Rational Design of a Potential New Nematicide Targeting Chitin Deacetylase.

In a previously published study, the authors devised a molecular topology QSAR (quantitative structure-activity relationship) approach to detect novel fungicides acting as inhibitors of chitin deacetylase (CDA). Several of the chosen compounds exhibited noteworthy activity. Due to the close relationship between chitin-related proteins present in fungi and other chitin-containing plant-parasitic species, the authors decided to test these molecules against nematodes, based on their negative impact on agriculture. From an overall of 20 fungal CDA inhibitors, six showed to be active against Caenorhabditis elegans. These experimental results made it possible to develop two new molecular topology-based QSAR algorithms for the rational design of potential nematicides with CDA inhibitor activity for crop protection. Linear discriminant analysis was employed to create the two algorithms, one for identifying the chemo-mathematical pattern of commercial nematicides and the other for identifying nematicides with activity on CDA. After creating and validating the QSAR models, the authors screened several natural and synthetic compound databases, searching for alternatives to current nematicides. Finally one compound, the N2-(dimethylsulfamoyl)-N-{2-[(2-methyl-2-propanyl)sulfanyl]ethyl}-N2-phenylglycinamide or nematode chitin deacetylase inhibitor, was selected as the best candidate and was further investigated both in silico, through molecular docking and molecular dynamic simulations, and in vitro, through specific experimental assays. The molecule shows favorable binding behavior on the catalytic pocket of C. elegans CDA and the experimental assays confirm potential nematicide activity.

Suppression of Chitin-Triggered Immunity by Plant Fungal Pathogens: A Case Study of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii.

Fungal pathogens are significant plant-destroying microorganisms that present an increasing threat to the world's crop production. Chitin is a crucial component of fungal cell walls and a conserved MAMP (microbe-associated molecular pattern) that can be recognized by specific plant receptors, activating chitin-triggered immunity. The molecular mechanisms underlying the perception of chitin by specific receptors are well known in plants such as rice and Arabidopsis thaliana and are believed to function similarly in many other plants. To become a plant pathogen, fungi have to suppress the activation of chitin-triggered immunity. Therefore, fungal pathogens have evolved various strategies, such as prevention of chitin digestion or interference with plant chitin receptors or chitin signaling, which involve the secretion of fungal proteins in most cases. Since chitin immunity is a very effective defensive response, these fungal mechanisms are believed to work in close coordination. In this review, we first provide an overview of the current understanding of chitin-triggered immune signaling and the fungal proteins developed for its suppression. Second, as an example, we discuss the mechanisms operating in fungal biotrophs such as powdery mildew fungi, particularly in the model species Podosphaera xanthii, the main causal agent of powdery mildew in cucurbits. The key role of fungal effector proteins involved in the modification, degradation, or sequestration of immunogenic chitin oligomers is discussed in the context of fungal pathogenesis and the promotion of powdery mildew disease. Finally, the use of this fundamental knowledge for the development of intervention strategies against powdery mildew fungi is also discussed.

Rational Design of Chitin Deacetylase Inhibitors for Sustainable Agricultural Use Based on Molecular Topology.

Fungicide resistance is a major concern in modern agriculture; therefore, there is a pressing demand to develop new, greener chemicals. Chitin is a major component of the fungal cell wall and a well-known elicitor of plant immunity. To overcome chitin recognition, fungal pathogens developed different strategies, with chitin deacetylase (CDA) activity being the most conserved. This enzyme is responsible for hydrolyzing the N-acetamido group in N-acetylglucosamine units of chitin to convert it to chitosan, a compound that can no longer be recognized by the plant. In previous works, we observed that treatments with CDA inhibitors, such as carboxylic acids, reduced the symptoms of cucurbit powdery mildew and induced rapid activation of chitin-triggered immunity, indicating that CDA could be an interesting target for fungicide development. In this work, we developed an in silico strategy based on QSAR (quantitative structure-activity relationship) and molecular topology (MT) to discover new, specific, and potent CAD inhibitors. Starting with the chemical structures of few carboxylic acids, with and without disease control activity, three predictive equations based on the MT paradigm were developed to identify a group of potential molecules. Their fungicidal activity was experimentally tested, and their specificity as CDA inhibitors was studied for the three best candidates by molecular docking simulations. To our knowledge, this is the first time that MT has been used for the identification of potential CDA inhibitors to be used against resistant powdery mildew strains. In this sense, we consider of special interest the discovery of molecules capable of stimulating the immune system of plants by triggering a defensive response against fungal species that are highly resistant to fungicides such as powdery mildew.

Suppression of Chitin-Triggered Immunity by a New Fungal Chitin-Binding Effector Resulting from Alternative Splicing of a Chitin Deacetylase Gene.

