Abstract
Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.
Highlights
Self-incompatibility (SI) is an elaborate mechanism that promotes outcrossing and maintains genetic diversity in many flowering plants [1]
The S-locus encodes the stigma determinant of SI, the S-locus receptor kinase gene (SRK), which is a membrane-anchored Ser/Thr kinase localized in the plasma membrane of stigmatic papilla cells [3], and the pollen determinant of SI, a small secreted peptide localized in the pollen coat, which is known as S-locus cysteine-rich protein (SCR) [4] or the S-locus protein 11 (SP11) [5]
We show that transgenic plants generated by RNA interference (RNAi)-based silencing and CRISPR/Cas9-based gene editing of BnaMLPKs exhibit partial and complete breakdown of SI, respectively
Summary
Self-incompatibility (SI) is an elaborate mechanism that promotes outcrossing and maintains genetic diversity in many flowering plants [1]. The plasma-membrane-tethered M-locus protein kinase (MLPK) is thought to interact with the activated SRK [10]. Another SRK interactor is the arm-repeat-containing protein ARC1, an E3 ubiquitin ligase [11], that can ubiquitinate Exo70A1 and direct this putative component of the exocyst for degradation by the proteasome [12]. The involvements of THL1/2, MLPK, ARC1, and Exo70A1 in SI have been reported only in a subset of Brassica species Their roles remain controversial [16,17] given studies in transgenic Arabidopsis thaliana self-incompatible SRK/SCR plants that found no evidence for the proposed roles of these genes in SI [16,18]
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