TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4.

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches' broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.

Citrus tristeza virus (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety "Westin".

Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus.