Transcriptional regulation, led by transcription factors (TFs) such as those of the WRKY family, is a mechanism used by the organism to enhance or repress gene expression in response to stimuli. Here, we report on the genome-wide analysis of the Theobroma cacao WRKY TF family and also investigate the expression of WRKY genes in cacao infected by the fungus Moniliophthora perniciosa. In the cacao genome, 61 non-redundant WRKY sequences were found and classified in three groups (I to III) according to the WRKY and zinc-finger motif types. The 61 putative WRKY sequences were distributed on the 10 cacao chromosomes and 24 of them came from duplication events. The sequences were phylogenetically organized according to the general WRKY groups. The phylogenetic analysis revealed that subgroups IIa and IIb are sister groups and share a common ancestor, as well as subgroups IId and IIe. The most divergent groups according to the plant origin were IIc and III. According to the phylogenetic analysis, 7 TcWRKY genes were selected and analyzed by RT-qPCR in susceptible and resistant cacao plants infected (or not) with M. perniciosa. Some TcWRKY genes presented interesting responses to M. perniciosa such as Tc01_p014750/Tc06_p013130/AtWRKY28, Tc09_p001530/Tc06_p004420/AtWRKY40, Tc04_p016130/AtWRKY54 and Tc10_p016570/ AtWRKY70. Our results can help to select appropriate candidate genes for further characterization in cacao or in other Theobroma species.

The phylloplane is the first contact surface between Theobroma cacao and the fungus Moniliophthora perniciosa, which causes witches' broom disease (WBD). We evaluated the index of short glandular trichomes (SGT) in the cacao phylloplane and the effect of irrigation on the disease index of cacao genotypes with or without resistance to WBD, and identified proteins present in the phylloplane. The resistant genotype CCN51 and susceptible Catongo presented a mean index of 1,600 and 700 SGT cm, respectively. The disease index in plants under drip irrigation was reduced by approximately 30% compared with plants under sprinkler irrigation prior to inoculation. Leaf water wash (LWW) of the cacao inhibited the germination of spores by up to 98%. Proteins from the LWW of CCN51 were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by tandem mass spectrometry. The gel showed 71 spots and identified a total of 42 proteins (28 from the plant and 14 from bacteria). Proteins related to defense and synthesis of defense metabolites and involved in nucleic acid metabolism were identified. The results support the hypothesis that the proteins and water-soluble compounds secreted to the cacao phylloplane participate in the defense against pathogens. They also suggest that SGT can contribute to the resistance of cacao.

The leaf surface combines biochemical substances and pre-existing morphological structures, as well as the presence of microorganisms. This dynamic environment constitutes a plant's initial defense, as well as the first contact of phytopathogens during invasion. Spore germination starts on the phylloplane and is a fundamental process for fungal development, and hence the establishment of disease. In this review, we address the phylloplane's innate defense mechanisms and biochemical reactions involved in the early stage of phytopathogenic fungal development. The focus is present the pre-infection molecular and biochemical processes of the interaction between Theobroma cacao and Moniliophthora perniciosa, showing how the defense mechanisms of the phylloplane can act to inhibit proteins involved at the beginning of fungal spore germination. We conclude that the phylloplane of the cocoa resistant genotype to M. perniciosa has performed chemical compounds, pre-existing morphological structures and the presence of microorganisms that participate in the pre-infection defense of the plant. Also, the inhibition of proteins involved in the germination mechanism of M. perniciosa basidiospores by chemical and structural compounds present in the cocoa phylloplane may decrease the disease index. Therefore, understanding how the phylloplane defense acts in the fungal spore germination process is essential to develop pre-infection control strategies for cacao plants against witches' broom.