Glutamate Receptor-like (GLR) Family in Brassica napus: Genome-Wide Identification and Functional Analysis in Resistance to Sclerotinia sclerotiorum

Plant glutamate receptor-like channels (GLRs) are homologs of animal ionotropic glutamate receptors. GLRs are critical in various plant biological functions, yet their genomic features and functions in disease resistance remain largely unknown in many crop species. Here, we report the results on a thorough genome-wide study of the GLR family in oilseed rape (Brassica napus) and their role in resistance to the fungal pathogen Sclerotinia sclerotiorum. A total of 61 GLRs were identified in oilseed rape. They comprised three groups, as in Arabidopsis thaliana. Detailed computational analyses, including prediction of domain and motifs, cellular localization, cis-acting elements, PTM sites, and amino acid ligands and their binding pockets in BnGLR proteins, unveiled a set of group-specific characteristics of the BnGLR family, which included chromosomal distribution, motif composition, intron number and size, and methylation sites. Functional dissection employing virus-induced gene silencing of BnGLRs in oilseed rape and Arabidopsis mutants of BnGLR homologs demonstrated that BnGLR35/AtGLR2.5 positively, while BnGLR12/AtGLR1.2 and BnGLR53/AtGLR3.2 negatively, regulated plant resistance to S. sclerotiorum, indicating that GLR genes were differentially involved in this resistance. Our findings reveal the complex involvement of GLRs in B. napus resistance to S. sclerotiorum and provide clues for further functional characterization of BnGLRs.

Oilseed rape (Brassica napus) is one of the most important oil crops, yet the functions of GLRs in oilseed rape disease resistance remain unclear.In this study, we performed comprehensive computational and functional analyses of BnGLRs to clarify their genomewide constitution and sequence characteristics and their role in plant disease resistance against Sclerotinia sclerotiorum, one of the most important pathogens in oilseed rape.In total, 61 GLR genes were identified from the genome of the oilseed rape cultivar ZS11.Systematical analyses of the phylogeny, chromosomal distribution, domains and motifs, promoter cis-acting elements, pore morphology, post-translational modification (PTM) sites, and amino acid ligands for BnGLRs demonstrated family-conserved and group-specific characteristics of the BnGLR family.Virus-induced gene silencing (VIGS) analyses in oilseed rape and homolog gene mutant analyses in Arabidopsis revealed that BnGLR35 positively, while BnGLR12 and BnGLR53 negatively, modulated plant resistance to S. sclerotiorum.Our findings demonstrate the functional complexity of the GLR gene family in plant disease resistance.

Phylogeny of BnGLR and AtGLR Families
To elucidate the evolution of the BnGLR family, the maximum likelihood phylogenetic tree was generated for 20 AtGLR and 61 BnGLR proteins (Figure 1).Based on the phylogenetic tree, the BnGLR proteins were clearly clustered group-wide with AtGLRs into three major groups as Groups I, II, and III.The size and distribution of the three BnGLR groups were unequal to those of AtGLRs (Figure 1).Group I contained 19 BnGLRs (BnGLR1-BnGLR19) and 4 AtGLRs, Group II carried 17 BnGLRs (BnGLR20-BnGLR36) and 9 AtGLRs, while group III consisted of 25 BnGLRs (BnGLR37-BnGLR61) and 7 AtGLRs.This result demonstrated the significant and group-dependent expansion of BnGLRs in comparison with AtGLRs.
Intriguingly, multiple alignment of the ion channel domain by ClustalW revealed the existence of the conserved motif "SYTANLTS" in the C-terminus of motif 1 among BnGLRs, which was similar but not identical to the known functional motif "SYTANLAA" reported in two rat iGluRs and Rosaceae and Arabidopsis GLRs to be involved in ion exchange or transportation through the plasma membrane (Figure S1).Additionally, 12 amino acids, F ( 9), E (20), F (27,42,44), S (45), R (61), W (67), F (69), S (77), Y (78), and A (80), were identical in this domain of all BnGLRs (Figure S1).Distinct colors indicate different groups of GLRs, Group I in brown, Group II in green, and Group III in yellow.

