A cDNA fragment (FaPR4) encoding a class I pathogenesis-related protein 4 (PR-4) from Ficus awkeotsang was obtained by PCR cloning. Plant PR-4s were grouped into class I and II, differing by the presence of ChtBD and hinge. The predicted mature FaPR4 comprises N-terminal chitin-binding domain (ChtBD), hinge, Barwin domain and C-terminal extension. FaPR4-C, an N-terminal truncated form of FaPR4, was designed to mimic the structural feature of class II PR-4s. FaPR4 and FaPR4-C were over-expressed in yeast Pichia pastoris, and both recombinants exhibited RNase and anti-fungal activities. To our knowledge, it is the first report that FaPR4, a member of class I PR-4s has RNase activity as class II. FaPR4 possesses better anti-fungal activities toward Fusarium oxysporum and Sclerotium rolfsii than FaPR4-C. Heat-treated FaPR4 remained RNase and anti-fungal activities; while heat-treated FaPR4-C lost those activities. Therefore, ChtBD of FaPR4 may not only contribute to its anti-fungal but also improve the thermal stability of protein. It also implied the correlation of RNase activity with anti-fungal activity of FaPR4-C. Furthermore, FaPR4 was detected to have weak but significant chitinase activity, and its chitinase activity was reduced after heat treatment. The chitinase activity by FaPR4-C was much lower than FaPR4.
"Moreover, most of the PR-4 proteins have a signal peptide and some of them show transmembrane structure in the N-terminal region
[28-30]. Some PR-4 proteins also present a C-terminal extension domain involved in protein targeting to the vacuole
[31-33]. It has been shown that the barley Barwin protein is able to slightly interact with the oligosaccharide β-(1,4) tetramer of N-acetylglucosamine, an analog of chitin
[Show abstract][Hide abstract] 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.
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.
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.
"The mechanism whereby PR4 proteins inhibit fungal growth occurs through chitinase (Ponstein et al., 1994; Lu et al., 2012) as well as RNase and DNase (Caporale et al., 2004; Bertini et al., 2009; Guevara-Morato et al., 2010; Li et al., 2010; Lu et al., 2012) activities. Although the purified recombinant LcPR4a inhibited A. lentis growth, its mode of action remains unknown and will be the subject of future studies. "
[Show abstract][Hide abstract] ABSTRACT: A novel pathogenesis-related protein 4 (PR4) encoding gene, LcPR4a, was induced in Lens culinaris following Ascochyta lentis infection. LcPR4a encodes a predicted 146 amino acid protein of 15.8 kDa. The putative LcPR4a protein belongs to the class II PR4 family and has close phylogenetic affinity to PR4 proteins from related species. qPCR analysis revealed differential expression of the LcPR4a gene upon Ascochyta lentis infection in both resistant and susceptible cultivars. This, combined with preliminary in vitro antifungal assays of the recombinant protein expressed in E. coli, suggests the potential important role of LcPR4a in the defence response of lentil to Ascochyta lentis attack.
[Show abstract][Hide abstract] ABSTRACT: Crop plants have evolved an array of mechanisms to counter biotic and abiotic stresses. Many pathogenesis-related proteins are expressed by plants during the attack of pathogens. Advances in recombinant DNA technology and understanding of plant-microbe interactions at the molecular level have paved the way for isolation and characterization of genes encoding such proteins, including chitinases. Chitinases are included in families 18 and 19 of glycosyl hydrolases (according to www.cazy.org ) and they are further categorized into seven major classes based on their aminoacid sequence homology, three-dimensional structures, and hydrolytic mechanisms of catalytic reactions. Although chitin is not a component of plant cell walls, plant chitinases are involved in development and non-specific stress responses. Also, chitinase genes sourced from plants have been successfully over-expressed in crop plants to combat fungal pathogens. Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.
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