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

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.


Introduction
Self-incompatibility (SI) is an elaborate mechanism that promotes outcrossing and maintains genetic diversity in many flowering plants [1]. In the Brassicacease, SI is sporophytically regulated by a single genetic locus called the S-locus, at which multiple variants (now known as S haplotype) occur in any one species [2]. 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]. Biochemical studies have shown that SCR/SP11 is the ligand for SRK that are encoded in the same S haplotype [6,7]. This S haplotype-specific receptor-ligand interaction, and the resulting

Sequence Analysis of BnaMLPK Transcripts
Previous studies indicated that two isoforms of BrMLPK and BoMLPK are generated by alternative splicing [10,22]. We wondered if the alternative splicing still exists in BnaMLPKs. To confirm this hypothesis, we cloned the transcript sequences of BnaMLPK in S-70 using primers designed based on two isoforms of BrMLPK (BrMLPKf1 and BrMLPKf2) sequences (Table S2). Two different sequences were detected by Sanger sequencing (Table S1), which were transcribed by BnaA3.MLPK (Table S1; Figure 1A), named BnaA3.MLPKf1 and BnaA3.MLPKf2. BnaA3.MLPKf1 starts to transcribe from the first exon to the last exon, whereas the transcriptome of BnaA3.MLPKf2 initiates at the 475th base of the first intron ( Figure 1A). The sequence length of BnaMLPKf2 transcript is 54 bp longer than that of BnaMLPLf1, which was detected by PCR ( Figure 1B). However, other MLPK paralogues contain one transcript. Therefore, a total of five transcripts were obtained from the four BnaMLPK genes. The domain analysis result indicated that, except for BnaA3.MLPKf2, the other four BnaMLPK proteins contain a typical plant N-myristylation consensus sequence at the N-terminal [23,24] ( Figure 1C). The C-terminal of these five proteins is conserved, which contains a protein kinase domain ( Figure 1D).  Figure S1).

Sequence Analysis of BnaMLPK Transcripts
Previous studies indicated that two isoforms of BrMLPK and BoMLPK are generated by alternative splicing [10,22]. We wondered if the alternative splicing still exists in BnaMLPKs. To confirm this hypothesis, we cloned the transcript sequences of BnaMLPK in S-70 using primers designed based on two isoforms of BrMLPK (BrMLPKf1 and BrMLPKf2) sequences (Table S2). Two different sequences were detected by Sanger sequencing (Table S1), which were transcribed by BnaA3.MLPK (Table S1; Figure 1A), named BnaA3.MLPKf1 and BnaA3.MLPKf2. BnaA3.MLPKf1 starts to transcribe from the first exon to the last exon, whereas the transcriptome of BnaA3.MLPKf2 initiates at the 475th base of the first intron ( Figure 1A). The sequence length of BnaMLPKf2 transcript is 54 bp longer than that of BnaMLPLf1, which was detected by PCR ( Figure 1B). However, other MLPK paralogues contain one transcript. Therefore, a total of five transcripts were obtained from the four BnaMLPK genes. The domain analysis result indicated that, except for BnaA3.MLPKf2, the other four BnaMLPK proteins contain a typical plant N-myristylation consensus sequence at the N-terminal [23,24] ( Figure 1C). The C-terminal of these five proteins is conserved, which contains a protein kinase domain ( Figure 1D).

