The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca(2+) and Mg(2+) dependent-DNase activity and antifungal action on Moniliophthora perniciosa

Abstract

Background: The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b.

Results: TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae.

Conclusion: To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively.

Figure 1

Nucleotide and amino acid sequences of TcPR-4b. The signal peptide is underlined; the remaining sequence corresponds to the Barwin domain. The asterisk represents the ORF termination codon. The putative phosphorylation site is squared on the amino acid sequence. RT-qPCR primer position is indicated in gray.

Figure 2

Amino acid sequence alignment of TcPR-4b with other PR4 proteins identified from NCBI database. The class I PR4s used for alignment are: AtHEL (Arabdopsis thaliana, NP_187123.1), FaPR4 (Ficus pumila var. awkeotsang, ADO24163.1), CcPR-4 (Capsicum chinense, BAD11073), StWIN2 (Solanum tuberosum, P09762), NtCBP20 (Nicotiana tabacum, AAB29959.2). The class II PR-4 s used for the alignment were: MdPR-4 (Malus domestica, AFH74426.1), NtPR-4B (Nicotiana tabacum, P29063.1), CaPR-4 (Capsicum annuum, AAF63520.1), SlPR-4 (Solanum lycopersicum, NP_001234083.1), Wheatwin1 (Triticum aestivum, O64392.1), Wheatwin2 (Triticum aestivum, O64393.1), Barwin (Hordeum vulgare, 1BW3_A), OsPR-4b (Oryza sativa, AY435041), LrPR4 (Lycoris radiate, ACI31201.1). Gaps introduced to get the best alignment are indicated by (-), (*) represents identical amino acids between all sequences, (.) and (:) represent conserved substitutions and semi-conserved substitutions, respectively. Signal peptide, chitin-binding domain and vacuolar signal are indicated in gray scale. The Barwin domain is also indicated; the six conserved cysteines in the Barwin domain are highlighted in black.

Figure 3

Tridimensional structure of TcPR-4b obtained by homology modeling with the barley Barwin protein (PDB code 1BW3_A.pdb) as template using SWISS-MODEL. A. Alignment of TcPR-4b with the barley Barwin protein. B. Ribbon representation of the TcPR-4b structure. The secondary structures are indicated by different colors: α-helices in red, β-strands in green and P-loops in blue. Dots indicate conserved cysteine residues forming disulfide linkages C₄₉-C₈₁, C₇₀-C₁₀₄ and C₈₄-C₁₄₃. C. Molecular surface of TcPR-4b with the T₉₇ putative phosphorylation site indicated in yellow. D. Molecular surface position of conserved amino acids H₃₁, H₁₃₀, D₁₁₀ e R₂₇ which are highlighted in yellow. E. Molecular surface position of the regions 35-41 and 109-111 are indicated in pink.

Figure 4

Scheme of the PR-4 family in the Theobroma cacao genome database (CocoaGenDB). A. Struture of the PR-4 proteins from Theobroma cacao. ChtB: chitin-binding domain. B. Localization and organization of five of the PR-4 genes on Theobroma cacao chromosome 5. Grey arrows indicate the PR-4 gene position.

Figure 5

Bayesian phylogenetic analysis, using amino acid data. Bayesian consensus phylogram of PR-4 from T. cacao and other plant species. VS: vacuolar signal.

Figure 6

Relative expression of TcPR-4b in TSH1188 and Catongo meristems inoculated or not (control) with M. perniciosa. A. Representation of the plant symptoms and fungus phase during the infection time course in Catongo genotype. The harvesting times of inoculated plants are indicated on the top of the figure. (*) indicates the times that were harvested also in the non-inoculated (control) plants. B. RT-qPCR of TcPR4b. The control, used as calibrator (for this reason is always 1), corresponds to the average of the expression values of TcPR-4b in 5 non-inoculated samples in each genotype (see also Methods section). The results are the arithmetical mean of the repetitions ± standard error. Different letters indicate significant statistical difference between samples by the Scott-Knott test (P ≤ 0.01): lower case letters correspond to statistics between harvesting times for each genotype while upper case letters correspond to statistics between genotypes for each harvesting time. dai: days after inoculation; hai: hours after inoculation.

Figure 7

SDS-PAGE analysis of the recombinant TcPR-4b protein. M. Molecular weight marker (Thermo Scientific). Lane 1: pET28a without insert and without induction. Lane 2: pET28a without insert and 4 h after induction with 1 M of IPTG at 37°C. Lane 3: pET28a-TcPR-4b 1 h after induction with 1 M of IPTG at 37°C. Lane 4: pET28a-TcPR-4b 2 h after induction with 1 M of IPTG at 37°C. Lane 5: pET28a-TcPR-4b 3 h after induction with 1 M of IPTG at 37°C. Lane 6: pET28a-TcPR-4b 4 h after induction with 1 M of IPTG at 37°C. Lane 7: pET28a-TcPR-4b 1 h after induction with 0.4 M of IPTG at 37°C. Lane 8: pET28a-TcPR-4b 2 h after induction with 0.4 M of IPTG at 37°C. Lane 9: pET28a-TcPR-4b 3 h after induction with 0.4 M of IPTG at 37°C. Lane 10: pET28a-TcPR-4b 4 h after induction with 0.4 M of IPTG at 37°C. Lane 11: pET28a-TcPR-4b after induction overnight with 1 M of IPTG at 18°C. Lane 12: pET28a-TcPR-4b after induction overnight with 0.4 M of IPTG at 18°C. Lane 13: purified insoluble fraction of the recombinant TcPR-4b (15.43 kDa). The arrow indicates the recombinant TcPR-4b protein.

Figure 8

Ribonuclease activity of the recombinant TcPR-4b on tomato (Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

Figure 9

Deoxyribonuclease activity of recombinant TcPR-4b. A and B. DNase activity of the TcPR-4b tested against 1 μg of plasmidial DNA (pGEM-T® Easy Vector; Promega). C. DNase activity of the TcPR-4b tested against 1 μg of genomic DNA from Nicotiana tabacum. The incubation with TcPR-4b was made overnigth at 25°C. The boiling conditions were 10 min at 95°C. The following concentrations were used: 10 mM of MgCl₂, 10 mM of EDTA; 1 mM CaCl₂. gDNA: genomic DNA; pDNA: plasmidial DNA.

Figure 10

Chitinase activity of TcPR-4b. Blank: buffer A or B (see Methods section).

Figure 11

Action of TcPR-4b on dikaryotic M. perniciosa survival. A. Antifungal activity of recombinant TcPR-4b protein. PBS: phosphate buffered saline. B to E. ROS detection on dikaryotic M. perniciosa broken hyphae in absence (B and C; control -PBS) or in presence (C and E) of TcPR-4b (10 μg/ml). B and C, bars = 30 μm; D and E, bars = 10 μm. The arrows indicated the fluorescent foci.

Figure 12

Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A. Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B. Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl₂. The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl₂.