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

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 and whole-genome sequencing revealed that the mutant referred to here as lbr1 for LatB  resistant1 had a point mutation in the AT5G09810 gene, which encodes the vegetative actin (ACT) isoform ACT7. The cytosine to thymine mutation in exon two of lbr1 ACT7 led to substitution of proline to serine at position 34 (P34S) adjacent to the nucleotide-binding cleft of the ACT7 protein. Transgenic complementation with ACT7 wild type (ACT7WT) and ACT7 P34S (ACT7P34S) constructs confirmed that ACT7 is the causal gene for the lbr1 phenotype. ACT7P34S also rescued the seedling developmental defects and conferred partial resistance to LatB in recessive act7-5 mutants. Furthermore, expressing a P34S mutation in ACT2 (ACT2P34S), another vegetative ACT isoform, conferred partial LatB resistance to wild type. However, lbr1 roots were not resistant to other treatments that inhibit growth. 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.