Mutations adjacent to the nucleotide-binding cleft of Arabidopsis thaliana ACTIN7 confer resistance to the actin-disrupting compound latrunculin B

This is the authors' abstract. We don't add key points for this paper.

A forward genetic screen identified a dominant-negative Arabidopsis thaliana mutant resistant to growth inhibition caused by the actin-disrupting compound latrunculin B (LatB). Map-based cloning combined with whole-genome sequencing revealed that the mutant referred to here as lbr1 for L at B r esistant1 had a point mutation in the AT5G09810 gene, which encodes the vegetative actin (ACT) isoform ACT7. The cytosine to thymine mutation in the second exon of ACT7 of lbr1 led to substitution of proline to serine at position 34 (P34S) adjacent to the nucleotide-binding cleft of the ACT7 protein. Confirmation that ACT7 is the causal gene for the lbr1 phenotype was achieved through transgenic complementation with ACT7 wild type ( ACT7 WT ) and ACT7 P34S ( ACT7 P34S ) constructs. ACT7 P34 also rescued the seedling developmental defects and conferred partial resistance to LatB in the recessive act7-5 mutant. Furthermore, expressing a P34S mutation in ACT2 (ACT2 P34S ), another vegetative ACT isoform, conferred partial LatB resistance to wild type. Finally, site-directed mutagenesis of ACT7 amino acid residues forming putative hydrogen bonds with LatB, based on yeast and mammalian actin docking and structural analyses, reveals domains adjacent to the actin nucleotide-binding cleft crucial for LatB’s effects on the plant actin cytoskeleton. Highlight Characterization of the dominant-negative lbr1 mutant uncovers amino acid residues in the actin protein crucial for latrunculin’s mechanism of action in plants.