Domains and Gene Structures of BnGLRs
Domain composition analyses revealed that all BnGLRs contained a ligand-binding domain and an ion channel domain, a bacterial periplasmic substrate-binding protein domain (PBPe) (accession: SM000079), or a low complexity region (LCR) and 2-4 short transmembrane TMDs in "SMART" database, while a periplasmic_binding_protein_type_1 (PBP1) and a PBP2, or, more specifically, a PBP1_GABAb_receptor_plant (accession: cd06366) and a Glur_Plant (accession: cd13686) in the NCBI-CDD database.Remarkably, BnGLR21 carried a B3 domain (Table 1; Figures S2 and S3).Whether it is functional in regulating the transcription of target genes remains to be experimentally confirmed.
To study the GLR gene structure, the distribution of the exons and introns in BnGLR genes was identified.The number of introns ranged from 3 to 7. On average the introns were predicted to be 4.05, 4.47, and 5.52 in Group I, II, and III, respectively, indicating that the BnGLR genes of Group III contained more introns than those of the other groups (Table 1; Figure 3).Additionally, the size of introns also differed significantly in the BnGLR genes of different groups (Figure 3).Together, these results indicate that the gene structure of BnGLR genes is group-specific.

Domains and Gene Structures of BnGLRs
Domain composition analyses revealed that all BnGLRs contained a ligand-binding domain and an ion channel domain, a bacterial periplasmic substrate-binding protein domain (PBPe) (accession: SM000079), or a low complexity region (LCR) and 2-4 short transmembrane TMDs in "SMART" database, while a periplasmic_binding_protein_type_1 (PBP1) and a PBP2, or, more specifically, a PBP1_GABAb_receptor_plant (accession: cd06366) and a Glur_Plant (accession: cd13686) in the NCBI-CDD database.Remarkably, BnGLR21 carried a B3 domain (Table 1; Figures S2 and S3).Whether it is functional in regulating the transcription of target genes remains to be experimentally confirmed.
To study the GLR gene structure, the distribution of the exons and introns in BnGLR genes was identified.The number of introns ranged from 3 to 7. On average the introns were predicted to be 4.05, 4.47, and 5.52 in Group I, II, and III, respectively, indicating that the BnGLR genes of Group III contained more introns than those of the other groups (Table 1; Figure 3).Additionally, the size of introns also differed significantly in the BnGLR genes of different groups (Figure 3).Together, these results indicate that the gene structure of BnGLR genes is group-specific.

Tertiary Structure of BnGLR Proteins
To examine whether BnGLRs were possible transmembrane protein channels, the PoreWalker webserver was used to predict the tertiary structures of three BnGLR proteins representing each group, BnGLR12, BnGLR35, and BnGLR53.The pore structure and 3D geometry of these BnGLR proteins were shown to fit with a pore morphology that longitudinally passed through the extracellular to intracellular opening of the proteins (Figure 5A-F).
The pore size and constraints, which are considered as selective barriers, were predicted as shown in Figure 5A-F.Moreover, the Protter online tool was used for the structural visualization of these BnGLR proteins.The results showed that these BnGLR proteins were predicted to be membrane proteins with four transmembrane domains (Figure 5G-I).Together, these data support the BnGLR proteins to be transmembrane channels.

Post-Translational Modification Sites in BnGLRs 2.8.1. Phosphorylation
The attachment of the phosphate group to the serine, threonine, and tyrosine amino acids of BnGLR proteins was predicted.Generally, all BnGLR proteins contained all these three phosphorylation sites.The maximum putative phosphorylation serine (48) and threonine (17) were found in BnGLR41 and BnGLR33, respectively.Whereas BnGLR37, -38, -39, and -42 had the maximum number (13) of putative phosphorylation tyrosine in their protein sequences (Figure 6, Table S3).This result indicated that BnGLRs are likely phosphorylation-inducible proteins.

Glycosylation
There are four main categories of glycosylation based on the linkage between amino acids and sugars: N-linked glycans, O-linked glycans, GPI anchors, and C-mannosylation.Glycosylation analyses revealed the O-linked glycans in BnGLRs and AtGLRs, which were characterized by the interaction of a sugar with the hydroxyl group of serine or threonine.BnGLR50, BnGLR52, BnGLR54, and BnGLR55 carried the maximal four "Asn" residues, while 24 BnGLRs out of 61 did not contain any "Asn" residue (Figure 6, Table S3).This result implied that only some members of BnGLRs can be glycosylated.

Sumoylation
Sumoylation sites were characterized by a covalent bond of small ubiquitin-like modifiers to a specific lysine residue through an enzymatic action.All BnGLRs had sumo sites.A maximum of 17 sumoylation sites were predicted in BnGLR36.The positions and scores of these sumoylation sites are clearly marked in Figure 6 and Table S3.This result suggests that all BnGLRs can likely be sumoylated.

