Phylogenomics reveals the evolutionary origin of lichenization in chlorophyte algae

Mutualistic symbioses, such as lichens formed between fungi and green algae or cyanobacteria, have contributed to major transitions in the evolution of life and are at the center of extant ecosystems. However, our understanding of their evolution and function remains elusive in most cases. Here, we investigated the evolutionary history and the molecular innovations at the origin of lichens in green algae. We de novo sequenced the genomes or transcriptomes of 15 lichen-forming and closely-related non-lichen-forming algae and performed comparative phylogenomics with 22 genomes previously generated. We identified more than 350 functional categories significantly enriched in chlorophyte green algae able to form lichens. Among them, functions such as light perception or resistance to dehydration were shared between lichenizing and other terrestrial algae but lost in non-terrestrial ones, indicating that the ability to live in terrestrial habitats is a prerequisite for lichens to evolve. We detected lichen-specific expansions of glycosyl hydrolase gene families known to remodel cell walls, including the glycosyl hydrolase 8 which was acquired in lichenizing Trebouxiophyceae by horizontal gene transfer from bacteria, concomitantly with the ability to form lichens. Mining genome-wide orthogroups, we found additional evidence supporting at least two independent origins of lichen-forming ability in chlorophyte green algae. We conclude that the lichen-forming ability evolved multiple times in chlorophyte green algae, following a two-step mechanism which involves an ancestral adaptation to terrestrial lifestyle and molecular innovations to modify the partners cell walls.

Comparative Analysis of Sugar Metabolites and Their Transporters in Sugarcane Following Sugarcane mosaic virus (SCMV) Infection

Sugarcane mosaic virus (SCMV) is one of the major pathogens of sugarcane. SCMV infection causes dynamic changes in plant cells, including decreased photosynthetic rate, respiration, and sugar metabolism. To understand the basics of pathogenicity mechanism, we performed transcriptome and proteomics analysis in two sugarcane genotypes (Badila: susceptible to SCMV and B-48: SCMV resistant). Using Saccharum spontaneum L. genome as a reference, we identified the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) that participate in sugar metabolism, transport of their metabolites, and Carbohydrate Activating enZYmes (CAZymes). Sequencing data revealed 287 DEGs directly or indirectly involved in sugar metabolism, transport, and storage, while 323 DEGs are associated with CAZymes. Significant upregulation of glucose, sucrose, fructose, starch, and SWEET-related transcripts was observed in the Badila after infection of SCMV. B-48 showed resistance against SCMV with a limited number of sugar transcripts up-regulation at the post-infection stage. For CAZymes, only glycosyltransferase (GT)1 and glycosyl hydrolase (GH)17 were upregulated in B-48. Regulation of DEGs was analyzed at the proteomics level as well. Starch, fructose, glucose, GT1, and GH17 transcripts were expressed at the post-translational level. We verified our transcriptomic results with proteomics and qPCR data. Comprehensively, this study proved that Badila upregulated sugar metabolizing and transporting transcripts and proteins, which enhance virus multiplication and infectionl.

Current Perspectives on Chitinolytic Enzymes and Their Agro-Industrial Applications

Chitinases are a large and diversified category of enzymes that break down chitin, the world’s second most prevalent polymer after cellulose. GH18 is the most studied family of chitinases, even though chitinolytic enzymes come from a variety of glycosyl hydrolase (GH) families. Most of the distinct GH families, as well as the unique structural and catalytic features of various chitinolytic enzymes, have been thoroughly explored to demonstrate their use in the development of tailor-made chitinases by protein engineering. Although chitin-degrading enzymes may be found in plants and other organisms, such as arthropods, mollusks, protozoans, and nematodes, microbial chitinases are a promising and sustainable option for industrial production. Despite this, the inducible nature, low titer, high production expenses, and susceptibility to severe environments are barriers to upscaling microbial chitinase production. The goal of this study is to address all of the elements that influence microbial fermentation for chitinase production, as well as the purifying procedures for attaining high-quality yield and purity.

Differential Responses of Ceratitis Capitata to Infection by the Entomopathogenic Fungus Purpureocillium Lilacinum.

