The selenium-independent phospholipid hydroperoxide glutathione peroxidase from Theobroma cacao (TcPHGPX) protects plant cells against damages and cell death.

Proteins from the glutathione peroxidase (GPX) family, such as GPX4 or PHGPX in animals, are extensively studied for their antioxidant functions and apoptosis inhibition. GPXs can be selenium-independent or selenium-dependent, with selenium acting as a potential cofactor for GPX activity. However, the relationship of plant GPXs to these functions remains unclear. Recent research indicated an upregulation of Theobroma cacao phospholipid hydroperoxide glutathione peroxidase gene (TcPHGPX) expression during early witches' broom disease stages, suggesting the use of antioxidant mechanisms as a plant defense strategy to reduce disease progression. Witches' broom disease, caused by the hemibiotrophic fungus Moniliophthora perniciosa, induces cell death through elicitors like MpNEP2 in advanced infection stages. In this context, in silico and in vitro analyses of TcPHGPX's physicochemical and functional characteristics may elucidate its antioxidant potential and effects against cell death, enhancing understanding of plant GPXs and informing strategies to control witches' broom disease. Results indicated TcPHGPX interaction with selenium compounds, mainly sodium selenite, but without improving the protein function. Protein-protein interaction network suggested cacao GPXs association with glutathione and thioredoxin metabolism, engaging in pathways like signaling, peroxide detection for ABA pathway components, and anthocyanin transport. Tests on tobacco cells revealed that TcPHGPX reduced cell death, associated with decreased membrane damage and H2O2 production induced by MpNEP2. This study is the first functional analysis of TcPHGPX, contributing to knowledge about plant GPXs and supporting studies for witches' broom disease control.

Tc-cAPX, a cytosolic ascorbate peroxidase of Theobroma cacao L. engaged in the interaction with Moniliophthora perniciosa, the causing agent of witches' broom disease.

The level of hydrogen peroxide (H2O2) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H2O2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T. cacao APX involved in the molecular interaction of T. cacao-M. perniciosa. The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M. perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T. cacao (GenBank: ABR68691.2) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (rTc-cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H2O2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that rTc-cAPX is a homodimer in solution. Results indicate that the rTc-cAPX is correctly folded, stable and biochemically active. The purified rTc-cAPX presented biotechnological potential and is adequate for future structural and functional studies.

Polygalacturonases from Moniliophthora perniciosa are regulated by fermentable carbon sources and possible post-translational modifications.

We report the first molecular and in silico analysis of Monilophthora perniciosa polygalacturonases (PGs). Three MpPG genes (MpPG1, MpPG2 and MpPG3) were identified and analyzed at transcriptional level, by RT-qPCR, in dikaryotic M. perniciosa mycelium grown on solid-bran based medium and on liquid medium supplemented with different fermentable and non-fermentable carbon sources. The MpPG genes presented different expression patterns suggesting different individual regulation. However, all are mainly regulated by fermentable carbon sources (galactose and mannose). The integrated analysis of PG gene expression and systems biology (using MpG1 and MpG2 orthologs in Neurospora crassa, named NCU06961 and NCU02369, respectively) allowed identifying some possible mechanism of protein regulation during the necrotrophic fungal phase. MpPG1-NCU06961 and MpPG2-NCU02369 directly or indirectly interacted with central and highly connected proteins involved in protein synthesis and protein regulation associated to post-translational modifications, in cell wall metabolism, and in cellular metabolism related to energy production. This analysis also allowed the identification of key proteins for further studies of M. perniciosa development and/or for disease management, such as MpPG2, a pectin methylesterase, an acetolactate synthase and the small ubiquitin-like modifier SMT3-like.

Identification of new potential regulators of the Medicago truncatula-Sinorhizobium meliloti symbiosis using a large-scale suppression subtractive hybridization approach.

We set up a large-scale suppression subtractive hybridization (SSH) approach to identify Medicago truncatula genes differentially expressed at different stages of the symbiotic interaction with Sinorhizobium meliloti, with a particular interest for regulatory genes. We constructed 7 SSH libraries covering successive stages from Nod factor signal transduction to S. meliloti infection, nodule organogenesis, and functioning. Over 26,000 clones were differentially screened by two rounds of macroarray hybridizations. In all, 3,340 clones, corresponding to genes whose expression was potentially affected, were selected, sequenced, and ordered into 2,107 tentative gene clusters, including 767 MtS clusters corresponding to new M. truncatula genes. In total, 52 genes encoding potential regulatory proteins, including transcription factors (TFs) and other elements of signal transduction cascades, were identified. The expression pattern of some of them was analyzed by quantitative reverse-transcription polymerase chain reaction in wild-type and in Nod- M. truncatula mutants blocked before or after S. meliloti infection. Three genes, coding for TFs of the bHLH and WRKY families and a C2H2 zinc-finger protein, respectively, were found to be upregulated, following S. meliloti inoculation, in the infection-defective mutant lin, whereas the bHLH gene also was expressed in the root-hair-curling mutant hcl. The potential role of these genes in early symbiotic steps is discussed.