Methylation
The methylation sites in BnGLR proteins were predicted by using the online webserver, MASA.Methylation sites in lysine (Lys), arginine (Arg), and glutamate (Glu), but not in asparagine (N), were found in BnGLRs.The methylation site frequency was highest in Arg, followed by Lys, and lowest in Glu, which appeared only in six BnGLRs in Group III.Overall, the methylation sites in BnGLRs appeared to be site-and group-dependent.Group III BnGLRs contained more methylation sites, especially in Arg and Glu (Figure 6, Table S3).
Collectively, all BnGLRs carried phosphorylation and sumoylation sites, while glycosylation and methylation sites seemed to be BnGLR-dependent.Some Group I BnGLRs did not contain any methylation sites, while the absence of glycosylation site in BnGLRs was not group-dependent.

Glycosylation
There are four main categories of glycosylation based on the linkage between amino acids and sugars: N-linked glycans, O-linked glycans, GPI anchors, and C-mannosylation.Glycosylation analyses revealed the O-linked glycans in BnGLRs and AtGLRs, which were characterized by the interaction of a sugar with the hydroxyl group of serine or threonine.BnGLR50, BnGLR52, BnGLR54, and BnGLR55 carried the maximal four "Asn" residues, while 24 BnGLRs out of 61 did not contain any "Asn" residue (Figure 6, Table S3).This result implied that only some members of BnGLRs can be glycosylated.

Sumoylation
Sumoylation sites were characterized by a covalent bond of small ubiquitin-like modifiers to a specific lysine residue through an enzymatic action.All BnGLRs had sumo sites.A maximum of 17 sumoylation sites were predicted in BnGLR36.The positions and scores

Response of BnGLRs to the Pathogen Sclerotinia sclerotiorum
To analyze the response of BnGLR genes to the necrotrophic pathogen S. sclerotiorum (Ss), expression analysis was performed using quantitative real-time PCR (qRT-PCR).The primers that were used for expression analysis are listed in Table S1.The Ss was inoculated on oilseed rape leaves and sampled at 3, 6, and 12 h post-inoculation (hpi) and mock-inoculated samples were collected as a control.A total of 10 BnGLR genes, including BnGLR1, -5, -8, and -12 (Group I), BnGLR21, -26, and -35 (Group II), and BnGLR37, -53, and -59 (Group III) were selected, representing each group for expression analysis.Results showed that all genes were upregulated significantly in response to Ss inoculation.However, the extent of upregulation of BnGLR expression differed obviously.The expression was highest with a 23-fold peak enhancement at 12 hpi for BnGLR35, 20-fold at 6 hpi for BnGLR12, and 17-fold at 6hpi for BnGLR53, while it was lowest with a 5-fold peak enhance-ment at 6 hpi for BnGLR37 and 7~8-fold at 12 hpi for BnGLR1 and BnGLR21 (Figure 7).This result indicated that the upregulation of BnGLR expression was not group-dependent.Generally, BnGLRs were induced remarkably in response to Ss inoculation, indicating that these BnGLRs might be involved in resistance to Ss.The three BnGLR genes (BnGLR12, BnGLR35, and BnGLR53) that were expressed most highly in response to Ss were selected for further functional analysis in oilseed rape by reverse genetics technique Cabbage leaf curl virus (CaLCuV)-based virus-induced gene silencing (VIGS).The plants treated with empty vectors (EV) showed normal growth, suggesting that the CaLCuV-based vector infection did not obviously affect the vegetative growth of seedlings.BnGLR expression in the plants agro-infiltrated with PCVA-BnGLR12, PCVA-BnGLR35, and PCVA-BnGLR53 was significantly reduced (Figure 8A), indicating that these BnGLR genes were efficiently silenced.Compared with the plants treated with EV, the leaves of BnGLR35-silenced oilseed rape plants exhibited more severe necrosis (Figure 8B) with larger lesion areas (Figure 8C) and increased relative fungal biomass of S. sclerotiorum (Figure 8D), indicating that the silencing of BnGLR35 reduced oilseed rape resistance to Ss.In contrast, the BnGLR12and BnGLR53-silenced plants displayed milder disease symptoms (Figure 8B) with smaller lesion areas (Figure 8C) and lower S. sclerotiorum biomass (Figure 8D), indicating that the silencing of BnGLR12 and BnGLR53 enhanced oilseed rape resistance to Ss.These results demonstrated that BnGLR12, BnGLR35,

