SUMMARYVery long chain lipids are important components of the plant cuticle that establishes the boundary surface of aerial organs. In addition, these lipids were detected in the extracellular pollen coat (tryphine), where they play a crucial role in appropriate pollen-stigma communication. As such they are involved in the early interaction of pollen with the stigma. A substantial reduction in tryphine lipids was shown to compromise pollen germination and, consequently, resulted in male sterility. We investigated the role of two long-chain acyl-CoA synthetases (LACSs) in Arabidopsis with respect to their contribution to the production of tryphine lipids. LACS was shown to provide CoA-activated very long chain fatty acids (VLCFA-CoAs) to the pathways of wax biosynthesis. The allocation of sufficient quantities of VLCFA-CoA precursors should therefore be relevant to the generation of tryphine lipids. Here, we report on the identification of lacs1 lacs4 double knock-out mutant lines that were conditionally sterile and showed significant reductions in pollen coat lipids. Whereas the contributions of both LACS proteins to surface wax levels were roughly additive, their co-operation in tryphine lipid biosynthesis was clearly more complex. The inactivation of LACS4 resulted in increased levels of tryphine lipids accompanied by morphological anomalies of the pollen grains. The additional inactivation of LACS1 neutralized the morphological defects, decreased the tryphine lipids far below wild-type levels and resulted in conditionally sterile pollen.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-009-2269-0) contains supplementary material, which is available to authorized users.

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Verticillium longisporum is a host-specific vascular pathogen of oilseed rape (Brassica napus L.) that causes economic crop losses by impairing plant growth and inducing premature senescence. This study investigates whether plant damage through Verticillium stem striping is due to impaired plant water relations, whether V. longisporum affects responses of a susceptible B. napus variety to drought stress, and whether drought stress, in turn, affects plant responses to V. longisporum. Two-factorial experiments on a susceptible cultivar of B. napus infected or noninfected with V. longisporum and exposed to three watering levels (30, 60, and 100% field capacity) revealed that drought stress and V. longisporum impaired plant growth by entirely different mechanisms. Although both stresses similarly affected plant growth parameters (plant height, hypocotyl diameter, and shoot and root dry matter), infection of B. napus with V. longisporum did not affect any drought-related physiological or molecular genetic plant parameters, including transpiration rate, stomatal conductance, photosynthesis rate, water use efficiency, relative leaf water content, leaf proline content, or the expression of drought-responsive genes. Thus, this study provides comprehensive physiological and molecular genetic evidence explaining the lack of wilt symptoms in B. napus infected with V. longisporum. Likewise, drought tolerance of B. napus was unaffected by V. longisporum, as was the level of disease by drought conditions, thus excluding a concerted action of both stresses in the field. Although it is evident that drought and vascular infection with V. longisporum impair plant growth by different mechanisms, it remains to be determined by which other factors V. longisporum causes crop loss.