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Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.

Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B . cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG , which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro ; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates. Plant glucosinolates are important in defense against fungal pathogens. Here, the authors identify a major facilitator superfamily transporter protein of the pathogen Botrytis cinerea , mfsG , that plays a role in efflux and detoxification of glucosinolate-breakdown products during plant–pathogen interactions.

IQ Domain 1 (IQD1) is a novel Arabidopsis thaliana calmodulin-binding protein, which was found to be a positive regulator of glucosinolate (GS) accumulation and plant defense responses against insects. We demonstrate here that the IQD1 overexpressing line ( IQD1 OXP ) was also more resistant also to the necrotrophic fungus Botrytis cinerea , whereas an IQD1 knockout line ( iqd1-1 ) was much more sensitive. Furthermore, we showed that IQD1 is up-regulated by jasmonic acid (JA) and downregulated by salicylic acid (SA). A comparison of whole transcriptome expression between iqd1-1 and wild type plants revealed a substantial downregulation of genes involved in plant defense and hormone regulation. Further examination revealed a marked reduction of SA and increases in the levels of ethylene, JA and abscisic acid response genes in the iqd1-1 line. Moreover, quantification of SA, JA, and abscisic acids in IQD1 OXP and iqd1-1 lines relative to the wild type, showed a significant reduction in endogenous JA levels in the knockout line, simultaneously with increased SA levels. Relations between IQD1 OXP and mutants defective in plant-hormone response indicated that IQD1 cannot rescue the absence of NPR1 or impaired SA accumulation in the NahG line. IQD1 cannot rescue ein2 or eto1 mutations connected to the ethylene pathway involved in both defense responses against B. cinerea and in regulating GS accumulation. Furthermore, IQD1cannot rescue the aos , coi1 or jar1 mutations, all involved in the defense response against B. cinerea and it depends on JAR1 to control indole glucosinolate accumulation. We also found that in the B. cinerea , which infected the iqd1-1 mutant, the most abundant upregulated group of proteins is involved in the degradation of complex carbohydrates, as correlated with the sensitivity of this mutant. In summary, our results suggest that IQD1 is an important A. thaliana defensive protein against B. cinerea that is integrated into several important pathways, such as those involved in plant defense and hormone responses.

Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

Pseudozyma spp. are yeast-like fungi, classified in the Ustilaginales, which are mostly epiphytic or saprophytic and are not pathogenic to plants. Several Pseudozyma species have been reported to exhibit biological activity against powdery mildews. However, previous studies have reported that Pseudozyma aphidis, which can colonize plant surfaces, is not associated with the collapse of powdery mildew colonies. In this report, we describe a novel P. aphidis strain and study its interactions with its plant host and the plant pathogen Botrytis cinerea. This isolate was found to secrete extracellular metabolites that inhibit various fungal pathogens in vitro and significantly reduce B. cinerea infection in vivo. Moreover, P. aphidis sensitized Arabidopsis (Arabidopsis thaliana) plants' defense machinery via local and systemic induction of PATHOGENESIS-RELATED1 (PR1) and PLANT DEFENSIN1.2 (PDF1.2) expression. P. aphidis also reduced B. cinerea infection, locally and systemically, in Arabidopsis mutants impaired in jasmonic acid (JA) or salicylic acid (SA) signaling. Thus, in addition to direct inhibition, P. aphidis may inhibit B. cinerea infection via induced resistance in a manner independent of SA, JA, and Nonexpressor of PR1 (NPR1). P. aphidis primed the plant defense machinery and induced stronger activation of PDF1.2 after B. cinérea infection. Finally, P. aphidis fully or partially reconstituted PRl and PDF1.2 expression in npr1-1 mutant and in plants with the SA hydroxylase NahG transgene, but not in a jasmonate resistant1-1 mutant, after B. cinerea infection, suggesting that P. aphidis can bypass the SA/NPR1, but not JA, pathway to activate PR genes. Thus, either partial gene activation is sufficient to induce resistance, or the resistance is not directed solely through PR1 and PDF1.2 but probably through other pathogen-resistance genes or pathways as well.

