TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens.

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10-7 mol L-1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70-80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and ß-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL-1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

Phylogenetic Origin of Primary and Secondary Metabolic Pathway Genes Revealed by C. maxima and C. reticulata Diagnostic SNPs.

Modern cultivated Citrus species and varieties result from interspecific hybridization between four ancestral taxa. Among them, Citrus maxima and Citrus reticulata, closely associated with the pummelo and mandarin horticultural groups, respectively, were particularly important as the progenitors of sour and sweet oranges (Citrus aurantium and Citrus sinensis), grapefruits (Citrus paradisi), and hybrid types resulting from modern breeding programs (tangors, tangelos, and orangelos). The differentiation between the four ancestral taxa and the phylogenomic structure of modern varieties widely drive the phenotypic diversity's organization. In particular, strong phenotypic differences exist in the coloration and sweetness and represent important criteria for breeders. In this context, focusing on the genes of the sugar, carotenoid, and chlorophyll biosynthesis pathways, the aim of this work was to develop a set of diagnostic single-nucleotide polymorphism (SNP) markers to distinguish the ancestral haplotypes of C. maxima and C. reticulata and to provide information at the intraspecific diversity level (within C. reticulata or C. maxima). In silico analysis allowed the identification of 3,347 SNPs from selected genes. Among them, 1,024 were detected as potential differentiation markers between C. reticulata and C. maxima. A total of 115 SNPs were successfully developed using a competitive PCR technology. Their transferability among all Citrus species and the true citrus genera was very good, with only 0.87% of missing data. The ancestral alleles of the SNPs were identified, and we validated the usefulness of the developed markers for tracing the ancestral haplotype in large germplasm collections and sexually recombined progeny issued from the C. reticulata/C. maxima admixture gene pool. These markers will pave the way for targeted association studies based on ancestral haplotypes.