Investigating the role of a putative endolysin-like candidate effector protein in Verticillium longisporum virulence.

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.

Plant mitochondria and chloroplasts are targeted by the Rhizoctonia solani RsCRP1 effector.

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.

The Role of Glycoside Hydrolases in Phytopathogenic Fungi and Oomycetes Virulence.

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.

The RsRlpA Effector Is a Protease Inhibitor Promoting Rhizoctonia solani Virulence through Suppression of the Hypersensitive Response.

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.

A LysM effector protein from the basidiomycete Rhizoctonia solani contributes to virulence through suppression of chitin-triggered immunity.

Rhizoctonia solani is a fungal species that belongs to the fungal division Basidiomycota. It is a soil-borne pathogen that attacks a broad range of plant species and crops. Disease symptoms are commonly seen as damping off of seedlings and root rot, although it can infect plants at any developmental stage. Despite the severity of this disease, many aspects in R. solani infection biology remain unclear. Here we investigated the role of a LysM effector, previously predicted from the genome of a R. solani AG2-2IIIB strain that has sugar beet as a host. Gene expression analysis showed that RsLysM was highly induced upon sugar beet infection. When RsLysM was heterologously expressed in Cercospora beticola, necrotic lesion size and fungal colonization ability were increased, indicating a role in virulence. RsLysM displayed chitin-binding affinity and suppression of chitin-triggered immunity but could not protect hyphae from hydrolysis. Overall, this study is the first characterization of a LysM effector from Basidiomycota, suggesting that this necrotrophic fungal species relies on perturbation of chitin-triggered immunity to establish a successful infection.

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

BACKGROUND: Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed. RESULTS: The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species. CONCLUSIONS: The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

The immunophilin repertoire of Plasmodiophora brassicae and functional analysis of PbCYP3 cyclophilin.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases.

Detection of Verticillium species in Swedish soils using real-time PCR.

Verticillium species are soilborne plant pathogens, responsible for big yield losses worldwide. Here, we report improved procedures to generate DNA from Verticillium species imbedded in farm soils. Using new genomic sequence information, primers for V. dahliae, V. albo-atrum, V. tricorpus, and V. longisporum were designed. In a survey of 429 samples from intensively farmed soil of two Swedish regions, only V. dahliae and V. longisporum were identified. A bias towards V. longisporum (40%) was seen in the south, whereas V. dahliae was more frequent in the western region (19%). Analyses of soil and leaf samples from 20 sugar beet fields, where foliar wilting had been observed, revealed V. dahliae DNA in all leaf and soil samples and V. longisporum in 18 soil samples, illustrating host choice and longevity of the V. longisporum microsclerotia. This study demonstrates the applicability of new molecular diagnostic tools that are important for growers of variable crops.

Deglycosylating enzymes acting on N-glycans in fungi: Insights from a genome survey.

BACKGROUND: N-Glycosylation, one of the most prominent post-translational modifications of proteins, is found in all domains of life, i.e. eukaryotes, bacteria and archaea, and has been shown to play a crucial role in modulating the physicochemical/physiological properties of carrier proteins. Deglycosylating enzymes that act on N-glycans are widely used in analyzing the structures/functions of N-glycans. Fungi are known to produce various deglycosylating enzymes, some of which are fungi-specific. While such enzymes likely are biologically relevant in fungal biology, their properties as well as their functions have not been explored in detail. SCOPE OF REVIEW: In this review, we summarize the current knowledge of fungal deglycosylating enzymes and discuss their biological significance. MAJOR CONCLUSIONS: As of this writing, five types of deglycosylating enzymes that act on N-glycans are known to occur in fungi; (1) the cytosolic peptide: N-glycanase (PNGase), (2) the acidic PNGase, (3) the glycoside hydrolase family (GH) 85 endo-ß-N-acetylglucosaminidase (ENGase), (4) the GH18 cytosolic ENGase, and (5) the GH18 secreted ENGase. Interestingly, genome surveys indicate that the loss of cytosolic PNGase activity in certain fungi coincide with the occurrence of GH18 cytosolic ENGase, implying that the GH18 ENGase serves to replace the deglycosylation function of the cytosolic PNGase in those filamentous ascomycete fungi. GENERAL SIGNIFICANCE: Our review concludes that fungi promise to be valuable organisms for developing an understanding of the biological functions of PNGases/ENGases.

Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes.

BACKGROUND: Sugar beet (Beta vulgaris) is a crop cultivated for its high content in sugar, but it is vulnerable to many soil-borne pathogens. One of them is the basidiomycete Rhizoctonia solani. This fungal species has a compatibility system regulating hyphal fusions (anastomosis). Consequently, R. solani species are categorized in anastomosis groups (AGs). AG2-2IIIB isolates are most aggressive on sugar beet. In the present study, we report on the draft genome of R. solani AG2-2IIIB using the Illumina technology. Genome analysis, interpretation and comparative genomics of five sequenced R. solani isolates were carried out. RESULTS: The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb. In addition, two normalized EST libraries were sequenced. In total 20,790 of 21,980 AG2-2IIIB isotigs (transcript isoforms) were mapped on the genome with more than 95 % sequence identity. The genome of R. solani AG2-2IIIB was predicted to harbor 11,897 genes and 4908 were found to be isolate-specific. R. solani AG2-2IIIB was predicted to contain 1142 putatively secreted proteins and 473 of them were found to be unique for this isolate. The R. solani AG2-2IIIB genome encodes a high number of carbohydrate active enzymes. The highest numbers were observed for the polysaccharide lyases family 1 (PL-1), glycoside hydrolase family 43 (GH-43) and carbohydrate estarase family 12 (CE-12). Transcription analysis of selected genes representing different enzyme clades revealed a mixed pattern of up- and down-regulation six days after infection on sugar beets featuring variable levels of resistance compared to mycelia of the fungus grown in vitro. CONCLUSIONS: The established R. solani AG2-2IIIB genome and EST sequences provide important information on the gene content, gene structure and transcriptional activity for this sugar beet pathogen. The enriched genomic platform provides an important platform to enhance our understanding of R. solani biology.

Investigating the compatibility of the biocontrol agent Clonostachys rosea IK726 with prodigiosin-producing Serratia rubidaea S55 and phenazine-producing Pseudomonas chlororaphis ToZa7.

This study was carried out to assess the compatibility of the biocontrol fungus Clonostachys rosea IK726 with the phenazine-producing Pseudomonas chlororaphis ToZa7 or with the prodigiosin-producing Serratia rubidaea S55 against Fusarium oxysporum f. sp. radicis-lycopersici. The pathogen was inhibited by both strains in vitro, whereas C. rosea displayed high tolerance to S. rubidaea but not to P. chlororaphis. We hypothesized that this could be attributed to the ATP-binding cassette (ABC) proteins. The results of the reverse transcription quantitative PCR showed an induction of seven genes (abcB1, abcB20, abcB26, abcC12, abcC12, abcG8 and abcG25) from subfamilies B, C and G. In planta experiments showed a significant reduction in foot and root rot on tomato plants inoculated with C. rosea and P. chlororaphis. This study demonstrates the potential for combining different biocontrol agents and suggests an involvement of ABC transporters in secondary metabolite tolerance in C. rosea.

Identifying glycoside hydrolase family 18 genes in the mycoparasitic fungal species Clonostachys rosea.

