The complete chloroplast genome sequence of Psittacanthus schiedeanus (Cham. & Schltdl.) G.Don. (Santalales: Loranthaceae), the first plastome of a mistletoe species in the Psittacantheae tribe.

Psittacanthus schiedeanus (Cham. & Schltdl.) G.Don., 1834, is a mistletoe species in the Loranthaceae, characteristic of the canopy in cloud forest edges and widely distributed in northern Mesoamerica. Here, we report the complete chloroplast genome sequence of P. schiedeanus, the first for a species in the Psittacantheae tribe. The circularized quadripartite structure of the P. schiedeanus chloroplast genome was 122,586 bp in length and included a large single-copy region of 72,507 bp and two inverted repeats of 21,283 bp separated by a small single-copy region of 7,513 bp. The genome contained 112 genes, of which 96 are unique, including 65 protein-coding genes, 27 transfer RNA, and four ribosomal RNA. The overall GC content in the plastome of P. schiedeanus is 36.9%. Based on 43 published complete chloroplast genome sequences for species in the families Loranthaceae and Santalaceae (Santalales), the maximum-likelihood phylogenetic tree with high-support bootstrap values indicated that P. schiedeanus in the Psittacantheae tribe is sister to the tribe Lorantheae. The chloroplast genome provided in this study represents a valuable resource for genetic, phylogenetic and conservation studies of Psittacanthus species, and an important advance for unraveling the evolutionary history of these hemiparasitic plants.

Transcriptional Basis for Haustorium Formation and Host Establishment in Hemiparasitic Psittacanthus schiedeanus Mistletoes.

The mistletoe Psittacanthus schiedeanus, a keystone species in interaction networks between plants, pollinators, and seed dispersers, infects a wide range of native and non-native tree species of commercial interest. Here, using RNA-seq methodology we assembled the whole circularized quadripartite structure of P. schiedeanus chloroplast genome and described changes in the gene expression of the nuclear genomes across time of experimentally inoculated seeds. Of the 140,467 assembled and annotated uniGenes, 2,000 were identified as differentially expressed (DEGs) and were classified in six distinct clusters according to their expression profiles. DEGs were also classified in enriched functional categories related to synthesis, signaling, homoeostasis, and response to auxin and jasmonic acid. Since many orthologs are involved in lateral or adventitious root formation in other plant species, we propose that in P. schiedeanus (and perhaps in other rootless mistletoe species), these genes participate in haustorium formation by complex regulatory networks here described. Lastly, and according to the structural similarities of P. schiedeanus enzymes with those that are involved in host cell wall degradation in fungi, we suggest that a similar enzymatic arsenal is secreted extracellularly and used by mistletoes species to easily parasitize and break through tissues of the host.

Antifungal Effect of Copper Nanoparticles against Fusarium kuroshium, an Obligate Symbiont of Euwallacea kuroshio Ambrosia Beetle.

Copper nanoparticles (Cu-NPs) have shown great antifungal activity against phytopathogenic fungi, making them a promising and affordable alternative to conventional fungicides. In this study, we evaluated the antifungal activity of Cu-NPs against Fusarium kuroshium, the causal agent of Fusarium dieback, and this might be the first study to do so. The Cu-NPs (at different concentrations) inhibited more than 80% of F. kuroshium growth and were even more efficient than a commercial fungicide used as a positive control (cupric hydroxide). Electron microscopy studies revealed dramatic damage caused by Cu-NPs, mainly in the hyphae surface and in the characteristic form of macroconidia. This damage was visible only 3 days post inoculation with used treatments. At a molecular level, the RNA-seq study suggested that this growth inhibition and colony morphology changes are a result of a reduced ergosterol biosynthesis caused by free cytosolic copper ions. Furthermore, transcriptional responses also revealed that the low- and high-affinity copper transporter modulation and the endosomal sorting complex required for transport (ESCRT) are only a few of the distinct detoxification mechanisms that, in its conjunction, F. kuroshium uses to counteract the toxicity caused by the reduced copper ion.

Transcriptomic Analysis of the Host Response to Mild and Severe CTV Strains in Naturally Infected Citrus sinensis Orchards.

