Simulation of Fungal-Mediated Cell Death by Fumonisin B1 and Selection of Fumonisin B1-Resistant (fbr) Arabidopsis Mutants

Abstract

Fumonisin B1 (FB1), a programmed cell death-eliciting toxin produced by the necrotrophic fungal plant pathogen Fusarium moniliforme, was used to simulate pathogen infection in Arabidopsis. Plants infiltrated with 10 microM FB1 and seedlings transferred to agar media containing 1 microM FB1 develop lesions reminiscent of the hypersensitive response, including generation of reactive oxygen intermediates, deposition of phenolic compounds and callose, accumulation of phytoalexin, and expression of pathogenesis-related (PR) genes. Arabidopsis FB1-resistant (fbr) mutants were selected directly by sowing seeds on agar containing 1 microM FB1, on which wild-type seedlings fail to develop. Two mutants chosen for further analyses, fbr1 and fbr2, had altered PR gene expression in response to FB1. fbr1 and fbr2 do not exhibit differential resistance to the avirulent bacterial pathogen Pseudomonas syringae pv maculicola (ES4326) expressing the avirulence gene avrRpt2 but do display enhanced resistance to a virulent isogenic strain that lacks the avirulence gene. Our results demonstrate the utility of FB1 for high-throughput isolation of Arabidopsis defense-related mutants and suggest that pathogen-elicited programmed cell death of host cells may be an important feature of compatible plant-pathogen interactions.

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

“…Approximate 12 000 independent lines were screened for fbr mutants as described [20,31]. Briefly, pooled T2 seeds (20 lines/pool; approximate 10 seeds/line) were germinated and grown on MS medium containing 1 µM FB 1 for 1-2 weeks.…”

“…In higher plants, several studies suggested that fumonisin B 1 (FB 1 ) and Alternaria alternate lycopersici (AAL) toxin, two natural compounds that specifically inhibit ceramide synthase activity, cause apoptotic cell death in various species [15][16][17][18][19][20]. In Arabidopsis, FB 1 -induced PCD depends on multiple signaling pathways, including those of jasmonate, ethylene, and salicylic acid [19].…”

Identification of a Consensus DNA-Binding Site for the Arabidopsis thaliana SBP Domain Transcription Factor, AtSPL14, and Binding Kinetics by Surface Plasmon Resonance

“…The A. thaliana FB1-resistant (fbr6) mutant was originally identified by selecting for mutants capable of growth and development on media containing the fungal toxin fumonisin B1 (FB1). FB1 disrupts sphingolipid metabolism in eukaryotes (38) and induces PCD, dependent on transcription, translation, reversible protein phosphorylation, light, and hormone signaling pathways in A. thaliana (10,11). In addition to resistance to FB1, the fbr6 mutant also exhibits altered plant architecture, including elongated petioles and enhanced leaf margin serration (9).…”

“…We found significant variation with flow rate, suggesting a mass transfer limitation (data not shown). Varying concentrations of recombinant protein (5,10,15,20,25,30, and 50 nM) were injected at the higher flow rate (75 µL/min). The SPR response data were then fit to various models using BIAevaluation 3.0 software (Biacore).…”

“…Despite the essential nature of PCD, large gaps remain in our knowledge of the mechanistic details and molecular components controlling plant PCD. Therefore, to identify novel genes that might participate in plant PCD, we developed a mutant selection scheme for the model plant Arabidopsis thaliana using a PCD-inducing fungal toxin fumonisin B1 (FB1), to identify FB1-resistant (fbr) mutants (9)(10)(11). Misexpression of the AtSPL14 gene in the fbr6 mutant confers the ability to proliferate in the presence of FB1 and modifies normal plant architecture, linking the insensitivity to cell death to altered plant development (9).…”