Arabidopsis Mutants of BnGLR Orthologs Exhibited Altered Plant Resistance against S. sclerotiorum
To further explore the role of BnGLR genes in plant resistance against S. sclerotiorum, inoculation analyses were performed in leaves of 4-week-old plants of six A. thaliana T-DNA insertion mutants of BnGLR orthologs, atglr1.2(mutant of the ortholog of BnGLR12), atglr2.5 (mutant of the ortholog of BnGLR35), and atglr3.2(mutant of the ortholog of BnGLR53).Prior to inoculation analysis, these Arabidopsis mutants were confirmed by PCR analysis and only homozygous mutant plants were selected for functional analysis.Compared with the wild type Col-0 (28 mm 2 ), the leaves of both lines of atglr2.5 plants showed more severe necrosis (53 mm 2 ) with more DAB staining (Figure 9A) and larger lesion areas (Figure 9B), while the leaves of two lines each of atglr1.2(6 mm 2 ) and atglr3.2(8 mm 2 ) plants displayed less severe necrosis with less DAB staining (Figure 9A) and smaller lesion areas (Figure 9B).It revealed that atglr2.5 mutants exhibited increased susceptibility to S.

Arabidopsis Mutants of BnGLR Orthologs Exhibited Altered Plant Resistance against S. sclerotiorum
To further explore the role of BnGLR genes in plant resistance against S. sclerotiorum, inoculation analyses were performed in leaves of 4-week-old plants of six A. thaliana T-DNA insertion mutants of BnGLR orthologs, atglr1.2(mutant of the ortholog of BnGLR12), atglr2.5 (mutant of the ortholog of BnGLR35), and atglr3.2(mutant of the ortholog of BnGLR53).Prior to inoculation analysis, these Arabidopsis mutants were confirmed by PCR analysis and only homozygous mutant plants were selected for functional analysis.Compared with the wild type Col-0 (28 mm 2 ), the leaves of both lines of atglr2.5 plants showed more severe necrosis (53 mm 2 ) with more DAB staining (Figure 9A) and larger lesion areas (Figure 9B), while the leaves of two lines each of atglr1.2(6 mm 2 ) and atglr3.2(8 mm 2 ) plants displayed less severe necrosis with less DAB staining (Figure 9A) and smaller lesion areas (Figure 9B).It revealed that atglr2.5 mutants exhibited increased susceptibility to S. sclerotiorum, while atglr1.2and atglr3.2mutants displayed reduced susceptibility compared with the wild type Col-0.In addition, compared with the Col-0 plants, atglr3.2and atglr1.2mutants manifested the enhanced generation of SsNLP1-stimulated H 2 O 2 .Conversely, the atglr2.5 mutants exhibited reduced SsNLP1-triggered H 2 O 2 production (Figure 9C).These findings suggest that the GLR genes play a crucial role in plant resistance against Ss, probably via modulating ROS accumulation.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 16 of 23 sclerotiorum, while atglr1.2and atglr3.2mutants displayed reduced susceptibility compared with the wild type Col-0.In addition, compared with the Col-0 plants, atglr3.2and atglr1.2mutants manifested the enhanced generation of SsNLP1-stimulated H2O2.Conversely, the atglr2.5 mutants exhibited reduced SsNLP1-triggered H2O2 production (Figure 9C).These findings suggest that the GLR genes play a crucial role in plant resistance against Ss, probably via modulating ROS accumulation.This experiment was performed with three biological replicates, each yielding similar results.

Discussion
In this study, we identified 61 BnGLRs in the genome of oilseed rape cv.ZS11.A set of evidence supports them to be potentially functional GLRs.They all contained a ligandbinding domain and an ion channel domain (Table 1; Figure S2).Their ion channel domain bore the conserved motif "SYTANLTS" (Figure S1), which was reported to be involved in ion exchange or transportation through the plasma membrane in rat, Rosaceae, and Arabidopsis [9].Furthermore, the BnGLRs were predicted to be localized to plasma membrane

