Phytotoxic fungal secondary metabolites as herbicides.

Among the alternatives to synthetic plant protection products, biocontrol appears as a promising method. This review reports on the diversity of fungal secondary metabolites phytotoxic to weeds and on the approach generally used to extract, characterize, identify and exploit them for weed management. The 183 phytotoxic fungal secondary metabolites discussed in this review fall into five main classes of molecules: 61 polyketides, 53 terpenoids, 36 nitrogenous metabolites, 18 phenols and phenolic acids, and 15 miscellaneous. They are mainly produced by the genera Drechslera, Fusarium and Alternaria. The phytotoxic effects, more often described by the symptoms they produce on plants than by their mode of action, range from inhibition of germination to inhibition of root and vegetative growth, including tissue and organ alterations. The biochemical characterization of fungal secondary metabolites requires expertise and tools to carry out fungal cultivation and metabolite extraction, phytotoxicity tests, purification and fractionation of the extracts, and chemical identification procedures. Phytotoxicity tests are mainly carried out under controlled laboratory conditions (not always on whole plants), while effectiveness against targeted weeds and environmental impacts must be assessed in greenhouses and open fields. These steps are necessary for the formulation of effective, environment-friendly fungal secondary metabolites-derived bioherbicides using new technologies such as nanomaterials. © 2023 Society of Chemical Industry.

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. The aim was to develop a polycistronic system for heterologous expression of fungal BGCs in Saccharomyces cerevisiae. 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 S. 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 a 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.

Vitislactone, a non-canonical strigolactone exudated by grapevine rootstocks in response to nitrogen starvation.

Strigolactones are compounds produced by plant roots in response to nutrient deficiency, acting both as local and systemic signals to control development and nutrition. Strigolactones are exuded in the rhizosphere to positively influence interactions with beneficial microbes. LC-MS/MS analysis shows that two genetically distinct grapevine rootstocks exudate one or two non-canonical strigolactones when subjected to low nitrogen conditions. Gene expression profiles and orobanche seed germination assays confirm that the biosynthesis and exudation of non-canonical compounds is the preferred pathway. The first compound, corresponding to heliolactone or 6-epi-heliolactone, is only exuded by the rootstock showing lower shoot branching and a higher level of mycorrhization with arbuscular mycorrhizal fungi. The structure of the second compound exuded by both rootstocks was identified by NMR and LC-MS/MS analysis. It is a non-canonical strigolactone, which has never been identified in another species. This first identification of a natural compound with the potential to stimulate beneficial root-microbe interactions in grapevines opens new perspectives in viticulture.

Spatial consistency of cell growth direction during organ morphogenesis requires CELLULOSE SYNTHASE INTERACTIVE1.

Extracellular matrices contain fibril-like polymers often organized in parallel arrays. Although their role in morphogenesis has been long recognized, it remains unclear how the subcellular control of fibril synthesis translates into organ shape. We address this question using the Arabidopsis sepal as a model organ. In plants, cell growth is restrained by the cell wall (extracellular matrix). Cellulose microfibrils are the main load-bearing wall component, thought to channel growth perpendicularly to their main orientation. Given the key function of CELLULOSE SYNTHASE INTERACTIVE1 (CSI1) in guidance of cellulose synthesis, we investigate the role of CSI1 in sepal morphogenesis. We observe that sepals from csi1 mutants are shorter, although their newest cellulose microfibrils are more aligned compared to wild-type. Surprisingly, cell growth anisotropy is similar in csi1 and wild-type plants. We resolve this apparent paradox by showing that CSI1 is required for spatial consistency of growth direction across the sepal.

Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

Biostimulation can prime elicitor induced resistance of grapevine leaves to downy mildew.

Using plant defense elicitors to protect crops against diseases is an attractive strategy to reduce chemical pesticide use. However, development of elicitors remains limited because of variable effectiveness in the field. In contrast to fungicides that directly target pathogens, elicitors activate plant immunity, which depends on plant physiological status. Other products, the biostimulants, can improve certain functions of plants. In this study, the objective was to determine whether a biostimulant via effects on grapevine physiology could increase effectiveness of a defense elicitor. A new methodology was developed to study biostimulant activity under controlled conditions using in vitro plantlets. Both biostimulant and defense elicitor used in the study were plant extracts. When added to the culture medium, the biostimulant accelerated the beginning of plantlet growth and affected the shoot and root development. It also modified metabolomes and phytohormone contents of leaves, stems, and roots. When applied on shoots, the defense elicitor changed metabolite and phytohormone contents, but effects were different depending on whether plantlets were biostimulated or controls. Defense responses and protection against Plasmopara viticola (downy mildew agent) were induced only for plantlets previously treated with the biostimulant, Therefore, the biostimulant may act by priming the defense elicitor action. In this study, a new method to screen biostimulants active on grapevine vegetative growth was used to demonstrate that a biostimulant can optimize the efficiency of a plant defense elicitor.

The peptide SCOOP12 acts on reactive oxygen species homeostasis to modulate cell division and elongation in Arabidopsis primary root.

