Identification of novel transcriptional regulators of Zat12 using comprehensive yeast one-hybrid screens.

To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc-finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined an approximately 200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 small nuclear RNA (snRNA) processing protein, and dormancy/auxin-associated family protein 2 (DRM2). Altered expression of Zat12 during high light stress in the knockout mutants further indicated the involvement of these proteins in the regulation of Zat12. Using a state of the art microfluidic approach we showed that AtUSB1 and DRM2 can specifically bind dsDNA and were able to identify the preferred DNA-binding motif of all four proteins. Overall, the proteins identified in this work provide an important start point for charting the earliest signaling network of Zat12 and of other genes required for acclimation to abiotic stresses.

ASCORBATE PEROXIDASE6 protects Arabidopsis desiccating and germinating seeds from stress and mediates cross talk between reactive oxygen species, abscisic acid, and auxin.

A seed's ability to properly germinate largely depends on its oxidative poise. The level of reactive oxygen species (ROS) in Arabidopsis (Arabidopsis thaliana) is controlled by a large gene network, which includes the gene coding for the hydrogen peroxide-scavenging enzyme, cytosolic ASCORBATE PEROXIDASE6 (APX6), yet its specific function has remained unknown. In this study, we show that seeds lacking APX6 accumulate higher levels of ROS, exhibit increased oxidative damage, and display reduced germination on soil under control conditions and that these effects are further exacerbated under osmotic, salt, or heat stress. In addition, ripening APX6-deficient seeds exposed to heat stress displayed reduced germination vigor. This, together with the increased abundance of APX6 during late stages of maturation, indicates that APX6 activity is critical for the maturation-drying phase. Metabolic profiling revealed an altered activity of the tricarboxylic acid cycle, changes in amino acid levels, and elevated metabolism of abscisic acid (ABA) and auxin in drying apx6 mutant seeds. Further germination assays showed an impaired response of the apx6 mutants to ABA and to indole-3-acetic acid. Relative suppression of abscisic acid insensitive3 (ABI3) and ABI5 expression, two of the major ABA signaling downstream components controlling dormancy, suggested that an alternative signaling route inhibiting germination was activated. Thus, our study uncovered a new role for APX6, in protecting mature desiccating and germinating seeds from excessive oxidative damage, and suggested that APX6 modulate the ROS signal cross talk with hormone signals to properly execute the germination program in Arabidopsis.

Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines.

Colletotrichum coccodes (Wallr.) S. Hughes, the causal agent of black dot on potato and anthracnose on tomato, reduces yield and crop quality. We explored the role of secreted pectate lyase (PL), a cell wall-degrading enzyme, in the aggressiveness of C. coccodes. In vitro-cultivated highly aggressive isolates secreted immunologically detectable PL levels 6 h after transfer to secondary medium versus 12 h for mildly aggressive isolates, suggesting that secreted PL is a virulence factor. The gene encoding PL, CcpelA, was cloned and used for the genetic manipulation of highly (US-41 and Si-72) and mildly (Si-60) aggressive isolates. CcpelA gene-disrupted mutants showed reduced aggressiveness towards tomato fruits and impaired PL secretion and extracellular activity. Conversely, overexpression of CcpelA in the Si-60 isolate increased its aggressiveness and PL secretion. Comparison of CcpelA cloned from isolates US-41 and Si-60 revealed that both encode identical proteins, but differ in their promoters. Bioinformatics analysis for cis-acting elements suggested that the promoters of the US-41 and Si-60 isolates contain one and no AreA-binding site (GATA box), respectively. AreA has been suggested to be involved in fungal aggressiveness; therefore, CcpelA may be a key virulence factor in C. coccodes pathogenicity, and the differences in isolate aggressiveness might result from promoter activity. Quantitative reverse transcriptase-polymerase chain reaction analyses confirmed the higher level of CcpelA transcript in isolate US-41 versus Si-60.

Extracts of Inula viscosa Control Downy Mildew of Grapes Caused by Plasmopara viticola.

ABSTRACT The oily paste extracts of Inula viscosa leaves made with organic solvents were effective in controlling downy mildew caused by Plasmopara viticola in detached leaf tissues of grapes in growth chambers. Thin-layer chromatography of such extracts revealed the presence of 11 Rf regions of which four contained highly effective compounds against the disease. Two major inhibitory compounds, each comprising 10.6% of the total paste weight, were identified as tomentosin and costic acid. An emulsified concentrate formulation of the oily paste extracts provided excellent activity against the disease in the field. The effective dose (concentration) required for 90% control of the disease in treated shoots in the field was below 0.125% (paste in water). No seasonal fluctuations were observed in the control efficacy of six extracts made from I. viscosa leaves harvested during the period of May to July. In whole vines, treated and artificially inoculated, the paste concentration required for 90% control of the disease ranged between 0.30 to 0.37%, whereas in naturally infected vines it was 0.58%. It appears that I. viscosa is a valuable source for fungicidal preparations against downy mildew of grapes. This is the first report on the control of a disease with I. viscosa extracts under field conditions.

Control of Meloidogyne javanica by Formulations of Inula viscosa Leaf Extracts.

Inula viscosa is a perennial plant that is widely distributed in Mediterranean countries. Formulations of I. viscosa extracts were tested for their effectiveness in control of Meloidogyne javanica in laboratory, growth chamber, microplot, and field experiments. Oily pastes were obtained by extraction of dry leaves with a mixture of acetone and n-hexane or n-hexane alone, followed by evaporation of the solvents. Emulsifiable concentrate formulations of the pastes killed M. javanica juveniles in sand at a concentration of 0.01% (paste, w/w) or greater and reduced the galling index of cucumber seedlings as well as the galling index and numbers of nematode eggs on tomato plants in growth chamber experiments. In microplot experiments, the hexane-extract formulation at 26 g paste/m(2) reduced nematode infection on tomato plants in one of two experiments. In a field experiment, a reduction of 40% in root galling index by one of two formulations was observed on lettuce plants. The plant extracts have potential as a natural nematicide, although the formulations need improvement.