ABSTRACT
KEY MESSAGE: Expression of the TaMDC1 in transgenic tomato plants confer resistance to bacterial and fungal pathogens, as well as an insect pest and thus prove in planta function of the wheat cystatin. Cystatins are the polypeptides with cysteine proteinase inhibitory activities. Plant cystatins or phytocystatins are known to contribute to plant resistance against insect pests. Recently, increasing data proved that some of the phytocystatins also have antifungal activities in vitro. Here, we functionally characterized a wheat multidomain cystatin, TaMDC1, using in planta assays. Expression of TaMDC1 in wheat seedlings is up-regulated in response to methyl jasmonate and salicylic acid, indicating that TaMDC1 is involved in biotic stress responses mediated by these plant hormones. The TaMDC1 cDNA was integrated in tomato genome and expressed under cauliflower mosaic virus 35S promoter. Four transgenic plants that show high level of the transgene expression were selected by RNA gel blot and immunoblot analysis and utilized to assess biotic stress resistance against the bacterial pathogen Pseudomonas syringae, the fungal pathogens Botrytis cinerea and Alternaria alternata, and the insect pest Colorado potato beetle (CPB, Leptinotarsa decemlineata). Detached leaf inoculation assays revealed that the tomato plants expressing TaMDC1 showed high levels of resistance against P. syringae and A. alternata, and elevated tolerance against B. cinerea. Sustenance of L. decemlineata larvae to the transgenic plants demonstrated inhibition of CPB larvae growth. Inhibitory activity of TaMDC1 against selected pathogens was also demonstrated by in vitro assays with total protein extracted from transgenic tomato plants. Taken together, the presented data suggest that TaMDC1 is involved in a broad spectrum biotic stress resistance in planta.
Subject(s)
Cystatins/metabolism , Disease Resistance/genetics , Plant Diseases/immunology , Plant Growth Regulators/metabolism , Triticum/genetics , Acetates/metabolism , Animals , Anti-Bacterial Agents/metabolism , Antifungal Agents/metabolism , Botrytis/physiology , Coleoptera/physiology , Cyclopentanes/metabolism , Cystatins/genetics , Gene Expression , Larva , Solanum lycopersicum/genetics , Solanum lycopersicum/microbiology , Solanum lycopersicum/parasitology , Oxylipins/metabolism , Plant Diseases/microbiology , Plant Diseases/parasitology , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/microbiology , Plant Leaves/parasitology , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Pseudomonas syringae/physiology , Salicylic Acid/metabolism , Triticum/immunology , Triticum/microbiology , Triticum/parasitologyABSTRACT
1. Diamines (putrescine and cadaverine) and polyamines (spermidine and spermine) were extracted from tissues of the sea star Pycnopodia helianthoides, separated and quantitated using reversed-phase high-performance liquid chromatography (RP-HPLC). Simultaneous measurements of levels of protein and DNA and rates of incorporation of 14C-thymidine were carried out. 2. The most abundant polyamine in tissues was spermidine (0.3873-2.5282 nmol/mg tissue) followed by spermine (0.103-1.5517 nmol/mg tissue), putrescine (0.2096-0.5322 nmol/mg tissue) and cadaverine (0.022-0.6064 nmol/mg tissue). 3. An unknown molecule with derivatization and elution behaviour similar to that of polyamine standards was detected in all tissues. 4. Protein levels ranged from 20.47 mg/g tissue in the body wall to 48.44 mg/g tissue in the pyloric caecum. 5. DNA levels were lowest in the ovary (0.25 mg/g tissue) and highest in the testis (5.62 mg/g tissue). 6. Incorporation of 14C-thymidine was highest in the testis. Testicular tissue had the highest spermidine/spermine ratio (5.4). A significant correlation between the spermidine/spermine ratio and 14C-thymidine incorporation (expressed either as DPM/g tissue or DPM/mg protein) suggests that polyamines are implicated in the regulation of cell proliferation in the sea star P. helianthoides.