Phylogenetic and Tissue-Specific Expression Analysis of BnaMLPKs
We explored the evolutionary relationship of MLPK proteins from various species using phylogenetic analysis. Four major clades, Clade A1, Clade A2, Clade B1, and Clade B2, were obtained ( Figure 2A). BnaA3.MLPKf1 and BnaA3.MLPKf2 were in Clade B2, and BnaC3.MLPK was in Clade B1 ( Figure 2A). BnaA4.MLPK and BnaC4.MLPK were in Clade A2 ( Figure 2A). Then, quantitative real-time PCR (qRT-PCR) was performed to analyze the expression pattern of BnaMLPKs in different tissue. Both BnaA3.MLPK and BnaC3.MLPK are enriched in the stigma, but the expression of BnaA3.MLPK is much higher than BnaC3.MLPK ( Figure 2B). The expressions of BnaA4.MLPK and BnaC4.MLPK are very low in all tissues ( Figure 2B). Then, we tested different isoforms of BnaA3.MLPK in different tissues. The results indicate BnaA3.MLPKf2 is enriched in the stigma and petal, which is much higher than BnaA3.MLPKf1 ( Figure 2C). These results demonstrate that BnaA3.MLPK and BnaC3.MLPK might participate in the regulation of SI responses, and BnaA3.MLPKf2 is the major isoform expressed in the stigma.

Phylogenetic and Tissue-Specific Expression Analysis of BnaMLPKs
We explored the evolutionary relationship of MLPK proteins from various species using phylogenetic analysis. Four major clades, Clade A1, Clade A2, Clade B1, and Clade B2, were obtained ( Figure 2A). BnaA3.MLPKf1 and BnaA3.MLPKf2 were in Clade B2, and BnaC3.MLPK was in Clade B1 ( Figure 2A). BnaA4.MLPK and BnaC4.MLPK were in Clade A2 ( Figure 2A). Then, quantitative real-time PCR (qRT-PCR) was performed to analyze the expression pattern of BnaMLPKs in different tissue. Both BnaA3.MLPK and BnaC3.MLPK are enriched in the stigma, but the expression of BnaA3.MLPK is much higher than BnaC3.MLPK ( Figure 2B). The expressions of BnaA4.MLPK and BnaC4.MLPK are very low in all tissues ( Figure 2B). Then, we tested different isoforms of BnaA3.MLPK in different tissues. The results indicate BnaA3.MLPKf2 is enriched in the stigma and petal, which is much higher than BnaA3.MLPKf1 ( Figure 2C). These results demonstrate that BnaA3.MLPK and BnaC3.MLPK might participate in the regulation of SI responses, and BnaA3.MLPKf2 is the major isoform expressed in the stigma.

RNAi Knockdown of BnaMLPKs Partially Suppressed SI Response in B. napus
Though MLPK is a positive regulator of the SI response in B. rapa, the specific roles in B. napus remain unclear. To validate the function of BnaMLPKs in S-70 B. napus, we attempted to generate RNAi transgenic lines of BnaMLPKs using the stigma-specific SLR1 promoter to drive the expression of two hairpin RNA interference (hpRNAi) constructs [25], which were used to suppress the expression of BnaA3.MLPK/BnaC3.MLPK (RM1) and BnaA4.MLPK /BnaC4.MLPK (RM7) ( Figure S2). The expressions of all four BnaMLPKs were partially suppressed in four RNAi transgenic lines, ranging from 25% to 60% ( Figure 3A-D). Phenotypic analysis showed that the seed setting was partially rescued in bnmlpk-RNAi lines ( Figure 3E), indicating that BnaMLPKs positively regulate SI response.
detected by real-time quantitative reverse transcription PCR (qPCR). The actin gene was used as control. (C) Tissue-specific expression of the two isoforms BnaA3.MLPKf1 and BnaA3.MLPKf2 in different tissues of B. napus.

