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Totozafy, J. C.

Publications and source records attributed to Totozafy, J. C..

3 recordsLinked to original sources

Apoplast multi-omics profiling during fungal infection uncovers new players of basal and early induced immunity

The extracellular space in plant tissues, known as the apoplast, remains one of the least characterized cellular compartments. The apoplastic fluid (APF), akin to mammalian extracellular fluid, serves as the primary interface between pathogens and their host. It allows the exchange of signalling molecules, coordinates host cell responses, and enables the circulation of pathogen effectors that modulate the immune response. We describe here the first multi-omics analysis of the APF content just 6 h after the onset of A. thaliana infection with B. cinerea, a fast-killing necrotrophic fungus. By varying plant nitrogen nutrition, known to affect both plant defenses and pathogen virulence, we identify candidates that do not stand out under optimal conditions. Our analysis uncovers novel nitrogen-dependent mechanisms that regulate both basal and early induced apoplastic immunity, revealing the presence of previously unidentified, potentially protective apoplastic metabolites as well as the intercellular transport of nuclear proteins, thereby offering new insights into early apoplastic immune responses.

plant biology↗

Conjugation of dn-OPDA with amino acids inhibits its hormonal bioactivity in Marchantia polymorpha

Jasmonates are important phytohormones activating plant tolerance to biotic and abiotic stress, as well as different development processes. A conserved signalling pathway activated by distinct hormones in different plant species mediates these responses: dinor-12-oxo-phytodienoic acid (dn-OPDA) isomers in bryophytes and lycophytes, and JA-Ile in most vascular plants. The final responses depend, in many cases, on the accumulation of specialized metabolites. To identify novel compounds regulated by the dn-OPDA pathway in Marchantia, untargeted metabolomic analyses were carried out in response to dn-OPDA-regulated stress. A novel group of molecules were identified as dn-OPDA-amino acid conjugates (dn-OPDA-aas), and their accumulation after wounding and herbivory confirmed by targeted metabolic profiling in Marchantia and all species in which we previously found dn-iso-OPDA. Mutants in GRETCHEN-HAGEN 3A (MpGH3A) failed to accumulate dn-OPDA-aa conjugates, and showed a constitutive activation of the OPDA pathway and increased resistance to herbivory. Our results show that dn-iso-OPDA bioactivity is reduced by conjugation with amino acids. Therefore, a dichotomous role of jasmonate conjugation in land plants is highlighted: jasmonic acid (JA) conjugation with isoleucine (Ile) produce the bioactive JA-Ile in tracheophytes, whereas conjugation of dn-iso-OPDA with different amino acids disactivate the hormone in bryophytes and lycophytes.

plant biology↗

Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites

Transcriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in Saccharomyces cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for an halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.

microbiology↗