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.

Sara Pereira Menezes,Edson Mario De Andrade Silva,Carlos Priminho Pirovani,Karina Peres Gramacho,Fabienne Micheli,Livia Santos Lima Lemos,Abelmon Da Silva Gesteira,Aurizângela Oliveira De Sousa,Bruno Silva Andrade,Eline Matos Lima

doi:10.1186/1471-2229-14-161

Abstract

BackgroundThe 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.ResultsTcPR-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.ConclusionTo 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.

Highlights

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
Sequence analysis of TcPR-4b The TcPR-4b gene identified from T. cacao-M. perniciosa interaction library presents an Open reading frame (ORF) of 429 nucleotides encoding a protein of 142 amino acid residues (Figure 1)
The analysis of the TcPR-4b domains by comparison with other classification is based on the presence (class I) and PR-4s from various mono- and dicotyledonous species, showed that TcPR4-b presents a high identity with class II PR-4s from other plant species (Figure 2; Table 1) such as Malus domestica (86%; MdPR-4 accession number AFH74426.1), Nicotiana tabacum (84%; NtPR-4B accession number P29063.1), Solanum lycopersicum (78%; SlPR-4 accession number NP_001234083.1), Capsicum annuum (77%; CaPR-4 accession number AAF63520.1), Capsicum chinense (78%; chinense class II PR-4 (CcPR-4) accession number BAD11073), Triticum aestivum (75%; Wheatwin2 accession number O64393.1) and Lycoris radiate (75%; LrPR4 accession number ACI31201.1)

Summary

Introduction

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. The PR proteins are defined as plant proteins induced in pathological situations not necessary implied with a direct interaction with the pathogen [4,5]. Several characterized PR proteins showed enzymatic activities related to antimicrobial properties. Focusing on PR-4 proteins, several studies showed the involvement of this family in plant defense responses regulated by signal molecules, such as salicylic acid (SA), abscissic acid (ABA), jasmonate (JA) and ethylene (ET). The MdPR-4 gene expression was associated to the plant defense response against Botryosphaeria dothidea, through signalization pathways dependent of SA and JA, as well as to several physiological functions such as flower formation [26]

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