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
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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.
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