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Botrytis cinerea is a notorious pathogen causing pre- and post-harvest spoilage in many economically important crops. Excessive application of site-specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2, encoding an ATP-binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B. cinerea multidrug-resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Phospholipase A 2 (PLA 2 ) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA 2 (VlsPLA 2 ) in Verticillium longisporum, a fungal phytopathogen that mostly infects plants belonging to the Brassicaceae family, causing severe annual yield loss worldwide. Expression of the VlsPLA 2 gene, which encodes active PLA 2 , is highly induced during the interaction of the fungus with the host plant Brassica napus. Heterologous expression of VlsPLA 2 in Nicotiana benthamiana resulted in increased synthesis of certain phospholipids compared to plants in which enzymatically inactive PLA 2 was expressed (VlsPLA 2 ΔCD ). Moreover, VlsPLA 2 suppresses the hypersensitive response triggered by the Cf4/Avr4 complex, thereby suppressing the chitin-induced reactive oxygen species burst. VlsPLA 2 -overexpressing V. longisporum strains showed increased virulence in Arabidopsis plants, and transcriptomic analysis of this fungal strain revealed that the induction of the gene contributed to increased virulence. VlsPLA 2 was initially localized to the host nucleus and then translocated to the chloroplasts at later time points. In addition, VlsPLA 2 bound to the vesicle-associated membrane protein A (VAMPA) and was transported to the nuclear membrane. In the nucleus, VlsPLA 2 caused major alterations in the expression levels of genes encoding transcription factors and subtilisin-like proteases, which play a role in plant immunity. In conclusion, our study showed that VlsPLA 2 acts as a virulence factor, possibly by hydrolysing host nuclear envelope phospholipids, which, through a signal transduction cascade, may suppress basal plant immune responses., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Verticillium is a genus of ascomycete fungi that encompasses several plant pathogenic species, and cause severe annual yield losses on many economically important crops worldwide. One of the most important species in this genus, is V. longisporum, which causes disease mainly on plants in the Brassicaceae family. Genome analysis of V. longisporum strain VL1 revealed a number of candidate effector genes that may be associated with fungal virulence. One of these candidate effector-genes encodes a putative endolysin-like protein. Endolysins are hydrolytic enzymes that are secreted by bacteriophages and recently, they have been identified in fungal genomes as well. In this study, the potential role of this gene has been investigated in V. longisporum. Our data showed that this gene was highly induced in the fungus during Brassica napus infection and its overexpression significantly increased V. longisporum virulence, indicating an involvement in the fungal infection process., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacillus subtilis MBI600 is a commercialized plant growth-promoting bacterial species used as a biocontrol agent in many crops, controlling various plant pathogens via direct or indirect mechanisms. In the present study, a detailed transcriptomic analysis of cucumber roots upon response to the Bs MBI600 strain is provided. Differentially expressed genes (DEGs) analysis showed altered gene expression in more than 1000 genes at 24 and 48 h post-application of Bs MBI600. Bs MBI600 induces genes involved in ISR and SAR signaling. In addition, genes involved in phytohormone production and nutrient availability showed an upregulation pattern, justifying the plant growth promotion. Biocontrol ability of Bs MBI600 seems also to be related to the activation of defense-related genes, such as peroxidase, endo-1,3(4)-beta-glucanase, PR-4, and thaumatin-like. Moreover, KEGG enriched results showed that differentially expressed genes were classified into biocontrol-related pathways. To further investigate the plant's response to the presence of PGPR, a profile of polar metabolites of cucumber treated with Bs MBI600 was performed and compared to that of untreated plants. The results of the current study gave insights into the mechanisms deployed by this biocontrol agent to promote plant resistance, helping to understand the molecular interactions in this system.

