The small RNA-mediated gene silencing machinery is required in Arabidopsis for stimulation of growth, systemic disease resistance, and suppression of the nitrile-specifier gene NSP4 by Trichoderma atroviride.

Trichoderma atroviride is a root-colonizing fungus that confers multiple benefits to plants. In plants, small RNA (sRNA)-mediated gene silencing (sRNA-MGS) plays pivotal roles in growth, development, and pathogen attack. Here, we explored the role of core components of Arabidopsis thaliana sRNA-MGS pathways during its interaction with Trichoderma. Upon interaction with Trichoderma, sRNA-MGS-related genes paralleled the expression of Arabidopsis defense-related genes, linked to salicylic acid (SA) and jasmonic acid (JA) pathways. SA- and JA-related genes were primed by Trichoderma in leaves after the application of the well-known pathogen-associated molecular patterns flg22 and chitin, respectively. Defense-related genes were primed in roots as well, but to different extents and behaviors. Phenotypical characterization of mutants in AGO genes and components of the RNA-dependent DNA methylation (RdDM) pathway revealed that different sets of sRNA-MGS-related genes are essential for (i) the induction of systemic acquired resistance against Botrytis cinerea, (ii) the activation of the expression of plant defense-related genes, and (iii) root colonization by Trichoderma. Additionally, plant growth induced by Trichoderma depends on functional RdDM. Profiling of DNA methylation and histone N-tail modification patterns at the Arabidopsis Nitrile-Specifier Protein-4 (NSP4) locus, which is responsive to Trichoderma, showed altered epigenetic modifications in RdDM mutants. Furthermore, NSP4 is required for the induction of systemic acquired resistance against Botrytis and avoidance of enhanced root colonization by Trichoderma. Together, our results indicate that RdDM is essential in Arabidopsis to establish a beneficial relationship with Trichoderma. We propose that DNA methylation and histone modifications are required for plant priming by the beneficial fungus against B. cinerea.

Secretome Analysis of Arabidopsis-Trichoderma atroviride Interaction Unveils New Roles for the Plant Glutamate:Glyoxylate Aminotransferase GGAT1 in Plant Growth Induced by the Fungus and Resistance against Botrytis cinerea.

The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.

IPA-1 a Putative Chromatin Remodeler/Helicase-Related Protein of Trichoderma virens Plays Important Roles in Antibiosis Against Rhizoctonia solani and Induction of Arabidopsis Systemic Disease Resistance.

Trichoderma spp. are filamentous fungi that colonize plant roots conferring beneficial effects to plants, either indirectly through the induction of their defense systems or directly through the suppression of phytopathogens in the rhizosphere. Transcriptomic analyses of Trichoderma spp. emerged as a powerful method for identifying the molecular events underlying the establishment of this beneficial relationship. Here, we focus on the transcriptomic response of Trichoderma virens during its interaction with Arabidopsis seedlings. The main response of T. virens to cocultivation with Arabidopsis was the repression of gene expression. The biological processes of transport and metabolism of carbohydrates were downregulated, including a set of cell wall-degrading enzymes putatively relevant for root colonization. Repression of such genes reached their basal levels at later times in the interaction, when genes belonging to the biological process of copper ion transport were induced, a necessary process providing copper as a cofactor for cell wall-degrading enzymes with the auxiliary activities class. RNA-Seq analyses showed the induction of a member of the SNF2 family of chromatin remodelers/helicase-related proteins, which was named IPA-1 (increased protection of Arabidopsis-1). Sequence analyses of IPA-1 showed its closest relatives to be members of the Rad5/Rad16 and SNF2 subfamilies; however, it grouped into a different clade. Although deletion of IPA-1 in T. virens did not affect its growth, the antibiotic activity of Δipa-1 culture filtrates against Rhizoctonia solani diminished but it remained unaltered against Botrytis cinerea. Triggering of the plant defense genes in plants treated with Δipa-1 was higher, showing enhanced resistance against Pseudomonas syringae but not against B. cinerea as compared with the wild type.

TBRG-1 a Ras-like protein in Trichoderma virens involved in conidiation, development, secondary metabolism, mycoparasitism, and biocontrol unveils a new family of Ras-GTPases.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

Genome-wide identification of gene families related to miRNA biogenesis in Mangifera indica L. and their possible role during heat stress.

Mango is a popular tropical fruit that requires quarantine hot water treatment (QHWT) for postharvest sanitation, which can cause abiotic stress. Plants have various defense mechanisms to cope with stress; miRNAs mainly regulate the expression of these defense responses. Proteins involved in the biogenesis of miRNAs include DICER-like (DCL), ARGONAUTE (AGO), HYPONASTIC LEAVES 1 (HYL1), SERRATE (SE), HUA ENHANCER1 (HEN1), HASTY (HST), and HEAT-SHOCK PROTEIN 90 (HSP90), among others. According to our analysis, the mango genome contains five DCL, thirteen AGO, six HYL, two SE, one HEN1, one HST, and five putative HSP90 genes. Gene structure prediction and domain identification indicate that sequences contain key domains for their respective gene families, including the RNase III domain in DCL and PAZ and PIWI domains for AGOs. In addition, phylogenetic analysis indicates the formation of clades that include the mango sequences and their respective orthologs in other flowering plant species, supporting the idea these are functional orthologs. The analysis of cis-regulatory elements of these genes allowed the identification of MYB, ABRE, GARE, MYC, and MeJA-responsive elements involved in stress responses. Gene expression analysis showed that most genes are induced between 3 to 6 h after QHWT, supporting the early role of miRNAs in stress response. Interestingly, our results suggest that mango rapidly induces the production of miRNAs after heat stress. This research will enable us to investigate further the regulation of gene expression and its effects on commercially cultivated fruits, such as mango, while maintaining sanitary standards.

