RmCP, a cerato-platanin protein from Rigidoporus microporus, induces defense responses during interaction with Hevea brasiliensis

Abstract

Introduction: The rubber tree (Hevea brasiliensis) is susceptible to various fungal pathogens with Rigidoporus microporus being one of the most harmful. This fungus causes white root disease in rubber trees which can potentially lead to massive tree losses if left untreated. The use of elicitor proteins in enhancing host plant resistance represents a sustainable approach for disease control by reducing the use of chemical fungicides. Although cerato-platanin proteins (CPs) are recognized elicitors in many pathosystems, CP from R. microporus has not been functionally characterized, leaving its role in rubber-pathogen interactions unknown.

Methods: The coding sequence of the CP homolog RmCP was heterologously expressed in Escherichia coli and purified to homogeneity by two-steps purification method, namely, affinity and size-exclusion chromatography. Bioactivity was assessed by infiltrating micromolar concentrations of RmCP into leaves of the host (H. brasiliensis) and a model non-host (Nicotiana tabacum).

Results: Cell death (Trypan blue), reactive-oxygen species (DAB/NBT), callose deposition (aniline blue) and transcription of four defense-related genes (HbCDPK5, HbMAPK, HbPR3, HbEDS1) were monitored over 72 h. Purified RmCP migrated as a single band between 11 and 17 kDa band. Infiltration induced localized necrosis in N. tabacum within 48 h and in detached rubber leaves within 72 h. Both hosts accumulated H₂O₂ and O₂^-, and deposited callose. Additionally, significant up-regulation of HbCDPK5 and HbMAPK (early signaling), followed by strong induction of downstream effector genes, HbPR3 and HbEDS1 was observed in H. brasiliensis. These findings identify RmCP as the first basidiomycete CP shown to activate multilayer innate immunity in a latex-producing perennial.

Conclusion: The study extends the functional spectrum of the CP family beyond ascomycete models and provides a biochemically defined platform for developing protein-based priming agents to combat white-root disease in rubber plantations.

Keywords: Hevea brasiliensis; Rigidoporus microporus; cerato-platanin; elicitor; plant immunity; white root disease.

Figure 1

Purification of RmCP recombinant protein. (A) Elution profile of affinity chromatography using HisTrap™ HP affinity column (GE Healthcare Bio-Sciences AB, Sweden) coupled with ÄKTAprime plus system (GE Healthcare, United States) for the purification of RmCP from the soluble fraction of cell lysate. (B) Elution profile of gel filtration chromatography using HiPrep 16/60 Sephacryl S-200 High Resolution column (GE Healthcare Bio-Sciences AB, Sweden) coupled with ÄKTAprime plus system (GE Healthcare, USA) for the purification of RmCP from the pooled fractions from affinity chromatography. (C) SDS-PAGE analysis of purified RmCP. Clear migration of a single band of the His-tagged recombinant RmCP was observed between 11 kDa and 17 kDa.

Figure 2

The cell death-inducing activity of RmCP. (A) Nicotiana tabacum leaf and (B) Hevea brasiliensis leaf were infiltrated with RmCP and control buffer (0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2; CB) and photographed on the fifth day post infiltration (dpi). Leaf area infiltrated with RmCP displayed clear necrotic lesion while the area infiltrated with CB appeared clear of any lesion. (C) Microscopic observation of cell death in N. tabacum and H. brasiliensis leaves infiltrated with CB and RmCP. Leaves were stained with trypan blue at 24 h post infiltration (hpi) and observed under light microscope. Dead leaf cells were stained blue.

Figure 3

The accumulation of reactive oxygen species (ROS) and callose deposition in infiltrated N. tabacum and H. brasiliensis leaves. (A) Microscopic observation of H₂O₂ accumulation in N. tabacum and H. brasiliensis leaves. Leaves infiltrated with control buffer (0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2; CB) and RmCP were stained with 3,3′-diaminobenzidine (DAB) 24 h post infiltration (hpi) and photographed under light microscope. H₂O₂ accumulation indicated by brown DAB deposits observed in leaves infiltrated with RmCP but not in CB. (B) Microscopic observation of O₂⁻ accumulation in N. tabacum and H. brasiliensis leaves. Infiltrated leaves were stained with nitro blue tetrazolium (NBT). O₂⁻ accumulation indicated by blue precipitates of formazan observed in leaves infiltrated with RmCP but not in CB. (C) Microscopic observation of callose deposition in N. tabacum and H. brasiliensis leaves. Infiltrated leaves were stained with aniline blue and photographed under light microscope with UV filter. Callose deposition indicated by green fluorescence observed in leaves infiltrated with RmCP but not in CB.

Figure 4

Relative abundance of HbCDPK5 (calcium-dependent protein kinase 5), HbMAPK (mitogen-activated protein kinase), HbPR3 (chitinase), and HbEDS1 (enhanced disease susceptibility 1) transcripts in leaves treated with purified RmCP recombinant protein. HbUBC2a (ubiquitin-protein ligase 2a) and HbUBC4 (ubiquitin-protein ligase 4) were used as internal control to normalize the data. The relative transcriptional abundances of the defense-related genes were determined after 24, 48 and 72 h post application (hpa) of RmCP and compared to expression level in control leaves treated with control buffer (0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2). Error bars were calculated based on three replicates. Asterisks indicate significant differences (p < 0.05, t-test).