Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa.

In plant-pathogen interaction, the hydrogen peroxide (H2O2) may play a dual role: its accumulation inhibits the growth of biotrophic pathogens, while it could help the infection/colonization process of plant by necrotrophic pathogens. One of the possible pathways of H2O production involves oxalic acid (Oxa) degradation by apoplastic oxalate oxidase. Here, we analyzed the production of H2O2, the presence of calcium oxalate (CaOx) crystals and the content of Oxa and ascorbic acid (Asa)--the main precursor of Oxa in plants--in susceptible and resistant cacao (Theobroma cacao L.) infected by the hemibiotrophic fungus Moniliophthora perniciosa. We also quantified the transcript level of ascorbate peroxidase (Apx), germin-like oxalate oxidase (Glp) and dehydroascorbate reductase (Dhar) by RT-qPCR. We report that the CaOx crystal amount and the H2O2 levels in the two varieties present distinct temporal and genotype-dependent patterns. Susceptible variety accumulated more CaOx crystals than the resistant one, and the dissolution of these crystals occurred in the early infection steps and in the final stage of the disease in the resistant and the susceptible variety, respectively. High expression of the Glp and accumulation of Oxa were observed in the resistant variety. The content of Asa increased in the inoculated susceptible variety, but remained constant in the resistant one. The susceptible variety presented reduced Dhar expression. The role of H2O2 and its formation from Oxa via Apx and Glp in resistant and susceptible variety infected by M. perniciosa were discussed.

Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms.

Three cystatin open reading frames named TcCys1, TcCys2 and TcCys3 were identified in cDNA libraries from compatible interactions between Theobroma cacao (cacao) and Moniliophthora perniciosa. In addition, an ORF named TcCys4 was identified in the cDNA library of the incompatible interaction. The cDNAs encoded conceptual proteins with 209, 127, 124, and 205 amino acid residues, with a deduced molecular weight of 24.3, 14.1, 14.3 and 22.8 kDa, respectively. His-tagged recombinant proteins were purified from Escherichia coli expression, and showed inhibitory activities against M. perniciosa. The four recombinant cystatins exhibited K(i) values against papain in the range of 152-221 nM. Recombinant TcCYS3 and TcCYS4 immobilized in CNBr-Sepharose were efficient to capture M. perniciosa proteases from culture media. Polyclonal antibodies raised against the recombinant TcCYS4 detected that the endogenous protein was more abundant in young cacao tissues, when compared with mature tissues. A ~85 kDa cacao multicystatin induced by M. perniciosa inoculation, MpNEP (necrosis and ethylene-inducing protein) and M. perniciosa culture supernatant infiltration were detected by anti-TcCYS4 antibodies in cacao young tissues. A direct role of the cacao cystatins in the defense against this phytopathogen was proposed, as well as its involvement in the development of symptoms of programmed cell death.

High-affinity copper transport and Snq2 export permease of saccharomyces cerevisiae modulate cytotoxicity of PR-10 from Theobroma cacao.

A pathogenesis-related (PR) protein from Theobroma cacao (TcPR-10) was identified from a cacao-Moniliophthora perniciosa interaction cDNA library. Nucleotide and amino acid sequences showed homology with other PR-10 proteins having P loop motif and Betv1 domain. Recombinant TcPR-10 showed in vitro and in vivo ribonuclease activity, and antifungal activity against the basidiomycete cacao pathogen M. perniciosa and the yeast Saccharomyces cerevisiae. Fluorescein isothiocyanate-labeled TcPR-10 was internalized by M. perniciosa hyphae and S. cerevisiae cells and inhibited growth of both fungi. Energy and temperature-dependent internalization of the TcPR-10 suggested an active importation into the fungal cells. Chronical exposure to TcPR-10 of 29 yeast mutants with single gene defects in DNA repair, general membrane transport, metal transport, and antioxidant defenses was tested. Two yeast mutants were hyperresistant compared with their respective isogenic wild type: ctr3Delta mutant, lacking the high-affinity plasma membrane copper transporter and mac1Delta, the copper-sensing transcription factor involved in regulation of high-affinity copper transport. Acute exposure of exponentially growing yeast cells revealed that TcPR-10 resistance is also enhanced in the Snq2 export permease-lacking mutant which has reduced intracellular presence of TcPR-10.

Comparative gene expression study to identify genes possibly related to storage root formation in cassava.

Cassava storage roots result from swelling of adventitious roots by secondary growth. In the present study we aimed to gain insight into the molecular processes occurring during cassava storage root formation. We report a comparative gene expression study in adventitious and storage roots in order to identify genes possibly related to storage organ formation. Our results revealed five genes with higher expression levels in secondary xylem of storage roots than adventitious roots. Among them, the Mec1 gene coding for Pt2L4 glutamic acid-rich protein and a putative RING Zinc Finger and LEA protein genes were strongly induced in secondary xylem tissue.