Systematic Characterization of Brassica napus HIR Gene Family Reveals a Positive Role of BnHIR2.7 in Sclerotinia sclerotiorum Resistance

: Hypersensitive-induced response protein (HIR) is a class of plant immune proteins that play pivotal roles in Sclerotinia sclerotiorum (Lib.) de Bary resistance. However, there has been no systematic investigation and identiﬁcation of HIR genes in rapeseed ( Brassica napus L.). Hence, we identiﬁed 50 BnHIR genes and classiﬁed them into four groups. Subcellular localization prediction suggested that HIR proteins are mainly localized in the mitochondria. Cis -acting elements involved in light and diverse abiotic stress were found in the promoter regions of BnHIR . The majority of BnHIR genes in Groups 1/3/4 were expressed in most examined tissues, especially in leaves and siliques pericarp, while the BnHIR genes in Group 2 were not or had low expression in all detected tissues. In the case of S. sclerotiorum inoculation, HIR genes in Groups 1/3/4 were strongly induced, especially homologous genes in Group 1, which exhibited different expression patterns. Moreover, overexpression of BnHIR2.7 in Arabidopsis thaliana illustrated its prominent resistance to S. sclerotiorum . Our study provides insight into the evolutionary relationships of the HIR family genes in B. napus and lays the foundation for their resistance to S. sclerotiorum in B. napus .


Introduction
Sclerotinia Disease (SD) is a host non-specific soil-borne fungal disease mainly caused by Sclerotinia sclerotiorum (Lib.) de Bary. After completing one life cycle, the fungus can remain active for decades by producing long-lived dormant bodies or sclerotia to exacerbate the potential SD inoculum [1]. SD is a potential problem that threatens multiple traits in Brassica napus L., such as reducing oil content, changing fatty acid composition, and fungal toxicity, which seriously affects the quality of rapeseed oil and causes substantial economic losses. Although traditional fungicides have a certain positive effect on SD control, the host range of S. sclerotiorum still exists widely due to its long-lived capacity in soil. Therefore, breeding new varieties is highly urgent to resist S. sclerotiorum in rapeseed [2].
The influence of pathogenic bacteria or the stress of unfavorable environmental conditions can stimulate plant hypersensitive reactions (HR). Hypersensitive-induced reaction (HIR) is involved in plant HR as a hypersensitivity protein [3]. HIR proteins are a class of hypersensitive reactions (HR). These proteins are part of the PID (proliferation, ion, and

Overexpression Vector Construction and Plant Transformation
Total RNA was extracted from the leaves of B. napus and A. thaliana using an RNA extraction kit (Tiangen, Beijing, China). First-strand cDNA was synthesized using Prime-Script RT Master Mix Kit (TaKaRa, Dalian, China). Total DNA was extracted from B. napus and A. thaliana leaves using the CTAB method [29].
After qRT-PCR analysis, the OV-BnHIR2.7 line with the highest expression of BnHIR2.7 and the Athir2.7-Mu line with the lowest expression was selected for phenotype observation. Plant height was measured from the cotyledon nodes to the top of the main inflorescence at maturity. At 50 days of the seedling stage, five siliques from the bottom to the top of the main stem were selected to measure and count the length of the siliques.

Expression Analysis of BnHIR2.7 under the Infection of S. sclerotiorum
To confirm the function of BnHIR2.7 to S. sclerotiorum resistance, leaves of A. thaliana Col-0, OV-BnHIR2.7, and AtHIR2.7-Mu were selected for S. sclerotiorum inoculation experiments. Edge hyphae were excised using a 1 mm punch and carefully turned over to the front of normal leaves. The lesion area was measured and photographed every 12 h, with three replicates of each experiment. Each A. thaliana plant was inoculated on three leaves, and samples were collected every 12 h and immediately stored in liquid nitrogen. The Col-0, OV-BnHIR2.7, and Athir2.7-Mu leaves that had grown normally for 40 days were selected, and the three inoculated leaves of each treatment were stained with trypan blue to observe the leaf cells. Briefly, a 0.4% trypan blue staining solution was used to immerse leaves in trypan blue dye solution for 36 h and then leaves were decolorized using glacial acetic acid and methanol (3:1, v:v). Moreover, the gene expression levels of some marker genes in the SA and MeJA pathways were identified after inoculation with S. sclerotiorum.