Terpenes constitute a major part of secondary metabolites secreted by plants in the rhizosphere. However, their specific functions in fungal-plant interactions have not been investigated thoroughly. In this study we investigated the role of monoterpenes in interactions between oilseed rape (Brassica napus) and the soilborne pathogen Verticillium longisporum. We identified seven monoterpenes produced by B. napus, and production of α-pinene, β-pinene, 3-carene, and camphene was significantly increased upon fungal infection. Among them, β-pinene was chosen for further analysis. Transcriptome analysis of V. longisporum on exposure to β-pinene resulted in identification of two highly expressed pleotropic drug transporters paralog genes named VlAbcG1a and VlAbcG1b. Overexpression of VlAbcG1a in Saccharomyces cerevisiae increased tolerance to β-pinene, while deletion of the VlAbcG1a homologous gene in Verticillium dahliae resulted in mutants with increased sensitivity to certain monoterpenes. Furthermore, the VlAbcG1a overexpression   strain displayed an increased tolerance to β-pinene and increased virulence in tomato plants. Data from this study give new insights into the roles of terpenes in plant-fungal pathogen interactions and the mechanisms fungi deploy to cope with the toxicity of these secondary metabolites., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Rhizoctonia solani is a soil-borne fungus, which rarely produces any spores in culture. Hence, all inoculation procedures are based on mycelia, often as a coat on cereal kernels, placed in close vicinity to the plant to be infected. In this protocol, an inoculation method is described where the fungus is first allowed to infest a perlite-maize flour substrate for 10 days, followed by thorough soil mixing to generate uniform fungal distribution. Pre-grown seedlings are then replanted in the infested soil. Plant materials can be harvested, five (sugar beet) and ten days ( Arabidopsis ) post infection, followed by a rapid cleaning step ahead of any nucleic acid preparation. Commercial DNA or RNA extraction kits can be used or, if higher DNA yield is required, a CTAB extraction method. Our purpose was to develop a reliable and reproducible protocol to determine the infection levels in planta upon infection with R. solani . This protocol is less laborious compared to previous ones, improves the consistency of plant infection, reproducibility between experiments, and suits both a root crop and Arabidopsis . Graphic abstract: Plant infection Inoculation of R. solani Preparation of inoculumPCR analysis Overview of the R. solani infection procedure., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Phytopathogenic fungi need to secrete different hydrolytic enzymes to break down complex polysaccharides in the plant cell wall in order to enter the host and develop the disease. Fungi produce various types of cell wall degrading enzymes (CWDEs) during infection. Most of the characterized CWDEs belong to glycoside hydrolases (GHs). These enzymes hydrolyze glycosidic bonds and have been identified in many fungal species sequenced to date. Many studies have shown that CWDEs belong to several GH families and play significant roles in the invasion and pathogenicity of fungi and oomycetes during infection on the plant host, but their mode of function in virulence is not yet fully understood. Moreover, some of the CWDEs that belong to different GH families act as pathogen-associated molecular patterns (PAMPs), which trigger plant immune responses. In this review, we summarize the most important GHs that have been described in eukaryotic phytopathogens and are involved in the establishment of a successful infection.

Botrytis cinerea is a plant pathogen causing the gray mold disease in a plethora of host plants. The control of the disease is based mostly on chemical pesticides, which are responsible for environmental pollution, while they also pose risks for human health. Furthermore, B. cinerea resistant isolates have been identified against many fungicide groups, making the control of this disease challenging. The application of biocontrol agents can be a possible solution, but requires deep understanding of the molecular mechanisms in order to be effective. In this study, we investigated the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, a new commercialized biopesticide, the pathogen B. cinerea and their plant host. Our analysis showed that this biocontrol agent reduced B. cinerea mycelial growth in vitro, and was able to suppress the disease incidence on cucumber plants. Moreover, treatment with B. subtilis led to induction of genes involved in plant immunity. RNA-seq analysis of B. cinerea transcriptome upon exposure to bacterial secretome, showed that genes coding for MFS and ABC transporters were highly induced. Deletion of the Bcmfs1 MFS transporter gene, using a CRISP/Cas9 editing method, affected its virulence and the tolerance of B. cinerea to bacterial secondary metabolites. These findings suggest that specific detoxification transporters are involved in these interactions, with crucial role in different aspects of B. cinerea physiology., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The fungal species Rhizoctonia solani belongs to the Basidiomycota division and is a ubiquitous soil-borne pathogen. It is the main agent of the damping-off disease in seedlings and causes the root and crown rot disease in sugar beets. Plant pathogens deploy small secreted proteins, called effectors, to manipulate plant immunity in order to infect the host. Here, a gene (RsCRP1) encoded a putative effector cysteine-rich protein was cloned, expressed in Cercospora beticola and used for virulence assays. The RsCRP1 gene was highly induced upon the early-infection stage of sugar beet seedlings and disease was promoted. Confocal microscopy demonstrated localization to the chloroplasts and mitochondria upon transient expression of RsCRP1 in leaves of Nicotiana benthamiana. Further, this effector was unable to induce necrosis or to suppress hypersensitive response induced by the Avr4/Cf4 complex in N. benthamiana. Overall, these data indicate that RsCRP1 is a novel effector targeting distinct plant cell organelles in order to facilitate a successful infection at the early stages of the disease development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)