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Coffee leaf rust, caused by the fungus Hemileia vastatrix, has become a major concern for coffee-producing countries. Additionally, there has been an increase in the resistance of certain races of the fungus to fungicides and breeding cultivars, making producers use alternative control methods. In this work, we transplanted the leaf surface microbiota of rust-resistant coffee species (Coffea racemosa and Coffea stenophylla) to Coffea arabica and tested whether the new microbiota would be able to minimize the damage caused by H. vastatrix. It was seen that the transplant was successful in controlling rust, especially from C. stenophylla, but the protection depended on the concentration of the microbiota. Certain fungi, such as Acrocalymma, Bipolaris, Didymella, Nigrospora, Setophaeosphaeria, Simplicillium, Stagonospora and Torula, and bacteria, such as Chryseobacterium, Sphingobium and especially Enterobacter, had their populations increased and this may be related to the antagonism seen against H. vastatrix. Interestingly, the relative population of bacteria from genera Pantoea, Methylobacterium and Sphingomonas decreased after transplantation, suggesting a positive interaction between them and H. vastatrix development. Our findings may help to better understand the role of the microbiota in coffee leaf rust, as well as help to optimize the development of biocontrol agents., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

An efficient method of genomic DNA extraction that provides high quality and yield is a crucial pre-requisite and limiting factor in plant genetic analysis. However, pure genomic DNA can be challenging to obtain from some plant species due to their sugar and secondary metabolite contents. Lippia alba is an important aromatic and medicinal plant, chemically characterized by the presence of tannins, flavonoids, anthocyanins, and essential oils, which interfere with the extraction of pure genomic DNA. In this scenario, optimizing the extraction methods and minimizing the effects of these compounds are necessary. This study compares six plant DNA extraction protocols based on the CTAB method. The quality and quantity of DNA samples obtained were determined by physical appearance by electrophoresis in agarose gels and spectrophotometry. The results highlight the difficulty in obtaining pure and clear bands for all tested methods, except for the polyvinylpyrrolidone (PVP)-based protocol created by our team, which was the better option for obtaining high-quality genomic DNA of L. alba. We conclude that adding PVP-40 into DNA extraction buffers can optimize the DNA extraction of L. alba and indicate this protocol for DNA extraction from other aromatic plants., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The analysis of large-scale sequence data has revealed that plants over time recruit certain microbes that are efficient colonizers of the rhizosphere. This enrichment phenomenon is especially seen in annual crops, but we suggest that there could have been some type of enrichment in perennial crops such as coffee plants. To verify this hypothesis, we performed a metagenomic and chemical analysis in rhizosphere with three different plant ages (young, mature, and old) and cultivated on the same farm. We verified that from mature to old plants, there was a decrease in diversity, particularly Fusarium and Plenodomus, while there was an increase in Aspergillus, Cladosporium, Metarhizium, and Pseudomonas. We also detected that the abundance of anti-microbials and ACC-deaminase grows as plants age, although denitrification and carbon fixation had reduced abundances. In summary, we detected an enrichment in the microbial community, especially in the great increase in the participation of Pseudomonas, passing from 50% of the relative abundance as the plants get older. Such enrichment can occur through the dynamics of nutrients such as magnesium and boron., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Many Pseudomonas species promote plant growth and colonize a wide range of environments. The annotation of a Coffea arabica ESTs database revealed a considerable number of Pseudomonas sequences. To evaluate the genomic and physiology of Pseudomonas that inhabit coffee plants, fluorescent Pseudomonas from C. arabica root environment were isolated. Two of them had their genomes sequenced; one from rhizospheric soil, named as MNR3A, and one from internal part of the root, named as EMN2. In parallel, we performed biochemical and physiological experiments to confirm genomic analyses results. Interestingly, EMN2 has achromobactin and aerobactin siderophore receptors, but does not have the genes responsible for the production of these siderophores, suggesting an interesting bacterial competition strategy. The two bacterial isolates were able to degrade and catabolize plant phenolic compounds for their own benefit. Surprisingly, MNR3A and EMN2 do not contain caffeine methylases that are responsible for the catabolism of caffeine. In fact, bench experiments confirm that the bacteria did not metabolize caffeine, but were resistant and chemically attracted to it. Furthermore, both bacteria, most especially MNR3A, were able to increase growth of lettuce plants. Our results indicate MNR3A as a potential plant growth promoting bacteria., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Background: Pseudomonas spp. promotes plant growth and colonizes a wide range of environments. During the annotation of a Coffea arabica ESTs database, we detected a considerable number of contaminant Pseudomonas sequences, specially associated with leaves. The genome of a Pseudomonas isolated from coffee leaves was sequenced to investigate in silico information that could offer insights about bacterial adaptation to coffee phyllosphere. In parallel, several experiments were performed to confirm certain physiological characteristics that could be associated with phyllospheric behavior. Finally, in vivo and in vitro experiments were carried out to verify whether this isolate could serve as a biocontrol agent against coffee rust and how the isolate could act against the infection.  RESULTS: The isolate showed several genes that are associated with resistance to environmental stresses, such as genes encoding heat/cold shock proteins, antioxidant enzymes, carbon starvation proteins, proteins that control osmotic balance and biofilm formation. There was an increase of exopolysaccharides synthesis in response to osmotic stress, which may protect cells from dessication on phyllosphere. Metabolic pathways for degradation and incorporation into citrate cycle of phenolic compounds present in coffee were found, and experimentally confirmed. In addition, MN1F was found to be highly tolerant to caffeine. The experiments of biocontrol against coffee leaf rust showed that the isolate can control the progress of the disease, most likely through competition for resources., Conclusion: Genomic analysis and experimental data suggest that there are adaptations of this Pseudomonas to live in association with coffee leaves and to act as a biocontrol agent., (© 2022. The Author(s).)