Citrus tristeza virus (CTV) is an important threat to the global citrus industry, causing severe economic losses worldwide. The disease management strategies are focused on vector control, tree culling, and the use of resistant varieties and rootstocks. Sweet orange (Citrus sinensis) trees showing either severe or mild CTV symptoms have been observed in orchards in Veracruz, Mexico, and were probably caused by different virus strains. To understand these symptomatic differences, transcriptomic analyses were conducted using asymptomatic trees. CTV was confirmed to be associated with infected plants, and mild and severe strains were successfully identified by a polymorphism in the coat protein (CP) encoding gene. RNA-Seq analysis revealed more than 900 significantly differentially expressed genes in response to mild and severe strains, with some overlapping genes. Importantly, multiple sequence reads corresponding to Citrus exocortis viroid and Hop stunt viroid were found in severe symptomatic and asymptomatic trees, but not in plants with mild symptoms. The differential gene expression profiling obtained in this work provides an overview of molecular behavior in naturally CTV-infected trees. This work may contribute to our understanding of citrus-virus interaction in more natural settings, which can help develop strategies for integrated crop management.

Characterization of Two Fusarium solani Species Complex Isolates from the Ambrosia Beetle Xylosandrus morigerus.

Ambrosia beetles are insect vectors of important plant diseases and have been considered as a threat to forest ecosystems, agriculture, and the timber industry. Several factors have been suggested as promoters of the pathogenic behavior of ambrosia beetles; one of them is the nature of the fungal mutualist and its ability to establish an infectious process. In Mexico, Xylosandrus morigerus is an invasive ambrosia beetle that damages many agroecosystems. Herein, two different isolates from the X. morigerus ambrosia beetle belonging to the Fusarium genus are reported. Both isolates belong to the Fusarium solani species complex (FSSC) but not to the Ambrosia Fusarium clade (AFC). The two closely related Fusarium isolates are pathogenic to different forest and agronomic species, and the morphological differences between them and the extracellular protease profile suggest intraspecific variability. This study shows the importance of considering these beetles as vectors of different species of fungal plant pathogens, with some of them even being phylogenetically closely related and having different pathogenic abilities, highlighting the relevance of the fungal mutualist as a factor for the ambrosia complex becoming a pest.

Molecular evidence of the avocado defense response to Fusarium kuroshium infection: a deep transcriptome analysis using RNA-Seq.

Fusarium kuroshium is a novel member of the Ambrosia Fusarium Clade (AFC) that has been recognized as one of the symbionts of the invasive Kuroshio shot hole borer, an Asian ambrosia beetle. This complex is considered the causal agent of Fusarium dieback, a disease that has severely threatened natural forests, landscape trees, and avocado orchards in the last 8 years. Despite the interest in this species, the molecular responses of both the host and F. kuroshium during the infection process and disease establishment remain unknown. In this work, we established an in vitro pathosystem using Hass avocado stems inoculated with F. kuroshium to investigate differential gene expression at 1, 4, 7 and 14 days post-inoculation. RNA-seq technology allowed us to obtain data from both the plant and the fungus, and the sequences obtained from both organisms were analyzed independently. The pathosystem established was able to mimic Fusarium dieback symptoms, such as carbohydrate exudation, necrosis, and vascular tissue discoloration. The results provide interesting evidence regarding the genes that may play roles in the avocado defense response to Fusarium dieback disease. The avocado data set comprised a coding sequence collection of 51,379 UniGenes, from which 2,403 (4.67%) were identified as differentially expressed. The global expression analysis showed that F. kuroshium responsive UniGenes can be clustered into six groups according to their expression profiles. The biologically relevant functional categories that were identified included photosynthesis as well as responses to stress, hormones, abscisic acid, and water deprivation. Additionally, processes such as oxidation-reduction, organization and biogenesis of the cell wall and polysaccharide metabolism were detected. Moreover, we identified orthologues of nucleotide-binding leucine-rich receptors, and their possible action mode was analyzed. In F. kuroshium, we identified 57 differentially expressed genes. Interestingly, the alcohol metabolic process biological category had the highest number of upregulated genes, and the enzyme group in this category may play an important role in the mechanisms of secondary metabolite detoxification. Hydrolytic enzymes, such as endoglucanases and a pectate lyase, were also identified, as well as some proteases. In conclusion, our research was conducted mainly to explain how the vascular tissue of a recognized host of the ambrosia complex responds during F. kuroshium infection since Fusarium dieback is an ambrosia beetle-vectored disease and many variables facilitate its establishment.

Design of a diagnostic system based on molecular markers derived from the ascomycetes pan-genome analysis: The case of Fusarium dieback disease.

