Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem

Measuring PLD Activity In Vivo

Phospholipase D (PLD) hydrolyzes structural phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) into phosphatidic acid (PA) and free choline/ethanolamine. In plants, this activity can be stimulated by a wide variety of biotic and abiotic stresses (Li et al., Biochim Biophys Acta 1791:927-935, 2009; Testerink and Munnik, J Exp Bot 62(7):2349-2361, 2011). This chapter describes a protocol for the measurement of PLD activity in vivo. The protocol takes advantage of a unique property of PLD, i.e., its ability to substitute a primary alcohol, such as 1-butanol, for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of phosphatidylbutanol (PBut), which is a specific and unique reporter for PLD activity. The assay is highly sensitive for detecting PLD activity in vivo, following stimulation of intact plant cells, seedlings, and tissues, being a valuable method for studying the regulation of plant PLD activity in vivo

Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture

The original version is available at http://www.springerlink.com/content/100383/The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for five hours, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to 5 times in cultivar Pavon and up to 3 times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.http://www.springerlink.com/content/v302947634352057/fulltext.pdfPeer reviewe

Reassessing the role of phospholipase D in the Arabidopsis wounding response.

Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis (Arabidopsis thaliana) AtPLDalpha1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using (32)P(i)-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD-silenced tomato cell-suspension cultures. pldalpha1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldalpha1/delta double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly (Pieris rapae) grew equally well on wild-type and the pld knock-out mutants