Coffee is one of the most important commodities in the global market. Of the 130 species of Coffea , only Coffea arabica and Coffea canephora are actually cultivated on a large scale. Despite the economic and social importance of coffee, little research has been done on the coffee tree microbiome. To assess the structure and function of the rhizosphere microbiome, we performed a deep shotgun metagenomic sequencing of the rhizospheres of five different species, C. arabica, C. canephora, Coffea stenophylla, Coffea racemosa, and Coffea liberica. Our findings indicated that C. arabica and C. stenophylla have different microbiomes, while no differences were detected between the other Coffea species. The core rhizosphere microbiome comprises genera such as Streptomyces, Mycobacterium, Bradyrhizobium, Burkholderia, Sphingomonas, Penicillium, Trichoderma, and Rhizophagus, several of which are potential plant-beneficial microbes. Streptomyces and mycorrhizal fungi dominate the microbial communities. The concentration of sucrose in the rhizosphere seems to influence fungal communities, and the concentration of caffeine/theobromine has little effect on the microbiome. We also detected a possible relationship between drought tolerance in Coffea and known growth-promoting microorganisms. The results provide important information to guide future studies of the coffee tree microbiome to improve plant production and health. IMPORTANCE The microbiome has been identified as a fundamental factor for the maintenance of plant health, helping plants to fight diseases and the deleterious effects of abiotic stresses. Despite this, in-depth studies of the microbiome have been limited to a few species, generally with a short life cycle, and perennial species have mostly been neglected. The coffee tree microbiome, on the other hand, has gained interest in recent years as Coffea trees are perennial tropical species of enormous importance, especially for developing countries. A better understanding of the microorganisms associated with coffee trees can help to mitigate the deleterious effects of climate change on the crop, improving plant health and making the system more sustainable.

Coffea arabica is an allotetraploid of high economic importance. C. arabica transcriptome is a combination of the transcripts of two parental genomes (C. eugenioides and C. canephora) that gave rise to the homeologous genes of the species. Previous studies have reported the transcriptional dynamics of C. arabica. In these reports, the ancestry of homeologous genes was identified and the overall regulation of homeologous differential expression (HDE) was explored. One of these genes is part of the FRIGIDA-like family (FRL), which includes the Arabidopsis thaliana flowering-time regulation protein, FRIGIDA (FRI). As nonfunctional FRI proteins give rise to rapid-cycling summer annual ecotypes instead of vernalization-responsive winter-annuals, allelic variation in FRI can modulate flowering time in A. thaliana. Using bioinformatics, genomic analysis, and the evaluation of gene expression of homeologs, we characterized the FRL gene family in C. arabica. Our findings indicate that C. arabica expresses 10 FRL homeologs, and that, throughout flower and fruit development, these genes are differentially transcribed. Strikingly, in addition to confirming the expression of FRL genes during zygotic embryogenesis, we detected FRL expression during direct somatic embryogenesis, a novel finding regarding the FRL gene family. The HDE profile of FRL genes suggests an intertwined homeologous gene regulation. Furthermore, we observed that FLC gene of C. arabica has an expression profile similar to that of CaFRL genes.

The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao (Theobroma cacao) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Coffee is one of the most valuable agricultural commodities and the plants' leaves are the primary site of infection for most coffee diseases, such as the devastating coffee leaf rust. Therefore, the use of bacterial microbiota that inhabits coffee leaves to fight infections could be an alternative agricultural method to protect against coffee diseases. Here, we report the leaf-associated bacteria in three coffee genotypes over the course of a year, with the aim to determine the diversity of bacterial microbiota. The results indicate a prevalence of Enterobacteriales in Coffea canephora, Pseudomonadales in C. arabica 'Obatã', and an intriguing lack of bacterial dominance in C. arabica 'Catuaí'. Using PERMANOVA analyses, we assessed the association between bacterial abundance in the coffee genotypes and environmental parameters such as temperature, precipitation, and mineral nutrients in the leaves. We detected a close relationship between the amount of Mn and the abundance of Pseudomonadales in 'Obatã' and the amount of Ca and the abundance of Enterobacteriales in C. canephora. We suggest that mineral nutrients can be key drivers that shape leaf microbial communities.

Moniliophthora perniciosa is the causative agent of witches' broom disease, which devastates cacao cultures in South America. This pathogenic fungus infects meristematic tissues and derives nutrients from the plant apoplast during an unusually long-lasting biotrophic stage. To survive, the fungus produces proteins to suppress the plant immune response. Proteins of the PR-1 (pathogenesis-related 1)/CAP superfamily have been implicated in fungal virulence and immune suppression. The genome of M. perniciosa encodes 11 homologues of plant PR-1 proteins, designated MpPR-1 proteins, but their precise mode of action is poorly understood. In this study, we expressed MpPR-1 proteins in a yeast model lacking endogenous CAP proteins. We show that some members of the MpPR-1 family bind and promote secretion of sterols, whereas others bind and promote secretion of fatty acids. Lipid binding by purified MpPR-1 occurs with micromolar affinity and is saturable in vitro Sterol binding by MpPR-1 requires the presence of a flexible loop region containing aromatic amino acids, the caveolin-binding motif. Remarkably, MpPR-1 family members that do not bind sterols can be converted to sterol binders by a single point mutation in the caveolin-binding motif. We discuss the possible implications of the lipid-binding activity of MpPR-1 family members with regard to the mode of action of these proteins during M. perniciosa infections., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pathogenic fungi Moniliophthora perniciosa causes Witches' Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is found in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.