Discussion
In this study, we identified 61 BnGLRs in the genome of oilseed rape cv.ZS11.A set of evidence supports them to be potentially functional GLRs.They all contained a ligand-binding domain and an ion channel domain (Table 1; Figure S2).Their ion channel domain bore the conserved motif "SYTANLTS" (Figure S1), which was reported to be involved in ion exchange or transportation through the plasma membrane in rat, Rosaceae, and Arabidopsis [9].Furthermore, the BnGLRs were predicted to be localized to plasma membrane and their tertiary structure fitted with membrane channel morphology, as exemplified by BnGLR12, BnGLR35, and BnGLR53 (Figure 5) as reported for known other gene family (DTX) [30].Additionally, binding pockets of seven amino acids existed in all BnGLR proteins (Table S2) as reported typical GLRs [16].Nevertheless, whether these BnGLRs function as calcium channels awaits further experimental confirmation.
One of our findings in this study is the group-dependent characteristics of BnGLRs.Firstly, group-wide chromosomal distribution of BnGLRs is obviously uneven.Regardless of the uncertainty of the localization of BnGLR4 (Group I) and BnGLR34 (Group II), Group I BnGLRs are absent in chromosomes A03-A05, A07-A08, A10, C03-C06, and C08, Group II BnGLRs are lacking in chromosomes A01-A02, A06-A10, C01, and C05-C08, whereas Group III BnGLRs are missing in chromosomes A01-A02, A04-A06, C02, and C06 (Table 1).Secondly, the motif composition of BnGLRs differs group-wide.For example, motif 5 is absent from Group I BnGLRs while present in those of the other two groups (Figure 2).Thirdly, the gene structure of BnGLRs distinguishes group-wide.BnGLR genes of Group III carry more introns than those of the other groups.BnGLR genes of Group II generally contain a long first-intron, while those of the other groups do not (Figure 3).Finally, the methylation sites discriminate between the groups of BnGLRs.The methylation sites in Glu only exist in six BnGLRs of Group III.Group III BnGLRs also contain more methylation sites in Arg and Glu than other group BnGLRs (Figure 6, Table S3).Collectively, our findings clarify the differentiation of BnGLRs of different groups, which implies that the functions and mechanisms of BnGLRs of different groups may be distinct.In this context, it is intriguing that we indeed found the distinct function of BnGLRs from various groups-BnGLR12 from Group I, BnGLR35 from Group II, and BnGLR53 from Group III-in oilseed rape resistance to the necrotrophic fungal pathogen S. sclerotiorum (Figure 8).Additionally, our results on chromosomal distribution, protein motif, and gene structure of BnGLRs differ in some aspects from a previous report, which documented that Group I BnGLRs distributed in chromosomes A03, A05, A07, A08, C03-C06, and C08; Group II BnGLRs existed in A02, C06, and C07; and Group III BnGLRs presented in A01, A02, and C02.Motif 5 existed in Group I BnGLRs.Group II BnGLRs contained more introns (2-12 introns) than other groups BnGLRs [14].These differences might be due to the variety in the oilseed rape cultivars used in the two studies [14].
Interestingly, 9 out of the total 61 BnGLRs carried one to several CGCG-boxes (the CAMTA-binding sites in their promoters (Figure 4)).These included BnGLR12 from Group I, BnGLR34 and BnGLR35 from Group II, and BnGLR37, -38, -39, -53, -60, and -61 from Group III.This demonstrates that these BnGLRs might be directly regulated by CAMTA3, an essential transcription factor in the calcium signaling pathways.Notably, we found previously that CAMTA3 plays a role in plant disease resistance against Ss [31].Therefore, it is likely that CAMTA3 and these GLRs coordinate in plant resistance to Ss.
PTM is one of the most important ways to regulate protein functions.We systematically analyzed four types of PTMs, including phosphorylation, sumoylation, glycosylation, and methylation, in BnGLRs.Consequently, we found that all BnGLRs carry plenty of phosphorylation and sumoylation sites, while glycosylation and methylation sites only exist in some BnGLRs (Figure 6, Table S3).Interestingly, AtGLR3.6 and AtGLR3.7 were recently reported to be phosphorylated by calcium-dependent protein kinases at Serine-856 and Serine-860, respectively, thereby leading to salt tolerance [32,33], indicating that oilseed rape GLRs are also highly likely phosphorylated at Serine.It would be interesting to examine whether GLRs are also phosphorylated at Thr and Tyr in accordance with our prediction results.It also deserves to be confirmed whether GLRs are also sumoylated, glycosylated, and/or methylated following the clues from our study.
The function of GLRs in plant resistance to pathogens has only been studied to a limited extent mainly in the model plant Arabidopsis.For example, Group III AtGLRs were reported to play a role in plant resistance against the oomycete H. arabidopsidis [24], fungus B. cinerea [25], and bacterium P. syringae [26].In this study, we explored the role of BnGLRs in resistance to S. sclerotiorum, one of the most destructive pathogens in oilseed rape, which is one of the most important oil crop species.We found that three BnGLRs-BnGLR12 from Group I, BnGLR35 from Group II, and BnGLR53 from Group III-are highly responsive to S. sclerotiorum infection with an increase in expression by over 15-fold at 6 hpi (Figure 7).More importantly, VIGS analyses demonstrated that BnGLR12, BnGLR35, and BnGLR53 play distinct roles in oilseed rape resistance to Ss; BnGLR35 positively, while BnGLR12 and BnGLR53 negatively, regulate this resistance (Figure 8).Furthermore, inoculation analysis and ROS detection employing Arabidopsis mutants of the BnGLR gene homologs (BnGLR12/AtGLR1.2,BnGLR35/AtGLR2.5, and BnGLR53/AtGLR3.2) confirmed this conclusion and extended their roles in PAMP (SsNLP1)-triggered immunity (Figure 9).The response of Group III GLR, AtGLR3.2, and its ortholog in B. napus (BnGLR53) are distinguished from previous studies of AtGLRs from Group III, in which radish GLR homologs of AtGLR3.3 and AtGLR3.4play a positive role in resistance against B. cinerea and Alternaria brassicae [25,34], possibly due to the difference in the pathogens used for inoculation analyses.Together with these reports, our findings reveal the complex memberdependent functions of the GLR family in plant resistance to pathogens.The mechanisms underlying BnGLR-mediated plant immunity await further dissection.
For the measurement of physicochemical properties such as isoelectric point (pI) and amino acid (aa) composition, an online tool ProtParam server was used (http://web.expasy.org/protparam/, accessed on 12 November 2020) [14].