A key factor to take actions against phytosanitary problems is the accurate and rapid detection of the causal agent. Here, we develop a molecular diagnostics system based on comparative genomics to easily identify fusariosis and specific pathogenic species as the Fusarium kuroshium, the symbiont of the ambrosia beetle Euwallaceae kuroshio Gomez and Hulcr which is responsible for Fusarium dieback disease in San Diego CA, USA. We performed a pan-genome analysis using sixty-three ascomycetes fungi species including phytopathogens and fungi associated with the ambrosia beetles. Pan-genome analysis revealed that 2,631 orthologue genes are only shared by Fusarium spp., and on average 3,941 (SD ± 1,418.6) are species-specific genes. These genes were used for PCR primer design and tested on DNA isolated from i) different strains of ascomycete species, ii) artificially infected avocado stems and iii) plant tissue of field-collected samples presumably infected. Our results let us propose a useful set of primers to either identify any species from Fusarium genus or, in a specific manner, species such as F. kuroshium, F. oxysporum, and F. graminearum. The results suggest that the molecular strategy employed in this study can be expanded to design primers against different types of pathogens responsible for provoking critical plant diseases.

Novel findings to the biosynthetic pathway of magnoflorine and taspine through transcriptomic and metabolomic analysis of Croton draco (Euphorbiaceae).

BACKGROUND: Croton draco is an arboreal species and its latex as well as some other parts of the plant, are traditionally used in the treatment of a wide range of ailments and diseases. Alkaloids, such as magnoflorine, prevent early atherosclerosis progression while taspine, an abundant constituent of latex, has been described as a wound-healer and antitumor-agent. Despite the great interest for these and other secondary metabolites, no omics resources existed for the species and the biosynthetic pathways of these alkaloids remain largely unknown. RESULTS: To gain insights into the pathways involved in magnoflorine and taspine biosynthesis by C. draco and identify the key enzymes in these processes, we performed an integrated analysis of the transcriptome and metabolome in the major organs (roots, stem, leaves, inflorescences, and flowers) of this species. Transcript profiles were generated through high-throughput RNA-sequencing analysis while targeted and high resolution untargeted metabolomic profiling was also performed. The biosynthesis of these compounds appears to occur in the plant organs examined, but intermediaries may be translocated from the cells in which they are produced to other cells in which they accumulate. CONCLUSIONS: Our results provide a framework to better understand magnoflorine and taspine biosynthesis in C. draco. In addition, we demonstrate the potential of multi-omics approaches to identify candidate genes involved in the biosynthetic pathways of interest.

Genomic Signals of Adaptation towards Mutualism and Sociality in Two Ambrosia Beetle Complexes.

Mutualistic symbiosis and eusociality have developed through gradual evolutionary processes at different times in specific lineages. Like some species of termites and ants, ambrosia beetles have independently evolved a mutualistic nutritional symbiosis with fungi, which has been associated with the evolution of complex social behaviors in some members of this group. We sequenced the transcriptomes of two ambrosia complexes (Euwallacea sp. near fornicatus⁻Fusarium euwallaceae and Xyleborus glabratus⁻Raffaelea lauricola) to find evolutionary signatures associated with mutualism and behavior evolution. We identified signatures of positive selection in genes related to nutrient homeostasis; regulation of gene expression; development and function of the nervous system, which may be involved in diet specialization; behavioral changes; and social evolution in this lineage. Finally, we found convergent changes in evolutionary rates of proteins across lineages with phylogenetically independent origins of sociality and mutualism, suggesting a constrained evolution of conserved genes in social species, and an evolutionary rate acceleration related to changes in selective pressures in mutualistic lineages.

Impact of Rearing Conditions on the Ambrosia Beetle's Microbiome.

Ambrosia beetles, along with termites and leafcutter ants, are the only fungus-farming lineages within the tree of life. Bacteria harbored by ambrosia beetles may play an essential role in the nutritional symbiotic interactions with their associated fungi; however, little is known about the impact of rearing conditions on the microbiota of ambrosia beetles. We have used culture-independent methods to explore the effect of rearing conditions on the microbiome associated with Xyleborus affinis, Xyleborus bispinatus, and Xyleborus volvulus, evaluating different media in laboratory-controlled conditions and comparing wild and laboratory conditions. Our results revealed that rearing conditions affected the fungal and bacterial microbiome structure and had a strong influence on bacterial metabolic capacities. We propose that the rearing conditions influence the ambrosia-associated fungal and bacterial communities. Furthermore, bacterial microbiome flexibility may help beetles adapt to different substrates.

Environmental pH modulates transcriptomic responses in the fungus Fusarium sp. associated with KSHB Euwallacea sp. near fornicatus.