RNAi Knockdown of BnaMLPKs Partially Suppressed SI Response in B. napus
Though MLPK is a positive regulator of the SI response in B. rapa, the specific roles in B. napus remain unclear. To validate the function of BnaMLPKs in S-70 B. napus, we attempted to generate RNAi transgenic lines of BnaMLPKs using the stigma-specific SLR1 promoter to drive the expression of two hairpin RNA interference (hpRNAi) constructs [25], which were used to suppress the expression of BnaA3.MLPK/BnaC3.MLPK (RM1) and BnaA4.MLPK /BnaC4.MLPK (RM7) ( Figure S2). The expressions of all four BnaMLPKs were partially suppressed in four RNAi transgenic lines, ranging from 25% to 60% ( Figures 3A-D). Phenotypic analysis showed that the seed setting was partially rescued in bnmlpk-RNAi lines ( Figure 3E), indicating that BnaMLPKs positively regulate SI response.  The expression analysis of BnaMLPKs in hpRNAi transgenic lines. The total RNA was extracted from at least thirty stigmas for each pollination treatment. Data represent the average of three technical replicates (±SE). Similar expression results were acquired with three biological replicates. The actin gene was considered as the control reference. RM1-109 and RM1-127 indicate the two different positive plants from the RM1 hpRNAi construct. RM7-6 and RM7-56 denote the two transgenic plants from the RM7 hpRNAi construct. All four genes of MLPK were down-regulated in un-pollinated stigmas of RM1 and RM7 transgenic lines. The un-pollinated wild type S-70 was used as control. (E) Images of mature siliques from pistils of S-70, RM1-127, RM1-109, cm1-18, RM7-6, and RM7-56 lines following self-pollination. S-70 and cm1-18 were used as controls. Scale bar = 1 cm.

Knockout Mutant of BnaMLPKs Created Using CRISPR/Cas9 System Completely Breaks Down SI Response in B. napus
The SI response was partially suppressed by knock down expression of BnaMLPKs in RNAi lines, but which paralogue is the dominant gene remained unclear. To address this question, two sgRNAs were designed to target the conserved sequences among the four paralogous genes ( Figure S3A,B). By genetic transformation into S-70, we obtained six positive plants that were further confirmed by PCR using Cas9-specific primers ( Figure S3C). Next, Sanger sequencing was used to assess the editing efficiency. The four BnaMLPK genes were examined in the six transgenic plants. As a result, the mutation frequency at the sgRNA1 target site ranged from 83.3% to 100% for each of the four genes, and at sgRNA2 target site, ranged from 66.7% to 100.0% (Table 3). Among all types of mutations, 16.7% (4/24) were deletions, 12.5% (3/24) were insertions, and 13.9% (17/24) were combined mutations ( Figure S4). Both alleles for each gene might be mutated by CRISPR/Cas9, which could produce five genotypes: homozygote, bi-allele, heterozygote, chimera, and WT. To estimate the proportion of each genotype among the transgenic lines, we detected the mutation type of each targeted site using sequencing. All amplicons were analyzed by inserting them into a TA vector and sequencing 10 individual clones for each of the 48 amplicons. The genotype data are summarized in Table 4. The genotyping results showed that 10.4% (5/48) sites were homozygous and 43.8% (21/48) sites were bi-allelic. Thus, a total of 54.2% sites had defects in both alleles ( Table 4). The frequencies of heterozygotes and chimeras were 10.4% (5/48) and 25.0% (12/48), respectively. No mutations were found in 8.3% (4/48) of the sites.

Expression of SI-Related Genes Changed in bnamlpk Mutant
Previous studies reported that SRK, ARC1, THL1/2, Exo70A1 and GLO1 genes are mainly involved in the SI of B. napus [3,9,11,12,14,26]. So, we wanted to know whether the SC phenotype of bnamlpk is caused by changing the expression of SI-related genes. qRT-PCR was used to detect the relative expression levels of these genes in the stigmas of bnamlpk mutant and wild type (S-70) after un-pollination (up) and self-pollination (sp). Without pollination, SRK and ARC1 were suppressed in bnamlpk compared to the S-70 (Figures 6A,B). After self-pollination, these two genes were downregulated (SRK: ~1.4 fold, ARC1: ~1.2 fold) in S-70, but not in bnamlpk (Figures 6A,B). The expression of GLO1 significantly decreased (~2.3 fold) in S-70 after self-pollination ( Figure 6C). GLO1 was slightly up-regulated (~1.3 fold) in bnamlpk without self-pollination ( Figure 6C). The expression of the