Construction of BnGLR Phylogenic Tree
MegaX software was used to align the AtGLRs and BnGLRs by applying the clustal W function.For the construction of a phylogenetic tree in Mega X (v10.2.2) [35], the maximum likelihood (ML) algorithm with 1000 bootstrap replicates and partial deletion (95% site coverage as cut-off) were used for gaps and missing data.Based on their sequence similarity with AtGLRs in the phylogenetic tree, the members of the BnGLR family were named.

Identification of Motifs, Domains, and Gene Structure
Conserved motifs in the BnGLR proteins were identified using the web tool Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme-suite.org/tools/meme, accessed on 29 November 2020) [28].The parameters were as follows: the maximum number of motifs was set to 12, the motif width ranged from 6 to 50 (inclusive), and the site distribution was limited to 0 or 1 occurrence (of a contributing motif site) per sequence.Logo motif analysis was also performed to demonstrate the conservativeness of each group of BnGLR proteins using the weblogo3 (v3.7) platform [36].
The conserved domains of BnGLR proteins were predicted using the SMART [37] and CDD databases [38].
By matching the cDNA to the corresponding gene sequences, the Gene Structure Display Server (GSDS) was used [39] to analyze the CDS and exon/intron structures of the GLR genes.

Promoter Profiling of BnGLRs
To identify the potential cis-acting elements in BnGLRs, upstream (1.5 kb) sequence of BnGLRs were obtained from NCBI and were analyzed using the search scan program in the PLACE database [8].

Prediction of Amino Acid Ligands Interacting with BnGLR Proteins
To visualize the predicted physical interactions between amino acid ligands and receptor BnGLR proteins, PDB images of receptor proteins and amino acid ligands (Ala, Asn, Cys, Glu, Gly, Met, and Ser) were obtained from Phyre2 (Protein Homology/Analogy Recognition Engine V 2.0) and Pubchem (https://pubchem.ncbi.nlm.nih.gov/,accessed on 8 January 2021), respectively.Interactions were predicted by using the online tool Patchdock [41] and the complex (receptor-ligand complex) was obtained in PDB format, and the complex PDB image was visualized using LIGPLOT (v2.2) software for identification of binding pocket positions [42].

Post-Translational Modifications in BnGLR Proteins
The sites of PTMs, including methylation at lysine, arginine, and glutamate; Nglycosylation at asparagine; phosphorylation at serine, threonine, and tyrosine; and sumoylation were predicted by using online bioinformatics tools given below as described [43]

Plant Materials and Pathogen Inoculation Analysis
Brassica napus plants were grown in growth cabinets at 23 • C under a 14 h/10 h light/dark photoperiod [36].All Arabidopsis thaliana plants used in this study were of Col-0 ecotype background and were grown at 16 h photoperiod and 70% humidity at 21 ± 1 • C in the growth chamber [44].The following mutants were used in this study and were obtained through the AraShare (Fuzhou, China): atglr1.2(SALK_136614C, SALK_053535C), atglr2.5 (SALK_078407C, SALK_050593C), and atglr3.2(SALK_063873C, SALK_133700C).Homozygous plants were screened based on growth on selectable medium and PCR using a combination of gene-specific and transposon-or T-DNA-specific primers (Table S1).
Plant leaves were inoculated using Sclerotinia sclerotiorum (Ss) mycelial plugs.Fresh sclerotia of Ss strain UF1 were cultured at 23 • C on potato dextrose agar medium (PDA) to produce mycelia, which were transferred to new PDA plates and grown for 2 days.The PDA plugs containing young Ss mycelia were punched to inoculate the leaves of 30 d old plants [36].Area of disease lesions was measured using the ImageJ (v1.54e) software [36].For disease resistance evaluation, at least five plants for each genotype were examined and the experiments were conducted three times independently.