Phytopathogenic fungi have evolved mechanisms to manipulate plant defences, such as chitin-triggered immunity, a plant defensive response based on the recognition of chitin oligomers by plant-specific receptors. To cope with chitin resistance, fungal pathogens have developed different strategies to prevent chitin recognition, such as binding, breaking, or modifying immunogenic oligomers. In powdery mildew fungi, the activity of chitin deacetylase (CDA) is crucial for this purpose, since silencing of the CDA gene leads to a rapid activation of chitin signalling and the subsequent suppression of fungal growth. In this work, we have identified an unusually short CDA transcript in Podosphaera xanthii, the cucurbit powdery mildew pathogen. This transcript, designated PxCDA3, appears to encode a truncated version of CDA resulting from an alternative splicing of the PxCDA gene, which lacked most of the chitin deacetylase activity domain but retained the carbohydrate-binding module. Experiments with the recombinant protein showed its ability to bind to chitin oligomers and prevent the activation of chitin signalling. Furthermore, the use of fluorescent fusion proteins allowed its localization in plant papillae at pathogen penetration sites. Our results suggest the occurrence of a new fungal chitin-binding effector, designated CHBE, involved in the manipulation of chitin-triggered immunity in powdery mildew fungi.

Chitin Deacetylase, a Novel Target for the Design of Agricultural Fungicides.

Fungicide resistance is a serious problem for agriculture. This is particularly apparent in the case of powdery mildew fungi. Therefore, there is an urgent need to develop new agrochemicals. Chitin is a well-known elicitor of plant immunity, and fungal pathogens have evolved strategies to overcome its detection. Among these strategies, chitin deacetylase (CDA) is responsible for modifying immunogenic chitooligomers and hydrolysing the acetamido group in the N-acetylglucosamine units to avoid recognition. In this work, we tested the hypothesis that CDA can be an appropriate target for antifungals using the cucurbit powdery mildew pathogen Podosphaera xanthii. According to our hypothesis, RNAi silencing of PxCDA resulted in a dramatic reduction in fungal growth that was linked to a rapid elicitation of chitin-triggered immunity. Similar results were obtained with treatments with carboxylic acids such as EDTA, a well-known CDA inhibitor. The disease-suppression activity of EDTA was not associated with its chelating activity since other chelating agents did not suppress disease. The binding of EDTA to CDA was confirmed by molecular docking studies. Furthermore, EDTA also suppressed green and grey mould-causing pathogens applied to oranges and strawberries, respectively. Our results conclusively show that CDA is a promising target for control of phytopathogenic fungi and that EDTA could be a starting point for fungicide design.

RNA-seq analysis and fluorescence imaging of melon powdery mildew disease reveal an orchestrated reprogramming of host physiology.

The cucurbit powdery mildew elicited by Podosphaera xanthii is one of the most important limiting factors in cucurbit production. Our knowledge of the genetic and molecular bases underlying the physiological processes governing this disease is very limited. We used RNA-sequencing to identify differentially expressed genes in leaves of Cucumis melo upon inoculation with P. xanthii, using RNA samples obtained at different time points during the early stages of infection and their corresponding uninfected controls. In parallel, melon plants were phenotypically characterized using imaging techniques. We found a high number of differentially expressed genes (DEGs) in infected plants, which allowed for the identification of many plant processes that were dysregulated by the infection. Among those, genes involved in photosynthesis and related processes were found to be upregulated, whereas genes involved in secondary metabolism pathways, such as phenylpropanoid biosynthesis, were downregulated. These changes in gene expression could be functionally validated by chlorophyll fluorescence imaging and blue-green fluorescence imaging analyses, which corroborated the alterations in photosynthetic activity and the suppression of phenolic compound biosynthesis. The powdery mildew disease in melon is a consequence of a complex and multifaceted process that involves the dysregulation of many plant pathways such as primary and secondary metabolism.

Transformation by growth onto agro-infiltrated tissues (TGAT), a simple and efficient alternative for transient transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii.