Expression of SI-Related Genes Changed in bnamlpk Mutant
Previous studies reported that SRK, ARC1, THL1/2, Exo70A1 and GLO1 genes are mainly involved in the SI of B. napus [3,9,11,12,14,26]. So, we wanted to know whether the SC phenotype of bnamlpk is caused by changing the expression of SI-related genes. qRT-PCR was used to detect the relative expression levels of these genes in the stigmas of bnamlpk mutant and wild type (S-70) after un-pollination (up) and self-pollination (sp). Without pollination, SRK and ARC1 were suppressed in bnamlpk compared to the S-70 ( Figure 6A,B). After self-pollination, these two genes were down-regulated (SRK:~1.4 fold, ARC1: 1.2 fold) in S-70, but not in bnamlpk ( Figure 6A,B). The expression of GLO1 significantly decreased (~2.3 fold) in S-70 after self-pollination ( Figure 6C). GLO1 was slightly up-regulated (~1.3 fold) in bnamlpk without self-pollination ( Figure 6C). The expression of the other genes showed almost no differences between the mutant and wild-type with or without self-pollination ( Figure 6D-F other genes showed almost no differences between the mutant and wild-type with or without selfpollination ( Figure 6D-F). These results suggest that BnaMLPKs might regulate the expression of SRK, ARC1, and GLO1 to control the SI response in B. napus.

BnaMLPK is a Positive Regulator of SI Response in B. napus
SRK-mediated signaling has been reported in Brassica species [13,[27][28][29]. MLPK, a plasma membrane-tethered M-locus protein kinase, is phosphorylated by SRK and required for the SI signaling pathway in B. rapa [10,19]. However, the homolog of BrMLPK in Arabidopsis plays no role in the SI response in self-incompatible transgenic A. thaliana (SRKb-SCRb) [20]. Therefore, more evidence is required to clarify the roles of MLPK in the SI response of Brassicacea. In this study, knockdown and knock-out of BnaMLPKs was performed in self-compatible B. napus S-70, which broke down the SI response, suggesting that BnaMLPKs are positive regulators of the SI response.
The sgRNA that targeted the conserved sequences among the four paralogous genes in the CRISPR/Cas9 system could in theory generate single, double, triple, and quadruple mutants. However, no single mutant bnamlpk line was separated in the present transgenic materials, probably due to the high genome editing efficiency. The cm34-9 line showed a SC phenotype (Figure 4), the genotypes of BnaA3.MLPK and BnaC3.MLPK were homozygous mutations, and BnaA4.MLPK and BnaC4.MLPK were heterozygous mutations. Based on the expression pattern and phenotype of the mutant, we propose that BnaA3.MLPK and BnaC3.MLPK are the functional genes of SI in B. napus. However, which paralogue gene plays the dominant function in the SI response remains unknown. The single mutants of BnaA3.MLPK and BnaC3.MLPK could be isolated in the next generation, which would help us to answer this question.

BnaMLPK is a Positive Regulator of SI Response in B. napus
SRK-mediated signaling has been reported in Brassica species [13,[27][28][29]. MLPK, a plasma membrane-tethered M-locus protein kinase, is phosphorylated by SRK and required for the SI signaling pathway in B. rapa [10,19]. However, the homolog of BrMLPK in Arabidopsis plays no role in the SI response in self-incompatible transgenic A. thaliana (SRKb-SCRb) [20]. Therefore, more evidence is required to clarify the roles of MLPK in the SI response of Brassicacea. In this study, knock-down and knock-out of BnaMLPKs was performed in self-compatible B. napus S-70, which broke down the SI response, suggesting that BnaMLPKs are positive regulators of the SI response.
The sgRNA that targeted the conserved sequences among the four paralogous genes in the CRISPR/Cas9 system could in theory generate single, double, triple, and quadruple mutants. However, no single mutant bnamlpk line was separated in the present transgenic materials, probably due to the high genome editing efficiency. The cm34-9 line showed a SC phenotype (Figure 4), the genotypes of BnaA3.MLPK and BnaC3.MLPK were homozygous mutations, and BnaA4.MLPK and BnaC4.MLPK were heterozygous mutations. Based on the expression pattern and phenotype of the mutant, we propose that BnaA3.MLPK and BnaC3.MLPK are the functional genes of SI in B. napus. However, which paralogue gene plays the dominant function in the SI response remains unknown. The single mutants of BnaA3.MLPK and BnaC3.MLPK could be isolated in the next generation, which would help us to answer this question.