Gene Expression Analysis
A total of 10 BnGLR genes including BnGLR1, -5, -8, and -12 (Group I), BnGLR21, -26, and for expression analysis.One-month old oilseed rape leaves were sampled at 0, 3, 6, and 12 h post-inoculation with S. sclerotiorum.Total RNA was extracted using Trizol reagent (Vazyme, Nanjing, China) following the previous study [45].Quantitative real-time PCR (qRT-PCR) was performed using the StepOne Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) with SYBR Green PCR Master Mix (TaKaRa, Dalian, China).The relative fold changes were calculated using the 2 −∆∆Ct method as previously described [45], with two technical replicates for each of the three biological replicates.The housekeeping gene BnActin7 was used as an internal control.Primers used for qRT-PCR are listed in Table S1.The VIGS experiment with cabbage leaf curl virus was carried out as previously reported [46], with a few adjustments.(i) The gene-specific sequences of BnGLR12, BnGLR35, and BnGLR53 were selected to ensure gene-specific silencing.Primers containing the restriction site (Xba I) and homologous arms were designed (Table S1).(ii) Using first-strand cDNA from the ZS11 cultivar, the target sequences of BnGLRs were amplified.The digested material was kept at 37 • C overnight to obtain the digested PCVA vector (digested with Xba I) (NEB, Ipswich, MA, USA).Using the ClonExpress Entry One Step Cloning Kit (Vazyme), the target sequence and the digested PCVA were ligated for 15 min at 50 • C. (iii) After transforming the ligation mix into competent DH5α cells (Invitrogen, Darmstadt, Germany), the cells were plated on LB plates containing antibiotics (kanamycin at 50 mg/mL) at 37 • C for overnight.A single colony was chosen, shaken well in liquid LB (containing 50 mg/mL kanamycin), and then sequenced using gene-specific primers.The plasmids from empty PCVA, PCVB, PCVA-BnGLR12, PCVA-35, and PCVA-53 were purified using the FastPure EndoFree Plasmid Maxi Kit (Vazyme).(iv) Using the freeze-thaw transformation technique, plasmids were transformed into Agrobacterium tumefaciens (GV3101).Following that, single colonies were chosen and shaken for 48-72 h at 28 • C in liquid LB (containing 50 mg/mL of kanamycin and 50 mg/mL of rifampicin).The BnGLRs were sequenced using gene-specific primers and primers A and B for the PCVA and PCVB empty vectors, respectively, to confirm that the constructed vector was effectively transformed into Agrobacterium.A mixture of Agrobacterium culture and glycerol (1:3) was placed in a 2 mL cryotube to preserve bacteria for long-term preservation at −80 • C. For inoculation with PCVA/PCVB and PCVA-BnGLRs, ZS11 seedlings (2-3 leaf-stage) were used for VIGS experiments.Twoweek-old oilseed rape plants were infiltrated with a 1 mL syringe and the infiltrated plants were placed in the dark for 24 h.The seedlings were then placed in a chamber for the following 15-21 days for successful silencing.To confirm whether plants were truly silent, half of the leaves were checked for BnGLR gene silencing by qRT-PCR.The remaining half of the same leaves were used for Ss inoculation analyses using mycelial plugs.

ROS Detection
For quantitative ROS measurement, 3 mm diameter leaf disks were immersed in 50 µL of distilled water in a 96-well plate and left in the dark overnight.A 100 µL solution comprising 100 µM luminol (Sigma-Aldrich, St. Louis, MO, USA) and 1 µg of horseradish peroxidase was used as a substitute for water.A microplate luminometer was used to measure the luminescence of H 2 O 2 for 51 min after the addition of H 2 O 2 elicitor, the fungal PAMP, SsNLP1 (1 µM) (TITERTEK BERTHOLD, Pforzheim, Germany), as previously described [36].

Statistical Analysis
All experiments were performed with three biological replicates.Data were statistically analyzed to determine the significant differences by Student's t-test and shown as the mean ± SE using Graphpad Prism 8.0 [36].

