Reverse genetics analysis of the 55-kDa B regulatory subunit of 2A serine/threonine protein phosphatase (PP2A) related to self-incompatibility in Chinese cabbage

Abstract

The type 2A serine/threonine protein phosphatases (PP2As) are key components in regulating signal transduction and controlling cell metabolism. In particular, PP2A has a role in pollen-pistil interaction during pollination in Brassica. In this study, reverse genetics screening was used to obtain a T-DNA-inserted Brassica rapa mutant line with the self-compatible phenotype. The type 2A PP2A 55-kDa B regulatory subunit gene (PR55/B) in this mutant was knocked out by T-DNA insertion, and its expression level was decreased, which led to self-compatible phenotypes, such as seed setting by flower self-pollination. For functional analysis of this self-compatible mutant line, the full-length PR55/B gene related to the self-incompatibility mechanism was isolated and the down-regulation vector (pPPi) was constructed for introduction into Chinese cabbage. Chinese cabbage lines transformed with pPPi showed significantly decreased PR55/B mRNA accumulation, which yielded self-compatible phenotypes. These transgenic lines were found to set pods and seeds by the self-pollination in both buds and flowers. Down-regulation of PP2A, which is related to self-incompatibility signal transduction, led to higher levels of the phosphorylated S-locus receptor kinase (SRK) substrate, which phosphorylated SRK blocks activation of armadillo-repeat-containing 1 (ARC1). Thus, the self-incompatibility response by ARC1 was not activated, and this led to the self-compatible response. Finally, PP2A is proposed to be a significant factor in self-incompatibility of Chinese cabbage.