Alternative Splicing Involved in MLPK-Regulated SI in B. napus
Alternative splicing (AS) of eukaryotic transcripts is a mechanism that enables cells to generate vast protein diversities from a limited number of genes. Studies in several plants have indicated that tissue-specific AS mediates tissue differentiation and promotes specialized characteristics [30]. Alternative splicing at first exons (AFEs) has been well studied in mammals, which is suggested to contribute to the diversification of gene expression [31]. Transcript analysis showed that two isoforms of BnaA3.MLPK are generated by AFEs. The expression patterns of these two isoforms of BnaA3.MLPK are significantly different, indicating the regulation of AFEs in gene expression.
B. napus (AACC, 2n = 38), as an allotetraploid species, was developed by natural allopolyploidization between B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) [21]. MLPK plays an important role in the SI response of B. rapa [10], but not in transgenic A. thaliana (SRKb-SCRb) [10]. These results imply that the function of MLPK has been differentiated between Arabidopsis and Brassica during evolution [32]. BrMLPK was found to generate two transcripts, BrMLPLf1 and BrMLPKf2, by AFEs, both of which function in SI signal transduction in the stigma of B. rapa [10]. This study also demonstrates that BnaA3.MLPKf1 and BnaA3.MLPKf2 are the major isoforms of BnaA3.MLPK in B. napus. Further experiments are need to determine the functions of the two transcripts, such as a complementary experiment transforming two BnaA3.MLPK transcripts into the bnamlpk mutant by rescuing the SI phenotype.

BnaMLPKs Regulate SI Responses by Influencing the Expression of SCR-SRK Pathway Components
Several genes have been identified in the SRK-SCR pathway [33]. Although the regulatory mechanism of these genes is well-studied at the protein level, little has been reported about their regulatory mechanism at the transcription levels. We found the expressions of SRK and ARC1 were suppressed in bnamlpk before self-pollination ( Figure 6A,B). Down-regulated expressions of SRK and ARC1 prevented the SI signal transduction, resulting in pollen tubes growth and elongation. Without pollination, the self-compatible factor GLO1 was slightly induced in bnamlpk. These results imply that the inhibition of SRK and ARC1 and the maintained high level of GLO1 cause the breaking down of the SI response in bnamlpk. The mechanism through which MLPK regulates the expressions of SRK, ARC1, and GLO1 needs to be further investigated.
Given the similar sequences, expression patterns, and transcriptional processing of BnaMLPK and its B. rapa homologue BrMLPK, the expectation is that the BnaMLPK protein would be required for the SI response of B. napus and would have the features and function previously reported for the BrMLPK protein, i.e., plasma-membrane localization, interaction with the SRK receptor, and involvement in SRK-mediated signaling. Consistent with previous reports, we found that down-regulation or disruption of BnaMLPK caused partial or complete loss of SI, thus confirming a role of BnaMLPK in SI. However, our results show that the self-compatibility phenotype of bnmlpk mutants is associated with a drastic reduction in the level of SRK transcripts in un-pollinated stigmas, a reduction that is known to cause breakdown of SI [34,35]. Although we did not investigate the signaling role of the BnaMLPK protein, we did reveal a novel role of BnaMLPK that had not been reported for BrMLPK. BnaMLPK may act independently of SRK signaling to regulate the transcript levels of genes required for SI. Further analysis is required to understand how this SRK-independent signaling regulatory function is achieved and if it involves transcriptional control in the nucleus by as-yet-unknown transcription factors or post-transcriptional control of transcript stability in the cytoplasm.

