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Front Plant Sci. 2015 Jun 30;6:478. doi: 10.3389/fpls.2015.00478. eCollection 2015.

Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix.

Author information

1
Centro de Investigação das Ferrugens do Cafeeiro, Instituto de Investigação Científica Tropical Oeiras, Portugal ; Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa Lisboa, Portugal.
2
Centro de Investigação das Ferrugens do Cafeeiro, Instituto de Investigação Científica Tropical Oeiras, Portugal.
3
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (UNL) Oeiras, Portugal ; Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa Caparica, Portugal.
4
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (UNL) Oeiras, Portugal ; Instituto de Biologia Experimental e Tecnológica Oeiras, Portugal.
5
Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa Lisboa, Portugal.
6
Luxembourg Institute of Science and Technology Belvaux, Luxembourg.
7
Centro de Investigação das Ferrugens do Cafeeiro, Instituto de Investigação Científica Tropical Oeiras, Portugal ; Department de Fitossanidade, Universidade Federal de Pelotas Pelotas, Brasil.
8
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (UNL) Oeiras, Portugal.

Abstract

A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24-96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24-96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24-48 hai) and a late/specific one (72-96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.

KEYWORDS:

2-DE; ELISA assay; MALDI-TOF/TOF MS; antibody production; coffee leaf rust (CLR); cytology

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