Summary: 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 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.

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.

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.

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.