Plant Materials and Growth Condition
The self-incompatible B. napus S-70 species was separated from the self-incompatible S-1300, which was acquired by interspecific hybridization between the self-compatible B. napus line Huayou8 and the self-incompatible B. rapa Xishuibai [36]. S-70 and its relative transgenic lines were grown in the transgenic field of Huazhong Agriculture University, Wuhan, China, during B. napus growing season, or cultivated in a growth room under the light intensity of 100 µmol·m −2 s −1 with a 16/8 h light/dark photoperiod at 22 • C.

Sequence Cloning of BnaMLPKs
To clone the genome DNA (gDNA) or CDS region of MLPKs, the B. napus genomic DNA or total RNA was extracted from leaves or stigmas following the CTAB method [37] or plant mini RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Approximately 1 µg of total RNA was used for cDNA synthesis with the Thermo Scientific Revertaid First Strand cDNA Synthesis kit (Thermo, New York, USA). Primers M4CDS-F/M4CDS-R were used to amplify the four paralogues of the MLPK genes (Table S2), followed by standard PCR procedure: 98 • C for 2 min; 98 • C for 20 s, 55 • C for 20 s, 72 • C for 1.5 min, 35 cycles; 72 • C for 10 min; and 25 • C for 5 min. Two primer pairs (Table S2), BrMLPKf1-F/BrMLPKf1-R and BrMLPKf2-F/BrMLPKf2-R, were designed according to the BrMLPKf1/2 cDNA sequence [10,22], which were used to amplify the transcripts of BnaMLPK. PCR was performed using the following procedure: 98 • C for 2 min; 98 • C for 20 s, 55 • C for 20 s, 72 • C for 1 min, 35 cycles; 72 • C for 10 min; and 25 • C for 5 min. All PCR products were purified and ligated into the pMD18-T vector (Takara, Wuhan, China). Subsequently, the positive clones were confirmed via sequencing (TsingKe, Wuhan, China).

Tissue-Specific Expression Analysis of BanMLPKs
Total RNA from different tissues was extracted using a Plant Total RNA Isolation Kit (Sangon Biotech, Shanghai, China, No. SK8631) following the manufacturer's instructions. Approximately 1 µg of total RNA was used for cDNA synthesis using a PrimeScriptTM RT reagent kit (TaKaRa, Tokyo, Japan, Cat#RR047A). A 10 µL total volume of reaction mixture was used for qPCR, which contained 5 µL 2× SYBR Green master mix (Achard et al., 172-5124, BioRad), 0.5 µL 5× diluted cDNA, 0.25 µL of each primer, and 4 µL ddH 2 O. Amplification was performed using a CFX Connect™ system (Bio-rad, USA). The amplification program involved one cycle of 95 • C for 5 min, followed by 50 cycles of 95 • C for 15 s, 60 • C for 20 s, and 72 • C for 20 s. The fluorescent products were detected at the third step of each cycle. The expression level of each gene was calculated using the 2 −∆∆CT method. All analyses were repeated with three biological replicates. The actin gene (Gene-Bank accession no: AF111812) served as the internal control. All primers are listed in Table S2.

