Improved sulfur nutrition provides the basis for enhanced production of sulfur-containing defense compounds in Arabidopsis thaliana upon inoculation with Alternaria brassicicola

The antifungal activities of many sulfur-containing defense compounds suggest a connection between pathogen infection, primary sulfur metabolism and sulfate nutritional status of plants. This relationship was investigated using Arabidopsis thaliana plants that were cultivated under different sulfur regimes and challenged by Alternaria brassicicola. Plants grown with 500 μM sulfate were significantly less infected compared to plants grown on 50 μM sulfate. Upon infection, the formation of the sulfur-containing defense compound camalexin and the gene expression of the sulfur-rich defense peptide defensin were clearly enhanced in plants grown with an optimal compared to a sufficient sulfate supply in the growth medium. Elevated levels of sulfite and O-acetylserine and cysteine biosynthetic enzymes after infection indicated a stimulation of sulfur metabolism under the higher sulfate supply. The results suggest that, in addition to pathogen-triggered activation of sulfur metabolism and sulfur-containing defense compound formation, the sulfate nutritional status is sensed to contribute to plant defense.     

Identification of Alternaria brassicicola genes expressed in planta during pathogenesis of Arabidopsis thaliana

Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on cruciferous plants including economically important Brassica species. The purpose of this study was to identify fungal genes expressed during infection of Arabidopsis. In order to identify candidate genes involved in pathogenicity, we employed suppression subtractive hybridization (SSH) between RNA isolated from A. brassicicola spores incubated in water and on the leaf surface of the Arabidopsis ecotype Landsberg. Two populations of cDNA were created from total RNA extracted after 24h when approximately 80% of the spores had germinated either on the leaf surface or in water. Following SSH, expression of clones was examined using dot-blot macro-arrays and virtual Northern blots. 47 cDNA clones differentially expressed between Alternaria infected Arabidopsis leaves and spore germination in water were selected for sequencing. Seventy-seven percent (36) of the cDNAs had significant homology to fungal sequences from databases examined, including available fungal genomes, while 13% (11) had no homology to sequences in the databases. All 36 genes had significant matches with genes of fungal origin, while 11 genes did not have significant hits in the databases examined. Five sequences were expressed on the plant leaf surface but not during spore germination in water according to virtual Northern blots. These five cDNAs were predicted to encode a cyanide hydratase, arsenic ATPase, formate dehydrogenase, major Alternaria allergen, and one unknown. RT-PCR was used to examine the expression of these five genes during infection of Brassica oleraceae var. capitata (cabbage), in vitro growth in nutrient rich media, and infection of Arabidopsis thaliana. Four of these genes are expressed in the nutrient rich medium, while the unknown gene P3F2 was only expressed during plant infection. The results of this study provide the first insight into genes expressed during A. brassicicola infection of Brassica species that may be involved in fungal pathogenesis.     

Differential protein expression in response to the phytoalexin brassinin allows the identification of molecular targets in the phytopathogenic fungus Alternaria brassicicola

The effects of the cruciferous phytoalexin brassinin on the protein expression patterns of the phytopathogenic fungus Alternaria brassicicola were investigated. Cell-free protein extracts of mycelia of A. brassicicola induced with brassinin at 0.50 and 0.10 mm were fractionated, and the proteins in soluble fractions were separated by two-dimensional electrophoresis. Spots corresponding to differentially expressed proteins were digested and analysed by liquid chromatography-electrospray ionization-mass spectrometry. The number of differentially expressed proteins was significantly higher in mycelia treated with brassinin at 0.50 mm (96 protein spots) than in mycelia treated with brassinin at 0.10 mm (18 protein spots). The majority of differentially expressed proteins included proteins involved in metabolism, processing, synthesis and several heat shock proteins (HSPs). Brassinin concentrations below 0.30 mm induced HSP90, a protein involved in the regulation of morphogenetic signalling in fungi, suggesting that 0.30 mm is a minimal concentration of brassinin necessary for the protection of brassicas against A. brassicicola. These results reveal that HSP90 is a potential target for inhibition in stressed A. brassicicola and confirm that brassinin has strong detrimental effects on A. brassicicola, suggesting that its detoxification by the fungus suppresses an important defence layer of the plant.     

Arabidopsis thaliana plants differentially modulate auxin biosynthesis and transport during defense responses to the necrotrophic pathogen Alternaria brassicicola

? Although the role of auxin in biotrophic pathogenesis has been extensively studied, relatively little is known about its role in plant resistance to necrotrophs. ? Arabidopsis thaliana mutants defective in different aspects of the auxin pathway are generally more susceptible than wild-type plants to the necrotrophic pathogen Alternaria brassicicola. We show that A.?brassicicola infection up-regulates auxin biosynthesis and down-regulates the auxin transport capacities of infected plants, these effects being partially dependent on JA signaling. We also show that these effects of A.?brassicicola infection together lead to an enhanced auxin response in host plants. ? Application of IAA and MeJA together synergistically induces the expression of defense marker genes PDF1.2 (PLANT DEFENSIN 1.2) and HEL (HEVEIN-LIKE), suggesting that enhancement of JA-dependent defense signaling may be part of the auxin-mediated defense mechanism involved in resistance to necrotrophic pathogens. ? Our results provide molecular evidence supporting the hypothesis that JA and auxin interact positively in regulating plant resistance to necrotrophic pathogens and that activation of auxin signaling by JA may contribute to plant resistance to necrotrophic pathogens.     

Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea.

Reliable methods for disease severity assessment are of crucial importance in the study of plant pathogen interactions, either for disease diagnostic on the field or to assess phenotypical differences in plants or pathogen strains. Currently, most of the assays used in fungal disease diagnostic rely on visual assessment of the symptoms, lesion diameter measurement or spore counting. However, these tests are tedious and often cannot discriminate between slightly different levels of resistance. Besides, they are not well suited to assess fungal development in the early phases of the infection, before macroscopical symptoms are visible or before sporulation. We describe here a pathogenicity assay based on the relative quantification of fungal and plant DNA in infected Arabidopsis thaliana leaves by means of real-time quantitative PCR. We show that it allows to monitor quantitatively the growth of the fungi Alternaria brassicicola and Botrytis cinerea in a sensitive and reliable way. Although highly sensitive, this test also exhibits a high robustness, which is crucial to significantly discriminate between lines displaying slightly different levels of resistance. Therefore, it allows to assess fungal development from the very first stages of infection and provides a fast and very practical alternative to currently described assays for phenotyping either plant mutant lines or fungal strains.     

Climatic factors influencing spore production in Alternaria brassicae and Alternaria brassicicola

Sporulation in A. brassicae and A. brassicicola on naturally-infected leaf discs of oilseed rape and cabbage required humidities equal to or higher than 91.5% and 87% r.h. respectively. The optimum temperatures for sporulation were 18–24°C for A. brassicae and 20–30°C for A. brassicicola at which temperatures both fungi produced spores in 12–14 h. Above 24°C sporulation in A. brassicae was inhibited. At sub-optimal temperatures sporulation times for A. brassicicola were significantly longer than for A. brassicae with the differences increasing with decrease in temperature. Interrupting a 16-h wet period at 20°C with a period of 2 h at 70% or 80% r.h. did not affect sporulation in either fungus but a dry interruption of 3–4 h inhibited sporulation in both. Exposure of both fungi to alternating wet (18 h at 100% r.h., 20°C) and dry periods (6 or 30 h at 5565% r.h., 20°C) did not affect the concentration of spores produced in each wet period. Sporulation times were not affected by either the host type of the age of the host tissue. White light (136 W/m²) inhibited sporulation in A. brassicae with the degree of inhibition increasing with increasing light intensity. The effect of light on sporulation in A. brassicicola was not tested.     

Isolation of 12 polymorphic microsatellite loci in the phytopathogenic fungus Alternaria brassicicola

Twelve polymorphic microsatellite markers were isolated from the phytopathogenic fungus Alternaria brassicicola, the causal agent of black spot of crucifers. An enrichment protocol was used to isolate microsatellite loci, which were then analysed in a collection of 46 isolates sampled from seven different countries. The number of alleles detected in 12 loci ranged from two to 10 (mean 3.5). Investigation of cross‐species amplifications showed that the designed primers were specific to A. brassicicola.     

Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling

All tested accessions of Arabidopsis are resistant to the fungal pathogen Alternaria brassicicola. Resistance is compromised by pad3 or coi1 mutations, suggesting that it requires the Arabidopsis phytoalexin camalexin and jasmonic acid (JA)-dependent signaling, respectively. This contrasts with most well-studied Arabidopsis pathogens, which are controlled by salicylic acid-dependent responses and do not benefit from absence of camalexin or JA. Here, mutants with defects in camalexin synthesis (pad1, pad2, pad3, and pad5) or in JA signaling (pad1, coi1) were found to be more susceptible than wild type. Mutants with defects in salicylic acid (pad4 and sid2) or ethylene (ein2) signaling remained resistant. Plant responses to A. brassicicola were characterized using expression profiling. Plants showed dramatic gene expression changes within 12 h, persisting at 24 and 36 h. Wild-type and pad3 plants responded similarly, suggesting that pad3 does not have a major effect on signaling. The response of coi1 plants was quite different. Of the 645 genes induced by A. brassicicola in wild-type and pad3 plants, 265 required COI1 for full expression. It is likely that some of the COI1-dependent genes are important for resistance to A. brassicicola. Responses to A. brassicicola were compared with responses to Pseudomonas syringae infection. Despite the fact that these pathogens are limited by different defense responses, approximately 50% of the induced genes were induced in response to both pathogens. Among these, requirements for COI1 were consistent after infection by either pathogen, suggesting that the regulatory effect of COI1 is similar regardless of the initial stimulus.     

Phytotoxin production by Alternaria linicola and phytoalexin production by the linseed host

Alternaria linicola produced a wide range of secondary metabolites when grown in a defined culture medium. Reverse phase chromatography fractions produced disease-like symptoms on linseed cultivars and a range of non-host species indicating the presence of phytotoxic components. Characterised via thin layer chromatography, these included the non-host specific phytotoxins tenuazonic acid, alternariol monomethyl ether, tentoxin and two destruxin-type compounds (which closely resembled destruxin A and destruxin B). The identity of four of the compounds was confirmed by two dimensional thin layer chromatography and proton nuclear magnetic resonance spectroscopy. In a second experiment, Linum leaf material infected with conidia of A. linicola and blastospores of Melampsora lini was extracted using a facilitated diffusion extraction technique. The resultant extracts contained a number of compounds which were fungitoxic to Cladosporium cladospiroides and, to a lesser extent, Alternaria brassicicola. One such compound corresponded to the phytoalexin coniferyl alcohol. Quantitative differences in the amount of the fungitoxic compounds produced between the inoculated and uninoculated resistant and susceptible host genotype combinations suggested that the production of fungitoxic compounds was greater in response to attempted colonisation. On this basis it is proposed that phytoalexin production is a component of the resistance reaction. The results from these investigations are discussed in relation to recent research on the ecology of the pathogen and the possible roles of phytotoxin production by the pathogen and phytoalexin production by the host on disease development.     

Studies on the epidemiology of Alternaria brassicicola in Brassica oleracea seed production crops

Alternaria brassicicola lesions present on overwintered leaf litter of Brassica oleracea seed production crops produced high concentrations of spores in the spring, these were able to initiate new infections on foliage and subsequently on inflorescences and pods. A vertical disease gradient developed in maturing crops, the lowest pods becoming infected first and infection spreading slowly upwards. Spores were produced abundantly after 20 h leaf wetness at a mean temperature of 13°C or more. Their release was stimulated by a fall in relative humidity but inhibited at a constant high relative humidity resulting in a daily cycle in air spore concentrations with minimum numbers occurring in the early morning and maximum numbers in the early afternoon. For most of the growing season spore movement was restricted to within the crop, however, massive release of spores and subsequent distribution over a wide area occurred when the crop was cut and later threshed. Using semi-selective agar traps spores released at these times were detected up to 1800 m downwind of the parent crop and were instrumental in infecting nearby young crops destined for seed production in the following season.     

Metabolism of the phytoalexins camalexins,their bioisosteres and analogues in the plant pathogenic fungus Alternaria brassicicola

The metabolism of the phytoalexins camalexin (1), 1-methylcamalexin (10) and 6-methoxycamalexin (11) by Alternaria brassicicola and their antifungal activity is reported. This work establishes that camalexins are slowly biotransformed (ca. six days) to the corresponding indole-3-thiocarboxamides, which are further transformed to the indole-3-carboxylic acids. These metabolites are substantially less inhibitory to A. brassicicola than the parent camalexins, indicating that these enzyme-mediated transformations are detoxifications. In addition, analyses of the metabolism of synthetic isomers and bioisosteres of camalexin (1) indicate that isomers of camalexin in the thiazole ring are not metabolized. Based on these results, the potential intermediates that lead to formation of indole-3-thiocarboxamides are proposed.