virulent strain, presented humid chlorotic lesions only at 24 hpi, which spread over the leaf surface (indicated with a red arrow). However, leaves inoculated with the avirulent strain presented a limit lesion in the leaf starting at 8 hpi (indicated by red arrows).

Supplementary Fig. 2: SDS-PAGE analysis of total proteins of non-inoculated plants (Col-0), mock-inoculated plants with a 10-mM MgCl2 solution (M) and plants inoculated by virulent (V) and avirulent (A) pathogens, during 4, 8 and 24 h post inoculation (hpi). Mr: molecular marker. 20 μg of protein was charged in each line.

Supplementary Fig. 3: Representative Coomassie blue-stained 2-DE gel of non-inoculated plants. Gels were performed on 7 cm immobilized pH gradient (linear pH 3–10) strips. 300 μg of proteins were loaded. The TCA–acetone–phenol protocol extraction was used.

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Sghaier-Hammami, B., Redondo-López, I., Maldonado-Alconada, A.M. et al. A proteomic approach analysing the Arabidopsis thaliana response to virulent and avirulent Pseudomonas syringae strains. Acta Physiol Plant 34, 905–922 (2012). https://doi.org/10.1007/s11738-011-0888-4

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