BACKGROUND: The Ambrosia Fusarium Clade phytopathogenic Fusarium fungi species have a symbiotic relationship with ambrosia beetles in the genus Euwallacea (Coleoptera: Curculionidae). Related beetle species referred to as Euwallacea sp. near fornicatus have been spread in California, USA and are recognized as the causal agents of Fusarium dieback, a disease that causes mortality of many plant species. Despite the importance of this fungi, no transcriptomic resources have been generated. The datasets described here represent the first ever transcripts available for these species. We focused our study on the isolated species of Fusarium that is associated with one of the cryptic species referred to as Kuroshio Shot Hole Borer (KSHB) Euwallacea sp. near fornicatus. RESULTS: Hydrogen concentration is a critical signal in fungi for growth and host colonization, the aim of this study was to evaluate the effect of different pH conditions on growth and gene expression of the fungus Fusarium sp. associated with KSHB. An RNA-seq approach was used to compare the gene expression of the fungus grown for 2 weeks in liquid medium at three different pH levels (5.0, 6.0, and 7.0). An unbuffered treatment was included to evaluate the capability of the fungus to change the pH of its environment and the impact in gene expression. The results showed that the fungus can grow and modulate its genetic expression at different pH conditions; however, growth was stunted in acidic pH in comparison with neutral pH. The results showed a differential expression pattern in each pH condition even when acidic conditions prevailed at the end of the experiment. After comparing transcriptomics data from the three treatments, we found a total of 4,943 unique transcripts that were differentially expressed. CONCLUSIONS: We identified transcripts related to pH signaling such as the conserved PAL/RIM pathway, some transcripts related to secondary metabolism and other transcripts that were differentially expressed. Our analysis suggests possible mechanisms involved in pathogenicity in this novel Fusarium species. This is the first report that shows transcriptomic data of this pathogen as well as the first report of genes and proteins involved in their metabolism identifying potential virulence factors.

Draft Genome Sequence of the Phytopathogenic Fungus Fusarium euwallaceae, the Causal Agent of Fusarium Dieback.

Here, we report the genome of Fusarium euwallaceae strain HFEW-16-IV-019, an isolate obtained from Kuroshio shot hole borer (a Euwallacea sp.). These beetles were collected in Tijuana, Mexico, from elm trees showing typical symptoms of Fusarium dieback. The final assembly consists of 287 scaffolds spanning 48,274,071 bp and 13,777 genes.

Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae).

BACKGROUND: Calophyllum brasiliense is highlighted as an important resource of calanolides, which are dipyranocoumarins that inhibit the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1 RT). Despite having great medicinal importance, enzymes involved in calanolide, biosynthesis and the pathway itself, are still largely unknown. Additionally, no genomic resources exist for this plant species. RESULTS: In this work, we first analyzed the transcriptome of C. brasiliense leaves, stem, and roots using a RNA-seq strategy, which provided a dataset for functional gene mining. According to the structures of the calanolides, putative biosynthetic pathways were proposed. Finally, candidate unigenes in the transcriptome dataset, potentially involved in umbelliferone and calanolide (angular pyranocoumarin) biosynthetic pathways, were screened using mainly homology-based BLAST and phylogenetic analyses. CONCLUSIONS: The unigene dataset that was generated in this study provides an important resource for further molecular studies of C. brasiliense, especially for functional analysis of candidate genes involved in the biosynthetic pathways of linear and angular pyranocoumarins.

Exposure to hycanthone alters chromatin structure around specific gene functions and specific repeats in Schistosoma mansoni.

Schistosoma mansoni is a parasitic plathyhelminth responsible for intestinal schistosomiasis (or bilharzia), a disease affecting 67 million people worldwide and causing an important economic burden. The schistosomicides hycanthone, and its later proxy oxamniquine, were widely used for treatments in endemic areas during the twentieth century. Recently, the mechanism of action, as well as the genetic origin of a stably and Mendelian inherited resistance for both drugs was elucidated in two strains. However, several observations suggested early on that alternative mechanisms might exist, by which resistance could be induced for these two drugs in sensitive lines of schistosomes. This induced resistance appeared rapidly, within the first generation, but was metastable (not stably inherited). Epigenetic inheritance could explain such a phenomenon and we therefore re-analyzed the historical data with our current knowledge of epigenetics. In addition, we performed new experiments such as ChIP-seq on hycanthone treated worms. We found distinct chromatin structure changes between sensitive worms and induced resistant worms from the same strain. No specific pathway was discovered, but genes in which chromatin structure modifications were observed are mostly associated with transport and catabolism, which makes sense in the context of the elimination of the drug. Specific differences were observed in the repetitive compartment of the genome. We finally describe what types of experiments are needed to understand the complexity of heritability that can be based on genetic and/or epigenetic mechanisms for drug resistance in schistosomes.