Small secreted peptides have been described as key contributors to complex signalling networks that control plant development and stress responses. The Brassicaceae-specific PROSCOOP family encodes precursors of Serine riCh endOgenOus Peptides (SCOOPs). In Arabidopsis SCOOP12 has been shown to promote the defence response against pathogens and to be involved in root development. Here, we explore its role as a moderator of Arabidopsis primary root development. We show that the PROSCOOP12 null mutation leads to longer primary roots through the development of longer differentiated cells while PROSCOOP12 overexpression induces dramatic plant growth impairments. In comparison, the exogenous application of synthetic SCOOP12 peptide shortens roots through meristem size and cell length reductions. Moreover, superoxide anion (O2·-) and hydrogen peroxide (H2O2) production in root tips vary according to SCOOP12 abundance. By using reactive oxygen species scavengers that suppress the proscoop12 phenotype, we showed that root growth regulation by SCOOP12 is associated with reactive oxygen species metabolism. Furthermore, our results suggest that peroxidases act as potential SCOOP12 downstream targets to regulate H2O2 production, which in turn triggers cell wall modifications in root. Finally, a massive transcriptional reprogramming, including the induction of genes from numerous other pathways, including ethylene, salicylic acid, and glucosinolates biosynthesis, was observed, emphasizing its dual role in defence and development.

Xyloglucan Remodeling Defines Auxin-Dependent Differential Tissue Expansion in Plants.

Size control is a fundamental question in biology, showing incremental complexity in plants, whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Our results indicate that auxin-reliant growth programs affect the molecular complexity of xyloglucans, the major type of cell wall hemicellulose in eudicots. Auxin-dependent induction and repression of growth coincide with reduced and enhanced molecular complexity of xyloglucans, respectively. In agreement with a proposed function in growth control, genetic interference with xyloglucan side decorations distinctly modulates auxin-dependent differential growth rates. Our work proposes that auxin-dependent growth programs have a spatially defined effect on xyloglucan's molecular structure, which in turn affects cell wall mechanics and specifies differential, gravitropic hypocotyl growth.

The miR166-SlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato.

Fruit set is inhibited by adverse temperatures, with consequences on yield. We isolated a tomato mutant producing fruits under non-permissive hot temperatures and identified the causal gene as SlHB15A, belonging to class III homeodomain leucine-zipper transcription factors. SlHB15A loss-of-function mutants display aberrant ovule development that mimics transcriptional changes occurring in fertilized ovules and leads to parthenocarpic fruit set under optimal and non-permissive temperatures, in field and greenhouse conditions. Under cold growing conditions, SlHB15A is subjected to conditional haploinsufficiency and recessive dosage sensitivity controlled by microRNA 166 (miR166). Knockdown of SlHB15A alleles by miR166 leads to a continuum of aberrant ovules correlating with parthenocarpic fruit set. Consistent with this, plants harboring an Slhb15a-miRNA166-resistant allele developed normal ovules and were unable to set parthenocarpic fruit under cold conditions. DNA affinity purification sequencing and RNA-sequencing analyses revealed that SlHB15A is a bifunctional transcription factor expressed in the ovule integument. SlHB15A binds to the promoters of auxin-related genes to repress auxin signaling and to the promoters of ethylene-related genes to activate their expression. A survey of tomato genetic biodiversity identified pat and pat-1, two historical parthenocarpic mutants, as alleles of SlHB15A. Taken together, our findings demonstrate the role of SlHB15A as a sentinel to prevent fruit set in the absence of fertilization and provide a mean to enhance fruiting under extreme temperatures.

Effects of Arabidopsis wall associated kinase mutations on ESMERALDA1 and elicitor induced ROS.

Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP, and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests that mutations of the potential WAK EGF O-fucosylation site also abate the WAK2cTAP phenotype, yet only evidence for an N-linked but not O-linked sugar addition can be found. Moreover, a WAK locus deletion allele has no effect on the ability of esmd1 to suppress an adhesion deficiency, indicating WAKs and their modification are not a required component of the potential ESMD1 signaling mechanism involved in the control of cell adhesion. The WAK locus deletion does however affect the induction of ROS but not the transcriptional response induced by the elicitors Flagellin, Chitin and oligogalacturonides (OGs).

Mutation of an Arabidopsis Golgi membrane protein ELMO1 reduces cell adhesion.

Plant growth, morphogenesis and development involve cellular adhesion, a process dependent on the composition and structure of the extracellular matrix or cell wall. Pectin in the cell wall is thought to play an essential role in adhesion, and its modification and cleavage are suggested to be highly regulated so as to change adhesive properties. To increase our understanding of plant cell adhesion, a population of ethyl methanesulfonate-mutagenized Arabidopsis were screened for hypocotyl adhesion defects using the pectin binding dye Ruthenium Red that penetrates defective but not wild-type (WT) hypocotyl cell walls. Genomic sequencing was used to identify a mutant allele of ELMO1 which encodes a 20 kDa Golgi membrane protein that has no predicted enzymatic domains. ELMO1 colocalizes with several Golgi markers and elmo1-/- plants can be rescued by an ELMO1-GFP fusion. elmo1-/- exhibits reduced mannose content relative to WT but no other cell wall changes and can be rescued to WT phenotype by mutants in ESMERALDA1, which also suppresses other adhesion mutants. elmo1 describes a previously unidentified role for the ELMO1 protein in plant cell adhesion.

