The tomato Cf-9 gene confers resistance to races of the fungal pathogen Cladosporium fulvum expressing the Avr9 gene. cDNA amplified fragment length p

The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived

In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9–dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca²⁺ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9–dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.

Warm season N₂-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two ‘additional’ enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in the feedback inhibition of N₂-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. A discussion of the potential roles for ureides and manganese in the feedback inhibition of N₂-fixation under water limitation is presented. The existing data are examined in relation to potential changes in both aerial carbon and nitrogen supply under water deficit.

The tomato Cf-9 gene confers resistance to races of Cla-dosporium fulvum expressing the corresponding avirulence gene Avr9. The availability of transgenic tobacco lines carrying Cf-9, and the well-characterized 28 amino acid Avr9 elicitor, make this an excellent system to study resistance gene function. In this paper we establish tobacco suspension cultures derived from transgenic tobacco plants containing Cf-9, as well as from plants without Cf-9. Cultures derived from Cf9 tobacco produce active oxygen species (AOS) within 5 min of treatment with pure or synthetic Avr9. This enabled us to perform biochemical and pharmacological analysis in cell culture of the very earliest events in resistance gene function. In addition to AOS production, an increase in oxygen uptake was detected in the Avr9-treated Cf9 cells. Both phenomena were inhibited by low concentrations of diphenyleneiodonium (DPI). Additional pharmacological inhibitor studies suggest that uptake of calcium, activation of protein kinases, and probably phospholipase A₂ (PLA₂) activity are intermediates in the Cf-9- and Avr9-dependent signaling pathway that leads to AOS production. Interestingly, those defense responses did not result in plant cell death.

Summary: The Cf‐9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf‐9 function, we tagged the Cf‐9 protein with a triple myc epitope at either the amino‐ or carboxy‐terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf‐9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c‐myc antibodies, c‐myc : Cf‐9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf‐9 protein is highly glycosylated. Plasma membranes were isolated using two‐phase partitioning, and c‐myc : Cf‐9 was enriched in these fractions, indicating that Cf‐9 is a plasma membrane protein. This was confirmed by silver‐enhanced immunogold labelling of tobacco protoplasts carrying the amino‐terminal c‐myc tag; a higher labelling density was observed on the surface of protoplasts derived from c‐myc : Cf‐9 tobacco compared to untransformed control. The presence of Cf‐9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.