Biology subjects

Camagna, M.

Publications and source records attributed to Camagna, M..

10 recordsLinked to original sources

bioRxiv2025

Foliar application of mixed DAMP- and MAMP-oligosaccharides enhances root growth and fruit yield in okra and tomato in greenhouse experiments

Plants perceive diverse endogenous and microbial molecular patterns that shape their defense, growth, and productivity. Damage-associated molecular patterns (DAMPs) such as cello- and xylo-oligosaccharides, and microbe-associated molecular patterns (MAMPs) such as chitin-oligosaccharides, are known to promote both development and immunity. Here, we evaluated the combined application of these three oligosaccharides (Oligo-Mix) in okra and cherry tomato under greenhouse conditions. Regular foliar treatment with Oligo-Mix enhanced root elongation and biomass accumulation, and increased fruit number and yield in both crops. RNA-seq analysis of Oligo-Mix-treated tomato leaves showed coordinated induction of photosynthetic genes, components of the translational machinery, cell-wall remodeling factors, and enzymes involved in flavonoid/anthocyanin biosynthesis, indicating broad enhancement of metabolic capacity and protective secondary pathways. Together, these results demonstrate that Oligo-Mix functions as a biostimulant that supports both vegetative growth and fruit production, offering a sustainable approach to improving crop performance.

plant biology↗

bioRxiv2025

SMART platform for enzyme engineering: A novel single-molecule display approach for evolution and functional studies of D-amino acid oxidase

This study introduces SMART (Single Molecule Assay on Ribonucleic Acid by Translated Product), an innovative in vitro platform integrating mRNA display, next-generation sequencing, and bioinformatics to address several key challenges in enzyme engineering. The characteristic feature of SMART is its auxiliary unit, attached to the mRNA-displayed enzyme library to mediate the biotinylation of reactive enzyme variants and enable their enrichment by streptavidin beads pull-down. The unit comprises a hairpin single-stranded DNA that hybridizes with the mRNA and anchors a DNA-binding protein, single-chain Cro, which carries functional auxiliary molecules specific to the enzyme of interest. Here, we report on the establishment of SMART for the engineering of oxidases, specifically D-amino acid oxidase (DAAO) from Schizosaccharomyces pombe, with engineered ascorbate peroxidase 2 as its auxiliary enzyme. In a single selection round of a DAAO library with randomized catalytic residue Y232 and D-Alanine as the substrate, we identified several catalytically active enzyme variants with altered substrate specificity in a matter of days. This surpasses traditional enzyme engineering methods in speed, cost, library capacity, and precision. This research underscores the potential of SMART for the engineering of various oxidases, and other enzymes after the corresponding adjustment of the auxiliary unit.

bioengineering↗

bioRxiv2024

Mixed DAMP/MAMP oligosaccharides promote both growth and defense against fungal pathogens of cucumber

Plants recognize a variety of environmental molecules, thereby triggering appropriate responses to biotic or abiotic stresses. Substances containing microbes-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) are representative inducers of pathogen resistance and damage repair, thus treatment of healthy plants with such substances can pre-activate plant immunity and cell repair functions. In this study, the effects of DAMP/MAMP oligosaccharides mixture (Oligo-Mix) derived from plant cell wall (cello-oligosaccharide and xylo-oligosaccharide), and fungal cell wall (chitin-oligosaccharide) were examined in cucumber. Treatment of cucumber with Oligo-Mix promoted root germination and plant growth, along with increased chlorophyll contents in the leaves. Oligo-Mix treatment also induced typical defense responses such as MAP kinase activation and callose deposition in leaves. Pretreatment of Oligo-Mix enhanced disease resistance of cucumber leaves against pathogenic fungi Podosphaera xanthii (powdery mildew) and Colletotrichum orbiculare (anthracnose). Oligo-Mix treatment increased the induction of hypersensitive cell death around the infection site of pathogens, which inhibited further infection and the conidial formation of pathogens on the cucumber leaves. RNA-seq analysis revealed that Oligo-Mix treatment upregulated genes associated with plant structural reinforcement, responses to abiotic stresses and plant defense. These results suggested that Oligo-Mix has beneficial effects on growth and disease resistance in cucumber, making it a promising biostimulant for agricultural application.

plant biology↗

bioRxiv2024

Botrytis cinerea detoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.

microbiology↗

bioRxiv2023

Substrate profiling of human Transglutaminase 1 using cDNA display and next-generation sequencing