Clonostachysrosea is a mycoparasitic fungal species that is an efficient biocontrol agent against many plant diseases. During mycoparasitic interactions, one of the most crucial steps is the hydrolysis of the prey's fungal cell wall, which mainly consists of glucans, glycoproteins and chitin. Chitinases are hydrolytic enzymes responsible for chitin degradation and it is suggested that they play an important role in fungal-fungal interactions. Fungal chitinases belong exclusively to the glycoside hydrolase (GH) family 18.These GH18 proteins are categorized into three distinct phylogenetic groups (A, B and C), subdivided into several subgroups. In this study, we identified 14 GH18 genes in the C. rosea genome, which is remarkably low compared with the high numbers found in mycoparasitic Trichoderma species. Phylogenetic analysis revealed that C. rosea contains eight genes in group A, two genes in group B, two genes in group C, one gene encoding a putative ENGase (endo-ß-N-acetylglucosaminidase) and the ech37 gene, which is of bacterial origin. Gene expression analysis showed that only two genes had higher transcription levels during fungal-fungal interactions, while eight out of 14 GH18 genes were triggered by chitin. Furthermore, deletion of the C group chiC2 gene decreased the growth inhibitory activity of C. rosea culture filtrates against Botrytis cinerea and Rhizoctonia solani, although the biocontrol ability of C. rosea against B. cinerea was not affected. In addition, a potential role of the CHIC2 chitinase in the sporulation process was revealed. These results provide new information about the role of GH18 proteins in mycoparasitic interactions.

Endo-ß-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process.

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-ß-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.

Functional analysis of the C-II subgroup killer toxin-like chitinases in the filamentous ascomycete Aspergillus nidulans.

Chitinases are hydrolytic enzymes responsible for chitin polymer degradation. Fungal chitinases belong exclusively to glycoside hydrolases family 18 and they are categorized into three phylogenetic groups (A, B and C), which are further divided into subgroups (A-II to A-V, B-I to B-V and C-I to C-II). Subgroup C chitinases display similarity with the α/ß-subunit of the zymocin yeast killer toxin produced by Kluyveromyces lactis, suggesting a role of these enzymes in fungal-fungal interactions. In this study, we investigated the regulation and function of 4 Aspergillus nidulans subgroup C-II killer toxin-like chitinases by quantitative PCR and by constructing gene deletion strains. Our results showed that all 4 genes were highly induced during interactions with Botrytis cinerea and Rhizoctonia solani, compared to self-interactions. In addition, chiC2-2 and chiC2-3 were also induced during contact with Fusarium sporotrichoides, while none of these genes were induced during interactions with Phytophthora niederhauserii. In contrast, no difference in expression levels were observed between growth on glucose-rich media compared with media containing colloidal chitin, while all genes were repressed during growth on R. solani cell wall material. Phenotypic analysis of chitinase gene deletion strains revealed that B. cinerea biomass was significantly higher in culture filtrate derived from the ΔchiC2-2 strain compared to biomasses grown in media derived from A. nidulans wild type or the other chitinase gene deletion strains. The analysis also showed that all chitinase gene deletion strains displayed increased biomass production in liquid cultures, and altered response to abiotic stress. In summary, our gene expression data suggest the involvement of A. nidulans subgroup C-II chitinases in fungal-fungal interactions, which is further proven for ChiC2-2. In addition, lacking any of the 4 chitinases influenced the growth of A. nidulans.

Coexistence and expression profiles of two alternative splice variants of the pheromone receptor gene pre­1 in Neurospora crassa.

In this study, we show that two splice variants of the pheromone receptor gene (pre-1) transcript coexist in vegetative and reproductive tissues of the filamentous ascomycete fungus Neurospora crassa. The two splice variants differ by intron retention of the last intron, which is predicted to result in a premature stop codon and loss of 322 amino acids in the C-terminal cytosolic region of PRE-1. Using quantitative PCR, we show that expression of the variants is influenced by mating type (mat), with a higher proportion of intron-spliced transcripts in a mat A strain and a higher proportion of the intron-retained variant in a mat a strain. The intron-retained PRE-1 variant is predicted to lack 6 ubiquitination sites that may influence receptor function. In conclusion, N. crassa produce two pre-1 splice variants that display different transcription profiles.