Plasmid Construction and B. napus Transformation
The GENE-sgRNA plant expression vectors were constructed following a previously reported method with minor modifications [38]. The target sgRNA sequences were designed using the web server CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR;). Using pCBC-DT1T2 as the template, two AtU6 promoter-sgRNA-AtU6 terminator cassettes were amplified by PCR using the primers listed in Table S2. The PCR fragments were inserted into pKSE401 by Golden Gate Assembly [39], and confirmed by Sanger sequencing. These vectors were then used for plant transformation.
The hairpin RNA interference (hpRNAi) construct was reformed from the pCAMBIA2300 vector. The reformed construct contains a stigma-specific SLR1 promoter [40], an intron, and a fragment of poly A ( Figure S2). The sense fragment with nucleotides 908 to 1107 from the coding sequence of BnaA3.MLPK (CDS 908-1107 ), including KpnI at N-terminal and SacI at the C-terminal, was amplified and inserted between the intron and the poly A. Then, the reverse complement fragment of CDS 908-1107 with PstI at the N-terminal and XbaI at the C-terminal was developed and inserted between the SLR1 promoter and the intron. Similarly, the sense fragment with nucleotides 1007 to 1206 from the coding sequence of BnaC4.MLPK (CDS 1007-1206 ), including KpnI at the N-terminal and SacI at theC-terminal, was amplified and inserted between the intron and the poly A. Then, the reverse complement fragment of CDS 1007-1206 with PstI at the N-terminal and XbaI at the C-terminal was developed and inserted between the SLR1 promoter and the intron. The reconstructed vector was transformed into Agrobacterium GV3103 for plant transformation.

Mutant Screening and Validation of Genome Editing
To analyze the mutations caused by CRISPR/Cas9, genomic DNA was extracted from leaves using the CTAB method [37]. The flanking sequences of the CRISPR target sites were amplified by PCR using gene-specific primers (Table S2). Then, most of the amplicons were directly sequenced. To decode mutations, the online tool DSDecode (http://dsdecode.scgene.com/;) was used for chromatogram decoding. The sequences files and the reference gene sequences were uploaded to the server and analyzed using default settings. The results were aligned with the reference sequences to ensure that the mutations were in the sgRNA targeted sites. For complex mutations, the amplicons were first sub-cloned into the pGEM18-T vector (Cat#A3610, Takara, Tokyo, Japan), and about 10 clones for each amplicon were individually sequenced. To analyze the RNAi lines, we used specific primers to detect the positive lines including SLR1-RNAi function fragment (Table S2). All the transgenic plants, including CRISPR/Cas9 mutants, were transferred to soil for further analysis.

Aniline Blue Staining Assay
The aniline blue staining was performed as reported with minor modifications [42]. After 16 h of pollination, the pistils were collected from the flowers, and immediately fixed in 3:1 ethanol/ glacial acetic acid for 2 h. The samples were further softened with 1 M NaOH at 60 • C for 1.5 h and then washed three times with distilled water. Pollen tubes were then stained by 0.1% decolorized aniline blue (pH9-11, in 0.1 M K 3 PO 4 ) and placed in the dark for 6 h. The stained samples were observed under a fluorescence microscope (Ax10, Zeiss, Berlin, Germany) equipped with a UV filter set.

Self-Incompatibility Assay
Self-incompatibility phenotypic observation was performed [36]. At the flowering stage, the major inflorescence and two or three secondary ramifications were bagged for self-pollination after removing all blooming flowers artificially. The bags were shaken gently every two days to ensure sufficient self-pollination. The transgenic or wild-type flowers were cross pollinated after emasculation one day before anthesis. After two weeks, the bags were removed to allow the growth of seeds. The phenotypes were observed after maturation of seeds.

Conclusions
In this study, we cloned the BrMLPK homologous gene in B. napus, analyzed the transcripts in stigmas, and revealed their expression patterns in different tissues. We also demonstrated that BnaMLPKs positively regulate the SI response in the B. napus. Combined with the results from previous SI studies, we speculate that MLPK mediates the SI signaling pathway through regulating the expression of SRK, ARC1, and GLO1. These findings not only strengthen our understanding of the molecular roles of MLPK in the SI response of B. napus, but also directly contribute to the future study of the molecular mechanisms of SI in Brassica.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/13/3303/s1, Figure S1. RNA-silencing constructs used in this study; Figure S2. Information of CRISPR/Cas9 construct of editing BnMLPK; Figure S3. The editing information of BnMLPK in T 0 generation mutants; Figure S4. The editing information of BnaMLPKs in T0 generation mutants; Table S1. The gDNA and CDS sequence of BnMLPK; Table S2. Primers used in the study.