A major limitation of molecular studies in powdery mildew fungi (Erysiphales) is their genetic intractability. This is because they are obligate biotrophs. In these parasites, biotrophy is determined by the presence of haustoria, which are specialized structures of parasitism that play an essential role in the acquisition of nutrients and the deliverance of effectors. Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a major limitation for crop productivity. In a previous study using P. xanthii conidia, we showed, for the first time, the transformation of powdery mildew fungi by Agrobacterium tumefaciens. In this work, we hypothesized that the haustorium could also act as a natural route for the acquisition of DNA. To test our hypothesis, melon cotyledons were agro-infiltrated with A. tumefaciens that contained diverse transfer DNA (T-DNA) constructs harbouring different marker genes under the control of fungal promoters and, after elimination of the bacterium, the cotyledons were subsequently inoculated with P. xanthii conidia. Our results conclusively demonstrated the transfer of different T-DNAs from A. tumefaciens to P. xanthii, including two fungicide resistance markers (hph and tub2), a reporter gene (gfp) and a translational fusion (cfp-PxEC2). These results were further supported by the co-localization of translational fluorescent fusions of A. tumefaciens VirD2 and P. xanthii Rab5 proteins into small vesicles of haustorial and hyphal cells, suggesting endocytosis as the mechanism for T-DNA uptake, presumably by the haustorium. From our perspective, transformation by growth onto agro-infiltrated tissues (TGAT) is the easiest and most reliable method for the transient transformation of powdery mildew fungi.

The Functional Characterization of Podosphaera xanthii Candidate Effector Genes Reveals Novel Target Functions for Fungal Pathogenicity.

Podosphaera xanthii is the main causal agent of powdery mildew disease in cucurbits. In a previous study, we determined that P. xanthii expresses approximately 50 Podosphaera effector candidates (PECs), identified based on the presence of a predicted signal peptide and the absence of functional annotation. In this work, we used host-induced gene silencing (HIGS), employing Agrobacterium tumefaciens as a vector for the delivery of the silencing constructs (ATM-HIGS), to identify genes involved in early plant-pathogen interaction. The analysis of seven selected PEC-encoding genes showed that six of them, PEC007, PEC009, PEC019, PEC032, PEC034, and PEC054, are required for P. xanthii pathogenesis, as revealed by reduced fungal growth and increased production of hydrogen peroxide by host cells. In addition, protein models and protein-ligand predictions allowed us to identify putative functions for these candidates. The biochemical activities of PEC019, PEC032, and PEC054 were elucidated using their corresponding proteins expressed in Escherichia coli. These proteins were confirmed as phospholipid-binding protein, α-mannosidase, and cellulose-binding protein. Further, BLAST searches showed that these three effectors are widely distributed in phytopathogenic fungi. These results suggest novel targets for fungal effectors, such as host-cell plasma membrane, host-cell glycosylation, and damage-associated molecular pattern-triggered immunity.

Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens.

The obligate biotrophic fungal pathogen Podosphaera xanthii is the main causal agent of powdery mildew in cucurbit crops all over the world. A major limitation of molecular studies of powdery mildew fungi (Erysiphales) is their genetic intractability. In this work, we describe a robust method based on the promiscuous transformation ability of Agrobacterium tumefaciens for reliable transformation of P. xanthii. The A. tumefaciens-mediated transformation (ATMT) system yielded transformants of P. xanthii with diverse transferred DNA (T-DNA) constructs. Analysis of the resultant transformants showed the random integration of T-DNA into the P. xanthii genome. The integrations were maintained in successive generations in the presence of selection pressure. Transformation was found to be transient, because in the absence of selection agent, the introduced genetic markers were lost due to excision of T-DNA from the genome. The ATMT system represents a potent tool for genetic manipulation of P. xanthii and will likely be useful for studying other biotrophic fungi. We hope that this method will contribute to the development of detailed molecular studies of the intimate interaction established between powdery mildew fungi and their host plants.

The Podosphaera xanthii haustorium, the fungal Trojan horse of cucurbit-powdery mildew interactions.

The powdery mildew fungi are obligate biotrophic plant pathogens that develop a specialized structure for parasitism termed haustorium, which is responsible for nutrient uptake and factor exchange with the plant. In this work, we present a detailed microscopy analysis of the haustoria of the cucurbit powdery mildew fungus Podosphaera xanthii, a major limiting factor for cucurbit production worldwide. Despite being located inside plant epidermal cells, transmission electron microscopy (TEM) analysis showed the characteristic highly irregular outline of the extrahaustorial membrane that separates the extrahaustorial matrix of haustoria from the cytoplasm of the plant cell. TEM analysis also revealed the presence of some vesicles and electron-dense plaques of material surrounding the haustoria. In confocal microscopy analysis and aniline blue staining we found a positive correlation between haustorial development and deposition of callose, which is distributed as plaques around haustorial complex. In this study, a method for the isolation of P. xanthii haustoria was also adapted, which permitted the analysis of the formation of haustorial lobes and the visualization of vacuoles and the pool of vesicles inside the haustorial complex. Our findings suggested that the haustorial lobes were responsible for vesicular trafficking and most likely act as the main mediators of the fungus-plant dialogue. All of these findings were integrated into a model of the P. xanthii-host cellular interactions.