Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola

Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.     

Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.     

The cDNA microarray analysis using an Arabidopsis pad3 mutant reveals the expression profiles and classification of genes induced by Alternaria brassicicola attack

The hypersensitive response (HR) was induced in a wild-type Arabidopsis thaliana plant (Columbia) (Col-wt) by inoculation with Alternaria brassicicola that causes the development of small brown necrotic lesions on the leaves. By contrast, pad3-1 mutants challenged with A. brassicicola produced spreading lesions. The cell death in pad3-1 mutants could not inhibit the pathogen growth and development, although both production of H(2)O(2) and localized cell death were similar in Col-wt and pad3-1 plants after the inoculation. The difference between Col-wt and pad3-1 plants is defense responses after the occurrence of cell death. In other words, PAD3 is necessary for defense response to A. brassicicola. Therefore, we examined the changes in the expression patterns of ca. 7,000 genes by cDNA microarray analysis after inoculation with A. brassicicola. The cDNA microarrays were also done to analyze Arabidopsis responses after treatment with signal molecules, reactive oxygen species (ROS)-inducing compounds and UV-C. The results suggested that the pad3-1 mutation altered not only the accumulation of camalexin but also the timing of expression of many defense-related genes in response to the challenge with A. brassicicola. Furthermore, the plants integrate two or more signals that act together for promoting the induction of multiple defense pathways.     

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.     

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.     

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.     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Cytokinin-like substances and accumulation of labelled metabolites at infection sites of Alternaria brassicicola

Green islands were observed on mustard leaves beneath the infection drops containing germinating conidia of Alternaria brassicicola, when the surrounding uninfected tissue had yellowed due to senescence. Green island formation has been correlated with the secretion of cytokinin-like substances by the pathogens. A. brassicicola secreted cytokinin-like substances in a liquid synthetic medium and their application to detached host leaves evoked the formation of green islands in the dark. ¹⁴C studies confirmed that green islands act as metabolic sinks in which photosynthates are retained or accumulated. Cytokinin-like substances appear to be actively involved in infection and pathogenesis of A. brassicicola.     

Characterization of early,chitin-induced gene expression in Arabidopsis

Three genes (i.e., a zinc finger protein, a lectin-like protein, and AtMPK3), previously shown to respond to chitin elicitation in microarray experiments, were used to examine the response of Arabidopsis spp. to chitin addition. Maximum induction for all three genes was found upon addition of crab-shell chitin at 100 mg per liter. Threefold induction was found with a chitin concentration as low as 10(-4) mg per liter. The specificity of this response was examined using purified chitin oligomers (degree of polymerization = 2 to 8). The larger chitin oligomers (hexamer to octamer), were most effective in inducing expression of the three genes assayed. Gene induction was observed after the addition of 1 nM chitin octamer. The protein kinase inhibitors staurosporine and K252a effectively suppressed chitin-induced gene expression, while the protein phosphatase inhibitors calyculin A and okadaic acid induced the accumulation of mRNA in the absence of chitin. The phosphorylation event necessary for transmission of the chitin signal was completed within the first 20 min of chitin addition. The level of chitin-induced gene expression of the lectin-like protein and AtMPK3 was not significantly changed in mutants blocked in the jasmonic acid (JA, jar1)-, ethylene (ein2)-, or salicylic acid (SA, pad4, npr1, and eds5)-dependent pathway. In contrast, expression of mRNA for the zinc finger protein was reduced in the mutants affected in the JA- or SA-dependent pathway.