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 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.

Generation of Transgenic Self-Incompatible Arabidopsis thaliana Shows a Genus-Specific Preference for Self-Incompatibility Genes

“…The ability of the SCRb-SRKb transgenes to restore self-incompatibility in the C24 ecotype (i.e., no ARC1) supports the idea that C24 plants might employ other unknown self-incompatibility signaling players; for example, a strong calcium flux mediated by glutamate receptor-like channels has been shown to occur in self-incompatible C24 stigmas [33,35,40]. In addition, while MLPK is required for self-incompatibility both in B. rapa and B. napus [17,18], an A. thaliana homolog of MLPK (APK1b) was found to be unfunctional in regulating the transient self-incompatibility response observed in SCRb-SRKb Col-0 lines [42]. Finally, there are differences in the cellular responses that take place in the stigmatic papillae of Brassica and Arabidopsis species, such as autophagy and the trafficking of vesicles and multivesicular bodies [16,61].…”

“…These observations indicate that the transfer of self-incompatibility into A. thaliana is based on the phylogenetic relationships with the transgene donor, and it would be good to verify this by testing other combinations of SP11/SCR and SRK alleles from different Brassicaceae species. Since the Brassica-specific MLPK gene has been proven to be necessary for self-incompatibility in Brassica species [17,18], it would be worth transforming B. napus SCR-SRK-MLPK-ARC1 all together into A. thaliana to determine if MLPK is the missing component for reconstructing the Brassica self-incompatibility pathway. As well, the reciprocal experiment of transferring Arabidopsis SCR and SRK genes into self-compatible B. napus would be quite interesting in order to see if the Arabidopsis SCR and SRK genes can restore self-incompatibility in a Brassica species, or if the diversification of the self-incompatibility pathway extends in both directions.…”

“…SP11/SCR is recognized by SRK as a ligand in an S-haplotype-specific manner, and SRK is then autophosphorylated to induce various signaling cascades [13,14].Models of the downstream self-incompatibility signaling pathways have been proposed in Brassica species [15,16]. The M locus protein kinase (MLPK) was identified as a positive regulator of self-incompatible response, since the B. rapa mlpk mutant lost its self-incompatibility phenotype and knocking out the B. napus MLPK genes by CRISPR/Cas9 completely knocked out the self-incompatibility response [17,18]. MLPK is tethered on the plasma membrane and can be phosphorylated by SRK by forming a complex with SRK to mediate signal transduction in self-incompatibility response [19,20].…”

EbARC1, an E3 Ubiquitin Ligase Gene in Erigeron breviscapus, Confers Self-Incompatibility in Transgenic Arabidopsis thaliana

“…Recent research on B. napus reported that the SI response was partially broken down in the bnmlpk mutant due to downregulated expression of BnSRK and BnARC1 [43]. Overexpression of the GATA transcription factor BnA5.ZML1 could partially breakdown the SI response in B. napus by indirectly regulating SRK and ARC1 expression [44]. In the future, using EbARC1 as bait to screen a library will help to identify new SI-related proteins and describe the regulatory network of SI in E. breviscapus.…”

Development ofArabidopsis thalianatransformants showing the self-recognition activity ofBrassica rapa

“…Since knockout mutation of the MLPK gene in self-incompatible Brassica napus lines using the CRISPR/Cas9 technology caused defects in SI [35], and MLPK proteins in Escherichia coli were phosphorylated by recombinant SRK kinase domain proteins [36], MLPK is considered as a substrate of SRK and to function in SI. Therefore, to examine whether the expression of BrMLPK, BrSRK-9, and BrSCR-9 in A. thaliana plants causes SI, we introduced the AtS1 pro :BrMLPK construct into BrSRK-9+BrSCR-9 A. thaliana line #1, resulting in 13 transformants.…”