The Small GTPases in Fungal Signaling Conservation and Function.

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1-G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus-plant interactions is reviewed as well.

The clinical laboratory evolution in coccidioidomycosis detection: Future perspectives.

Coccidioidomycosis is a systemic disease caused by the fungi Coccidioides immitis and C. posadasii. It is a prevalent disease in arid regions with high temperatures and low precipitations in America. Coccidioidomycosis is a highly endemic disease of US-Mexico border states but commonly underdiagnosed. The diagnosis of coccidiomycosis is not easy due to the lack of specific symptoms; it is usually an integral approach, including clinical laboratory tests as an essential part of the diagnosis. Nevertheless, despite various laboratory tests available, affordability can be a limitation, mainly in developing countries. This review's objectives are 1) to learn the different laboratory approaches that arose and their application for clinical diagnosis; 2) to discuss their advantages and weaknesses, and finally, 3) propose what is on the horizon for future advances in clinical laboratory diagnosis of coccidioidomycosis. It has been a long way in laboratory tests evolution to detect coccidioidomycosis from tissue microscopy to Real-Time PCR. However, there is a delay in technology adoption for Coccidioides spp. detection in the clinical laboratory. The molecular Point of Care Testing (POCT) technology has reached us in our trench while research in PCR variants stills on-going. None of the currently existing scientific literature in coccidioidomycosis research has mentioned it. However, this trend in infectious and non-infectious disease diagnosis will continue in that way in order to offer better options for an easy and fast diagnosis. Undoubtedly, the implementation of molecular POCT for Coccidioides spp. would save resources in health care attention and improve access to diagnostic tools.

Genome-Wide Identification of Mango (Mangifera indica L.) Polygalacturonases: Expression Analysis of Family Members and Total Enzyme Activity During Fruit Ripening.

Mango (Mangifera indica L.) is an important commercial fruit that shows a noticeable loss of firmness during ripening. Polygalacturonase (PG, E.C. 3.2.1.15) is a crucial enzyme for cell wall loosening during fruit ripening since it solubilizes pectin and its activity correlates with fruit softening. Mango PGs were mapped to a genome draft using seventeen PGs found in mango transcriptomes and 48 bonafide PGs were identified. The phylogenetic analysis suggests that they are related to Citrus sinensis, which may indicate a recent evolutive divergence and related functions with orthologs in the tree. Gene expression analysis for nine PGs showed differential expression for them during post-harvest fruit ripening, MiPG21-1, MiPG14, MiPG69-1, MiPG17, MiPG49, MiPG23-3, MiPG22-7, and MiPG16 were highly up-regulated. PG enzymatic activity also increased during maturation and these results correlate with the loss of firmness observed in mango during post-harvest ripening, between the ethylene production burst and the climacteric peak. The analysis of PGs promoter regions identified regulatory sequences associated to ripening such as MADS-box, ethylene regulation like ethylene insensitive 3 (EIN3) factors, APETALA2-like and ethylene response element factors. During mango fruit ripening the action of at least these nine PGs contribute to softening, and their expression is regulated at the transcriptional level. The prediction of the tridimensional structure of some PGs showed a conserved parallel beta-helical fold related to polysaccharide hydrolysis and a modular architecture, where exons correspond to structural elements. Further biotechnological approaches could target specific softening-related PGs to extend mango post-harvest shelf life.

Mango (Mangifera indica L.) cv. Kent fruit mesocarp de novo transcriptome assembly identifies gene families important for ripening.

Fruit ripening is a physiological and biochemical process genetically programmed to regulate fruit quality parameters like firmness, flavor, odor and color, as well as production of ethylene in climacteric fruit. In this study, a transcriptomic analysis of mango (Mangifera indica L.) mesocarp cv. "Kent" was done to identify key genes associated with fruit ripening. Using the Illumina sequencing platform, 67,682,269 clean reads were obtained and a transcriptome of 4.8 Gb. A total of 33,142 coding sequences were predicted and after functional annotation, 25,154 protein sequences were assigned with a product according to Swiss-Prot database and 32,560 according to non-redundant database. Differential expression analysis identified 2,306 genes with significant differences in expression between mature-green and ripe mango [1,178 up-regulated and 1,128 down-regulated (FDR ≤ 0.05)]. The expression of 10 genes evaluated by both qRT-PCR and RNA-seq data was highly correlated (R = 0.97), validating the differential expression data from RNA-seq alone. Gene Ontology enrichment analysis, showed significantly represented terms associated to fruit ripening like "cell wall," "carbohydrate catabolic process" and "starch and sucrose metabolic process" among others. Mango genes were assigned to 327 metabolic pathways according to Kyoto Encyclopedia of Genes and Genomes database, among them those involved in fruit ripening such as plant hormone signal transduction, starch and sucrose metabolism, galactose metabolism, terpenoid backbone, and carotenoid biosynthesis. This study provides a mango transcriptome that will be very helpful to identify genes for expression studies in early and late flowering mangos during fruit ripening.