Human transglutaminase 1 regulates skin development and is linked with several disease conditions with yet fully unknown mechanisms. To uncover all of its roles in health and disease, the understanding of protein substrates and their reactivity with transglutaminase 1 is necessary. To gain insight into the substrate profile of human transglutaminase 1, this study uses an in vitro selection system based on the cDNA display technology to screen two displayed peptide libraries differing in the number and position of mutated sites relative to the reactive glutamine, in terms of their reactivity to transglutaminase 1. Analysis of the selected DNA pools of by next-generation sequencing and in-house bioinformatics methods revealed a detailed transglutaminase 1 substrate profile indicating preferred and non-preferred amino acid sequences. We have identified a peptide with sequence AEQHKLPSKWPF showing high reactivity to human transglutaminase 1 and low reactivity to transglutaminase 2 and transglutaminase 3. The position weight matrix consisting of per residue amino acid enrichment factors of all selected peptides was used to search human proteins by our in-house search algorithm and identify highly scoring sequence motifs in their primary structure. The search identified six already known transglutaminase 1 substrate proteins as highly scored hits as well as a list of candidate substrates that are under investigation.

bioengineering↗

bioRxiv2023

Induction of plant disease resistance by mixed oligosaccharide elicitors prepared from plant cell wall and crustacean shells

Basal plant immune responses are activated by the recognition of the conserved pathogen-associated molecular patterns (PAMPs), or breakdown molecules released from plants after damage by pathogen infection, so-called danger-associated molecular patterns (DAMPs). While chitin-oligosaccharide (CHOS), a primary component of the fungal cell wall, is most known as PAMP, plant cell wall-derived oligosaccharides, cello-oligosaccharides (COS) from cellulose and xylo-oligosaccharide (XOS) from hemicellulose, are representative DAMPs, which activate signaling steps similar to PAMP-induced immunity to elicit defenses and provide protection against pathogens. In this study, elicitor activities of COS prepared from cotton linters, XOS prepared from corn cobs as well as chitin-oligosaccharide (CHOS) from crustacean shells were comparatively investigated. In Arabidopsis, treatment of COS, XOS or CHOS triggered typical defense responses such as reactive oxygen species (ROS) production, activation of MAP kinases phosphorylation, callose depositions, and activation of the promoter for defense-related transcription factor WRKY33. When COS, XOS and CHOS were used at concentrations with similar activity in inducing ROS production and callose depositions, CHOS was particularly highly potent in activating the MAPK kinases and WRKY33 promoters. Among the COS and XOS with different degrees of polymerization (DP), cellotriose (DP3) and xylotetraose (DP4) showed the highest activity for the activation of WRKY33 promoter. Simultaneous treatment of COS, XOS and CHOS leads to a strong transcriptional change for defense-related genes, and gene ontology (GO) enrichment analysis of RNAseq data revealed that a mixture of three oligosaccharide (oligo-mix) effectively activate the plants disease resistance. In practice, treatment of the oligo-mix enhanced the resistance of tomato to powdery mildew, but plant growth was not inhibited but rather tended to be promoted, providing evidence that mixed oligosaccharides have beneficial effects on improving disease resistance in plants, making them a promising class of compounds for practical application.

plant biology↗

bioRxiv2022

Functional characterization of Botrytis cinerea ABC transporter gene BcatrB in response to phytoalexins produced in plants belonging to families Solanaceae, Brassicaceae and Fabaceae

Botrytis cinerea, a generalist fungal pathogen of economically important crop species, has been shown to exhibit reduced sensitivity to fungicides and plant toxins. Specifically, previous reports indicate B. cinereas efficacy in tolerating a wide array of phytoalexins, toxic plant metabolites that play key role in plant immune defense strategies. Previously, we have shown that a distinct set of genes was induced in B. cinerea when treated with phytoalexins derived from different plant species such as rishitin (tomato and potato), capsidiol (tobacco and bell pepper) or resveratrol (grape and blueberry). In this study, we focused on the functional analyses of B. cinerea genes induced by rishitin treatment. B. cinerea can metabolize rishitin to at least 4 oxidized forms. Heterologous expression of rishitin-induced B. cinerea genes in the plant symbiotic fungus, Epichloe festucae, revealed that oxidoreductase (Bcin08g04910) and cytochrome P450 (Bcin16g01490) genes are involved in the oxidation of rishitin. BcatrB is an exporter of structurally unrelated anti-microbial compounds such as resveratrol, camalexin and fungicide fenpicionil. Expression of BcatrB is upregulated by rishitin, but not by structurally resembling capsidiol. BcatrB knock out transformants ({Delta}bcatrB) showed enhanced sensitivity to rishitin, but not to capsidiol. Likewise,{Delta} bcatrB showed reduced virulence on tomato fruits (which produce rishitin), but showed full virulence on Nicotiana benthamiana (which mainly produces capsidiol), suggesting that B. cinerea distinguishes phytoalexins and activates expression of appropriate transporter genes during the infection. Activation of BcatrB promoter was detected using P_BcatrB:GFP transformant during the B. cinerea infection in plant tissues. Surveying of 26 plant species across 13 families revealed that the BcatrB promoter is mainly activated during the infection of plants belonging to the Solanaceae, Fabaceae and Brassicaceae families. The BcatrB promoter is activated by the treatment with Fabaceae phytoalexins medicarpin and glyceollin, and{Delta} bcatrB showed reduced virulence on red clover, which produces medicarpin. These results suggest that BcatrB plays a critical role in the strategy employed by B. cinerea to bypass the plant innate immune responses in a wide variety of important crops belonging to the Solanaceae, Brassicaceae and Fabaceae families.