Verticillium longisporum phospholipase VlsPLA2 is a virulence factor that targets host nuclei and modulates plant immunity.

Phospholipase A2 (PLA2 ) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA2 (VlsPLA2 ) in Verticillium longisporum, a fungal phytopathogen that mostly infects plants belonging to the Brassicaceae family, causing severe annual yield loss worldwide. Expression of the VlsPLA2 gene, which encodes active PLA2 , is highly induced during the interaction of the fungus with the host plant Brassica napus. Heterologous expression of VlsPLA2 in Nicotiana benthamiana resulted in increased synthesis of certain phospholipids compared to plants in which enzymatically inactive PLA2 was expressed (VlsPLA2 ΔCD ). Moreover, VlsPLA2 suppresses the hypersensitive response triggered by the Cf4/Avr4 complex, thereby suppressing the chitin-induced reactive oxygen species burst. VlsPLA2 -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. VlsPLA2 was initially localized to the host nucleus and then translocated to the chloroplasts at later time points. In addition, VlsPLA2 bound to the vesicle-associated membrane protein A (VAMPA) and was transported to the nuclear membrane. In the nucleus, VlsPLA2 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 VlsPLA2 acts as a virulence factor, possibly by hydrolysing host nuclear envelope phospholipids, which, through a signal transduction cascade, may suppress basal plant immune responses.

Functional analysis of glycoside hydrolase family 18 and 20 genes in Neurospora crassa.

Glycoside hydrolase family 18 contains hydrolytic enzymes with chitinase or endo-N-acetyl-ß-D-glucosaminidase (ENGase) activity, while glycoside hydrolase family 20 contains enzymes with ß-N-acetylhexosaminidase (NAGase) activity. Chitinases and NAGases are involved in chitin degradation. Chitinases are phylogenetically divided into three main groups (A, B and C), each further divided into subgroups. In this study, we investigated the functional role of 10 Neurospora crassa genes that encode chitinases, 2 genes that encode ENGases and 1 gene that encode a NAGase, using gene deletion and gene expression techniques. No phenotypic effects were detected for any of the studied group A chitinase gene deletions. Deletion of the B group member chit-1 resulted in reduced growth rate compared with the wild type (WT) strain. In combination with the presence of a predicted glycosylphosphatidylinositol anchor motif in the C-terminal of chit-1, indicating cell wall localization, these data suggest a role in cell wall remodeling during hyphal growth for chit-1. Deletion of the ENGase gene gh18-10 resulted in reduced growth rate compared with WT, increased conidiation, and increased abiotic stress tolerance. In addition, Δgh18-10 strains displayed lower secretion of extracellular proteins compared to WT and reduced levels of extracellular protease activity. The connection between gh18-10 ENGase activity and the endoplasmic reticulum associated protein degradation process, a stringent quality control of glycoprotein maturation, is discussed. N. crassa group C chitinase genes gh18-6 and gh18-8 were both induced during fungal-fungal interactions. However, gh18-6 was only induced during interspecific interactions, while gh18-8 displayed the highest induction levels during self-self interactions. These results provide new information on functional differentiation of fungal chitinases.

Rhizoctonia solani Infection Assay of Young Sugar Beet and Arabidopsis plantlets.

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 infectionInoculation of R. solaniPreparation of inoculumPCR analysis Overview of the R. solani infection procedure.

Root Transcriptional and Metabolic Dynamics Induced by the Plant Growth Promoting Rhizobacterium (PGPR) Bacillus subtilis Mbi600 on Cucumber Plants.

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.

A Verticillium longisporum pleiotropic drug transporter determines tolerance to the plant host ß-pinene monoterpene.

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, ß-pinene, 3-carene, and camphene was significantly increased upon fungal infection. Among them, ß-pinene was chosen for further analysis. Transcriptome analysis of V. longisporum on exposure to ß-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 ß-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 ß-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.