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.
Highlights
Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix Berkeley and Broome, is the most important disease of Coffea arabica L
In the leaves of the resistant samples, the penetration hypha (Figure 1A) was the fungal growth stage observed with higher frequency during the all time-course of the experiment reaching about 55% at 24 hai and 50% at 96 hai, while haustorial mother cell (HMC) with haustorium (HMC/h) only reached 15% of infection sites at 72 hai, and did not exceed 22% at 96 hai (Figure 1D); at this stage the fungus stop growth and died
In the leaves of the susceptible samples, the penetration hypha was the most representative stage at 24 hai (61%) and 48 hai (41%) but, later on, the HMC/h greatly increased in frequency (40% at 72 hai and 44% at 96 hai), being responsible for the successful fungal growth (Figures 1B,D)
Summary
Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix Berkeley and Broome, is the most important disease of Coffea arabica L. Rust fungi interact intimately with the plant host cells (by means of haustoria, highly specialized intracellular hyphae) modifying plant metabolism to serve the fungus nutrient needs for completion of its life cycle. This mode of interaction involves a prolonged and effective suppression of the host immune system and, at the same time, the induction of specific host genes for establishing biotrophy (Schulze-Lefert and Panstruga, 2003; Voegele and Mendgen, 2003). It is in the apoplast where the pathogen and plant first contact, and the primary defenses are activated (Agrawal et al, 2010; Floerl et al, 2012; Delanois et al, 2014; Guerra-Guimarães et al, 2014)
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