microbiology↗

bioRxiv2022

Botrytis cinerea identifies host plants via the recognition of antifungal capsidiol to induce expression of a specific detoxification gene

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in over 400 plant species, but it is not known how this pathogen evolved this polyxenous nature. B. cinerea can metabolize a wide range of phytoalexins, including the stilbenoid, resveratrol, and the sesquiterpenoids capsidiol in tobacco, and rishitin in potato and tomato. In this study, we analyzed the metabolism of sesquiterpenoid phytoalexins by B. cinerea. Capsidiol was dehydrogenated to capsenone which was then further oxidized, while rishitin was directly oxidized to epoxy-or hydroxy-rishitins indicating that B. cinerea has separate mechanisms to detoxify structurally similar sesquiterpenoid phytoalexins. RNAseq analysis revealed that a distinct set of genes were induced in B. cinerea when treated with capsidiol or rishitin, suggesting that B. cinerea can distinguish structurally similar phytoalexins to activate appropriate detoxification mechanisms. The gene most highly upregulated by capsidiol treatment encoded a dehydrogenase, designated Bccpdh. Heterologous expression of Bccpdh in a capsidiol-sensitive plant symbiotic fungus, Epichloe festucae, resulted in an acquired tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea {Delta}bccpdh mutants became relatively sensitive to capsidiol. The{Delta} bccpdh mutant showed reduced virulence on the capsidiol producing Nicotiana and Capsicum species but remained fully pathogenic on potato and tomato. Homologs of Bccpdh are not found in taxonomically distant Ascomycota fungi but not in related Leotiomycete species, suggesting that B. cinerea acquired the ancestral Bccpdh by horizontal gene transfer, thereby extending the pathogenic host range of this polyxenous pathogen to capsidiol-producing plant species. Significance StatementB. cinerea can metabolize a wide range of phytoalexins, however, the extent to which phytoalexin detoxification contributes to pathogenicity is largely unknown. In this study, we have shown that B. cinerea recognizes structurally resembling sesquiterpenoid phytoalexins, rishitin and capsidiol, to activate appropriate detoxification mechanisms. We identify Bccpdh, encoding a dehydrogenase for capsidiol detoxification, which is upregulated in B. cinerea exclusively during the infection of capsidiol producing plant species, and is required to exert full virulence. Analysis of the Bccpdh locus implicates that the gene was acquired via horizontal gene transfer. This work highlights that the polyxenous plant pathogen B. cinerea can distinguish its host plants by its anti-microbial compounds, to activate appropriate mechanisms for enhanced virulence.

microbiology↗

bioRxiv2021

AP2/ERF transcription factor NbERF-IX-33 is involved in the regulation of phytoalexin production for the resistance of Nicotiana benthamiana to Phytophthora infestans.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, transient induction of ethylene production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated to capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies), and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

plant biology↗

bioRxiv2021

Two structurally different oomycete MAMPs induce distinctive plant immune responses.

Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes, microbe-associated molecular patterns (MAMPs), are sensed by plant cells as non-self molecules via pattern recognition receptors. While MAMPs of bacterial and fungal origin have been identified, reports on oomycete MAMPs are relatively limited. This study aimed to identify MAMPs from an oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato as markers, two structurally different groups of elicitors, namely ceramides and diacylglycerols were identified. P. infestans ceramides (Pi-Cer A, B and D) induced ROS production, while diacylglycerol (Pi-DAG A and B), containing eicosapentaenoic acid (EPA) as a substructure, induced phytoalexins production in potato. The molecular patterns in Pi-Cers and Pi-DAGs essential for defense induction were identified as 9-methyl-4,8-sphingadienine (9Me-Spd) and 5,8,11,14-tetraene-type fatty acid (5,8,11,14-TEFA), respectively. These structures are not found in plants, but in oomycetes and fungi, indicating that they are microbe molecular patterns recognized by plants. When Arabidopsis was treated with Pi-Cer D and EPA, partially overlapping but different sets of genes were induced. Furthermore, expression of some genes is upregulated only after the simultaneous treatment with Pi-Cer D and EPA, indicating that plants combine the signals from simultaneously recognized MAMPs to adapt the defense response to pathogens.

plant biology↗