Overexpression of a Cytochrome P450 Monooxygenase Involved in Orobanchol Biosynthesis Increases Susceptibility to Fusarium Head Blight.

Fusarium Head Blight (FHB) is a cereal disease caused primarily by the ascomycete fungus Fusarium graminearum with public health issues due to the production of mycotoxins including deoxynivalenol (DON). Genetic resistance is an efficient protection means and numerous quantitative trait loci have been identified, some of them related to the production of resistance metabolites. In this study, we have functionally characterized the Brachypodium distachyon BdCYP711A29 gene encoding a cytochrome P450 monooxygenase (CYP). We showed that BdCYP711A29 belongs to an oligogenic family of five members. However, following infection by F. graminearum, BdCYP711A29 is the only copy strongly transcriptionally induced in a DON-dependent manner. The BdCYP711A29 protein is homologous to the Arabidopsis thaliana MAX1 and Oryza sativa MAX1-like CYPs representing key components of the strigolactone biosynthesis. We show that BdCYP711A29 is likely involved in orobanchol biosynthesis. Alteration of the BdCYP711A29 sequence or expression alone does not modify plant architecture, most likely because of functional redundancy with the other copies. B. distachyon lines overexpressing BdCYP711A29 exhibit an increased susceptibility to F. graminearum, although no significant changes in defense gene expression were detected. We demonstrate that both orobanchol and exudates of Bd711A29 overexpressing lines stimulate the germination of F. graminearum macroconidia. We therefore hypothesize that orobanchol is a susceptibility factor to FHB.

Pectin Dependent Cell Adhesion Restored by a Mutant Microtubule Organizing Membrane Protein.

The cellulose- and pectin-rich plant cell wall defines cell structure, mediates defense against pathogens, and facilitates plant cell adhesion. An adhesion mutant screen of Arabidopsis hypocotyls identified a new allele of QUASIMODO2 (QUA2), a gene required for pectin accumulation and whose mutants have reduced pectin content and adhesion defects. A suppressor of qua2 was also isolated and describes a null allele of SABRE (SAB), which encodes a previously described plasma membrane protein required for longitudinal cellular expansion that organizes the tubulin cytoskeleton. sab mutants have increased pectin content, increased levels of expression of pectin methylesterases and extensins, and reduced cell surface area relative to qua2 and Wild Type, contributing to a restoration of cell adhesion.

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon.

BACKGROUND: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. RESULTS: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. CONCLUSION: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope-labelled internal standards.

Guanosine tetraphosphate (G4P) and guanosine pentaphosphate (G5P) are signalling nucleotides found in bacteria and photosynthetic eukaryotes that are implicated in a wide-range of processes including stress acclimation, developmental transitions and growth control. Measurements of G4P/G5P levels are essential for studying the diverse roles of these nucleotides. However, G4P/G5P quantification is particularly challenging in plants and algae due to lower cellular concentrations, compartmentalization and high metabolic complexity. Despite recent advances the speed and accuracy of G4P quantification in plants and algae can still be improved. Here, we report a new approach for rapid and accurate G4P quantification which relies on the use of synthesized stable isotope-labelled as internal standards. We anticipate that this approach will accelerate research into the function of G4P signaling in plants, algae and other organisms.

Specialized phenolic compounds in seeds: structures, functions, and regulations.

Plants produce a huge diversity of specialized metabolites (SM) throughout their life cycle that play important physiological and ecological functions. SM can protect plants and seeds against diseases, predators, and abiotic stresses, or support their interactions with beneficial or symbiotic organisms. They also have strong impacts on human nutrition and health. Despite this importance, the biosynthesis and biological functions of most of the SM remain elusive and their diversity and/or quantity have been reduced in most crops during domestication. Seeds present a large number of SM that are important for their physiological, agronomic, nutritional or industrial qualities and hence, provide interesting models for both studying biosynthesis and producing large amounts of specialized metabolites. For instance, phenolics are abundant and widely distributed in seeds. More specifically, flavonoid pathway has been instrumental for understanding environmental or developmental regulations of specialized metabolic pathways, at the molecular and cellular levels. Here, we summarize current knowledge on seed phenolics as model, and discuss how recent progresses in omics approaches could help to further characterize their diversity, regulations, and the underlying molecular mechanisms involved.

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

The Proline-Rich Family Protein EXTENSIN33 Is Required for Etiolated Arabidopsis thaliana Hypocotyl Growth.

Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.

CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) Mediates Plant Resistance against Pseudomonas syringae.

Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis.

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.