Functional Analysis of M-Locus Protein Kinase Revealed a Novel Regulatory Mechanism of Self-Incompatibility in Brassica napus L.

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 <i>Brassica napus</i> is not well understood. Here, we report that the M-locus protein kinase (<i>MLPK</i>) <i>BnaMLPKs</i>, the functional homolog of <i>BrMLPKs</i> in <i>Brassica rapa</i>, controls SI in <i>B. napus</i>. We identified four paralogue <i>MLPK</i> genes in <i>B. napus</i>, including <i>BnaA3.MLPK</i>, <i>BnaC3.MLPK</i>, <i>BnaA4.MLPK</i>, and <i>BnaC4.MLPK</i>. Two transcripts of <i>BnaA3.MLPK</i>, <i>BnaA3.MLPKf1</i> and <i>BnaA3.MLPKf2</i>, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that <i>BnaA3.MLPK</i>, especially <i>BnaA3.MLPKf2</i>, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of <i>BnaMLPKs</i> in <i>B. napus</i> 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 <i>BnaMLPKs</i> in regulating the SI response of <i>B. napus</i>. We found that the expression of SI positive regulators S-locus receptor kinase (<i>SRK</i>) and Arm-Repeat Containing 1 (<i>ARC1</i>) are suppressed in <i>bnmlpk</i> mutant, whereas the self-compatibility (SC) element Glyoxalase I (<i>GLO1</i>) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of <i>MLPK</i> in the SI of <i>B. napus.</i>