Plant pathogens challenge our efforts to maximize crop production due to their ability to rapidly develop resistance to pesticides. Fungal biocontrol agents have become an important alternative to chemical fungicides, due to environmental concerns related to the latter. Here we review the complex modes of action of biocontrol agents in general and epiphytic yeasts belonging to the genus Pseudozyma specifically and P. aphidis in particular. Biocontrol agents act through multiple direct and indirect mechanisms, which are mainly based on their secretions. We discuss the direct modes of action, such as antibiosis, reactive oxygen species-producing, and cell wall-degrading enzyme secretions which can also play a role in mycoparasitism. In addition, we discuss indirect modes of action, such as hyperbiotrophy, induced resistance and growth promotion based on the secretion of effectors and elicitors from the biocontrol agent. Due to their unique characteristics, epiphytic yeasts hold great potential for use as biocontrol agents, which may be more environmentally friendly than conventional pesticides and provide a way to reduce our dependency on fungicides based on increasingly expensive fossil fuels. No less important, the complex mode of action of Pseudozyma-based biocontrol agents can also reduce the frequency of resistance developed by pathogens to these agents.

The need to supply food for a growing world population, while protecting the environment by using sustainable agriculture, presents a major challenge, in terms of decreasing the use of chemical fertilizers and pesticides. The application of Pseudozyma aphidis cells onto crop plants considerably reduced infection by several pathogens, as well as significantly enhancing plant growth and yield. It was demonstrated in the current work that P. aphidis can enhance cucumber and tomato growth, both in vitro and in the greenhouse. P. aphidis was active when applied either via spray treatment or by seed coating. A significant promotion effect of P. aphidis on tomato and cucumber growth, flowering, and tomato yield and ripening, as compared to control plants, was demonstrated. Specifically, a 10–30% increase in tomato and cucumber plant height and leaf number, a 45% increase in average fruit weight, a 120% increase in tomato fruit ripening, and a flowering time advanced by two weeks were demonstrated. In conclusion, P. aphidis can significantly enhance the growth and yield of crop plants and can, thus, be used as bio-fertilizer.

Background Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium. Findings We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids. Conclusions The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.

The plant cell cuticle serves as the first barrier protecting plants from mechanical injury and invading pathogens. The cuticle can be breached by cutinase-producing pathogens and the degradation products may activate pathogenesis signals in the invading pathogens. Cuticle degradation products may also trigger the plant's defense responses. Botrytis cinerea is an important plant pathogen, capable of attacking and causing disease in a wide range of plant species. Arabidopsis thaliana shn1-1D is a gain-of-function mutant, which has a modified cuticular lipid composition. We used this mutant to examine the effect of altering the whole-cuticle metabolic pathway on plant responses to B. cinerea attack. Following infection with B. cinerea, the shn1-1D mutant discolored more quickly, accumulated more H2O2, and showed accelerated cell death relative to wild-type (WT) plants. Whole transcriptome analysis of B. cinerea-inoculated shn1-1D vs. WT plants revealed marked upregulation of genes associated with senescence, oxidative stress and defense responses on the one hand, and genes involved in the magnitude of defense-response control on the other. We propose that altered cutin monomer content and composition of shn1-1D plants triggers excessive reactive oxygen species accumulation and release which leads to a strong, unique and uncontrollable defense response, resulting in plant sensitivity and death.

Background Botrytis cinerea is a haploid necrotrophic ascomycete which is responsible for 'grey mold' disease in more than 200 plant species. Broad molecular research has been conducted on this pathogen in recent years, resulting in the sequencing of two strains, which has generated a wealth of information toward developing additional tools for molecular transcriptome, proteome and secretome investigations. Nonetheless, transformation protocols have remained a significant bottleneck for this pathogen, hindering functional analysis research in many labs. Results In this study, we tested three different transformation methods for B. cinerea : electroporation, air-pressure-mediated and sclerotium-mediated transformation. We demonstrate successful transformation with three different DNA constructs using both air-pressure- and sclerotium-mediated transformation. Conclusions These transformation methods, which are fast, simple and reproducible, can expedite functional gene analysis of B. cinerea .