Figure 1 .
Figure 1.Phylogenic tree for AtGLR and BnGLR proteins.The maximum-likelihood (ML) phylogenetic tree of the AtGLR and BnGLR proteins was constructed by MEGA X software (v10.2.2) with 1000 bootstrap replicates.Dotted lines represent the AtGLRs, whereas plain lines stand for BnGLRs.Distinct colors indicate different groups of GLRs, Group I in brown, Group II in green, and Group III in yellow.

Figure 2 .
Figure 2. Conserved motifs among BnGLR proteins.(A) Logos of the predicted motifs.The BnGLRspecific motif logos were generated by using weblogo3 (v3.7).The bit scores for each position in the

Figure 2 .
Figure 2. Conserved motifs among BnGLR proteins.(A) Logos of the predicted motifs.The BnGLRspecific motif logos were generated by using weblogo3 (v3.7).The bit scores for each position in the sequence are indicated to the left.(B) Motif profile of BnGLRs.The conserved motifs identified by MEME [28] are indicated by different colors.The consensus of each motif is provided.

Figure 3 .
Figure 3.The exon-intron structures of 61 BnGLR genes.The constituents of BnGLR genes are indicated in differentially colored shapes.CDS: green box; upstream/downstream: red box; introns: black lines.

Figure 4 .
Figure 4. Cis-acting elements in BnGLR gene promoters.Major stress responsive cis-elements predicted in BnGLRs are shown.The types of cis-elements are provided.The bubble heat map showing the number of elements was prepared using the TB-tool (v2.084) [29].

Figure 5 .
Figure 5. Pore morphology, dimensions, and topology of BnGLR proteins.(A-C) Protein tertiary structures of BnGLRs.Pore structure of three BnGLRs are shown; (D-F) pore diameter profile of BnGLRs.Pore dimensions were obtained using the PoreWalker software (v1.0); (G-I) topology of BnGLRs.Secondary structure of three BnGLRs was visualized by the online tool Protter (v1.0).The numbers count the transmembrane domains.

Figure 7 .
Figure 7. Transcriptional regulation of BnGLRs in response to S. sclerotiorum infection in oilseed rape plants.Gene expression was detected by qRT-PCR, in which BnActin7 gene was used as reference.This experiment was performed with three biological replicates, each yielding similar results.The asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n.s.-not significant) of BnGLR gene expression in four time points (0, 3, 6, 12 h post-inoculation (hpi)) when statistically analyzed by Student's t-test.2.10.Silencing of BnGLR12, BnGLR35, and BnGLR53 Distinctly Altered Oilseed Rape Resistance against S. sclerotiorum

Figure 9 .
Figure 9. Arabidopsis mutants of BnGLR orthologs exhibited altered plant resistance against S. sclerotiorum.(A) Disease symptoms at 24 h post-inoculation (hpi).Leaves before DAB staining (upper panels) and after staining (lower panels) are shown.(B) Lesion areas at 24 hpi.(C) SsNLP1-triggered ROS in atglr plants.ROS was measured in Col-0 and atglr leaf disks after addition of 1 µM SsNLP1.The dynamics of ROS production within 50 min after SsNLP1 addition is shown.ROS production at each time point was calculated as mean values of total photon counts.Data were statistically analyzed by Student's t-test (n = 8) and shown as the mean ± SE.The asterisks indicate significant differences (** p ≤ 0.01, *** p ≤ 0.001) of lesion areas between Col-0 and atglr mutants.Scale bar: 1 cm.This experiment was performed with three biological replicates, each yielding similar results.

Figure 9 .
Figure 9. Arabidopsis mutants of BnGLR orthologs exhibited altered plant resistance against S. sclerotiorum.(A) Disease symptoms at 24 h post-inoculation (hpi).Leaves before DAB staining (upper panels) and after staining (lower panels) are shown.(B) Lesion areas at 24 hpi.(C) SsNLP1-triggered ROS in atglr plants.ROS was measured in Col-0 and atglr leaf disks after addition of 1 µM SsNLP1.The dynamics of ROS production within 50 min after SsNLP1 addition is shown.ROS production at each time point was calculated as mean values of total photon counts.Data were statistically analyzed by Student's t-test (n = 8) and shown as the mean ± SE.The asterisks indicate significant differences (** p ≤ 0.01, *** p ≤ 0.001) of lesion areas between Col-0 and atglr mutants.Scale bar: 1 cm.This experiment was performed with three biological replicates, each yielding similar results.

Table 1 .
BnGLRs identified in this study.