Ceratitis capitata (medfly), is one of the most injurious pests of fruits with quarantine importance because of its extremely wide host range. The use of entomopathogenic fungi constitutes a promising approach for potential applications in integrated pest management. Nonetheless, developing methods of insect control can also involve the use of fungal machinery to produce metabolic disturbance that can increase its effectiveness by producing a detrimental effect on insect development. Insect species, such as Ceratitis capitata, depend on reproduction potential, nutrient reserves, metabolic activities and immune response for their survival. Accordingly, the purpose of this study is to use the entomopathogenic fungus Purpureocillium lilacinum to investigate, its sublethal effects on Ceratitis capitata. Laboratory bioassays were conducted on medfly V8 strain. The bioassays were monitored to determine the virulence of P. lilacinum on the fruit fly. P. lilacinum was tested against 5 days-old males and females, through abdominal topical applications. Following the fungal inoculation, we showed (i) a significant increase of sugar amount in tissues, (ii) a significant decrease in carbohydrase activities, digestive glycosyl hydrolase and proteinase activities in whole midguts of treated flies, (iii) an over-expression of Takeout and Attacin-A genes induced by infection. Moreover, the up-regulations observed for relish, cecropin 1, ceratotox-A and defensin genes are due to physiological mechanisms occurring during infection.

FoQDE2-dependent milRNA promotes Fusarium oxysporum f. sp. cubense virulence by targeting a glycosyl hydrolase coding gene at transcriptional level

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence and hypersensitivity to hydrogen peroxide (H2O2). These results indicate that milRNA biosynthesis is crucial for Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly downregulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR analysis. Through deletion and overexpression of milR-87 in the wild-type Foc strain, functions of milR-87 were studied. The results showed that milR-87 is crucial for Foc virulence in infection process. We furthermore identified a glycosyl hydrolase-coding gene, FOIG_15013, as the direct target of milR-87. The FOIG_15013 deletion mutant displayed a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases.Author summaryThe fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. As a typical representative of F. oxysporum species complex, the pathogen has been widely concerned. However, pathogenesis of Foc is not fully elucidated. In particular, pathogenic regulatory mechanism of the microRNA like small RNAs (milRNAs) found in Foc is unknown. Here, we found that FoQDE2, one Argonaute coding gene, and two Dicer coding genes FoDCL1 and FoDCL2, which are involved in milRNA biosynthesis, are significantly induced during the early infection stage of Foc. The results suggested that the milRNAs biosynthesis mediated by these genes may play an active role in the infection process of Foc. Based on this assumption, we subsequently found a FoQDE2-dependent milRNA (milR-87) and identified its target gene. Functional analysis showed that FoQDE2, miR-87 and its target gene were involved in the pathogenicity of Foc in different degree. The studies help us gain insight into the pathogenesis with FoQDE2, milR-87, and its target gene as central axis in Foc. The identified pathogenicity-involved milRNA provides an active target for developing novel and efficient biocontrol agents against Panama disease.

Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N′-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.

Transcriptomic Analysis of Late-Ripening Sweet Orange Fruits (Citrus sinensis) after Foliar Application of Glomalin-Related Soil Proteins

Glomalin, one of the glycoproteins generated in the spores and hyphae of arbuscular mycorrhizal (AM) fungi, has multiple functions in plants and soil, while the role of foliar spray of easily extractable glomalin-related soil proteins (EE-GRSP) in citrus fruits is not well defined. Our study aimed to use referenced transcriptome sequencing to uncover the mechanism and the role of exogenous EE-GRSP in two late-ripening varieties of sweet orange (Citrus sinensis) fruits including Navel Lane Late (LW) and Rohde Red Valencia (XC). The 1804 and 1861 differentially expressed genes were identified in fruits of LW and XC, respectively, following foliar spray of EE-GRSP. Photosynthesis ranked second in the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolism in the LW variety, and carbon fixation in photosynthetic organizations ranked first in KEGG metabolism in the XC variety. The response to foliar spray of EE-GRSP affected the fruit starch and sucrose metabolism of KEGG, with 15 (10 up-regulated and 5 down-regulated) and 13 (2 up-regulated and 11 down-regulated) differentially expressed genes identified in the LW and XC variety, respectively. Cs5g19060 (sucrose phosphate synthase 4) was activated and reduced by EE-GRSP on XC and LW, respectively. Cs1g18220 (β-fructofuranosidase) and Cs2g12180 (glycosyl hydrolase family 9) genes were up-regulated and down-regulated in LW and XC, respectively. These results established the involvement of molecular signaling in response to foliar spray of EE-GRSP activating fruit sugar metabolism is dependent on citrus varieties.

Fucosylated human milk oligosaccharide foraging within the species Bifidobacterium pseudocatenulatum is driven by glycosyl hydrolase content and specificity

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum , is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs) we observed two distinct groups of B. pseudocatenulatum , isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO binding specificity of the Family 1 solute binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain’s distinct HMO consumption pattern. Taken together, this data indicates the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ . IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breast fed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.