Identification and Characterization of the B. napus HIR Family
We identified 50 HIR genes with the conserved SPFH domain in B. napus. The putative HIR genes in B. napus were named according to their orthologs in A. thaliana (Table 1). Most AtHIR genes in B. napus have multiple homologous genes, and most AtHIR genes in B. napus have multiple homologs. However, in the subfamilies PHB5, PHB7, and HIR4, no homologues of A. thaliana genes were found in B. napus, indicating that the subfamily may have been lost. Among the 50 BnHIR proteins, the lengths ranged from 227 (BnHIR1) to 535aa (BnHIR1.3), and the MW ranged from 25.97 (BnHIR1) to 58.02 kDa (BnHIR1.3). Interestingly, the isoelectric point distribution of BnHIR proteins is wide, ranging from 4.99 (BnHIR1) to 9.55 (BnPHB2.5). Some proteins have transmembrane structures, such as BnPHB1.3, BnPHB1.5, and BnPHB1.7. Subcellular localization indicated that the majority of BnHIR proteins were localized in mitochondria and a few in the nucleus (Table 1).

Phylogenetic Analysis of HIR Proteins in B. napus
The maximal likelihood (ML) phylogenetic tree containing 68 HIR proteins from B. napus (50) and A. thaliana (18) was constructed to explore the phylogenetic relationships. The bootstrap value showed that B. napus HIR proteins can be divided into four groups: Group 1 (HIR1/2/3/4/7 clade), Group 2 (HIR5/6 clade), Group 3 (PHB1 clade), and Group 4 (PHB2/3/4 clades). Group 1 and Group 4 are the largest clades containing 26 members, and Group 3 is the smallest clade containing 4 members. In particular, HIR5/6 formed an independent clade during evolution, while HIR1/2/3/4/7 became a clade. In addition, some genes in the HIR2 subfamily correspond to Arabidopsis AtHIR1, which then combine to form a larger clade ( Figure 1). The evolutionary relationships among the rapeseed HIR genes are consistent with those of A. thaliana, except for the BnHIR1 clade.
The bootstrap value showed that B. napus HIR proteins can be divided into four groups: Group 1 (HIR1/2/3/4/7 clade), Group 2 (HIR5/6 clade), Group 3 (PHB1 clade), and Group 4 (PHB2/3/4 clades). Group 1 and Group 4 are the largest clades containing 26 members, and Group 3 is the smallest clade containing 4 members. In particular, HIR5/6 formed an independent clade during evolution, while HIR1/2/3/4/7 became a clade. In addition, some genes in the HIR2 subfamily correspond to Arabidopsis AtHIR1, which then combine to form a larger clade ( Figure 1). The evolutionary relationships among the rapeseed HIR genes are consistent with those of A. thaliana, except for the BnHIR1 clade.

Gene Structure and Conserved Motif Analysis
To analyze the exon/intron structure of the BnHIR and AtHIR genes, we compared the CDS with their corresponding genomic sequences. The results showed that most

Gene Structure and Conserved Motif Analysis
To analyze the exon/intron structure of the BnHIR and AtHIR genes, we compared the CDS with their corresponding genomic sequences. The results showed that most BnHIR genes contained 3-6 exons, and some genes contained more than 8 exons, such as BnHIR7 subfamily genes ( Figure 2a). Genes from the same subfamily presented similar gene structures. Most BnHIR genes have a long exon at the 3 ends of their genomic sequence. Furthermore, the gene structure of BnHIR is highly similar to their corresponding AtHIR.
To better investigate structural diversity among BnHIR proteins, MEME was used to analyze 15 conserved motifs identified in the full-length BnHIR protein ( Figure 2b). Motif 2 or motif 3 was observed in all 50 BnHIR proteins. Multiple motifs were observed in almost all BnHIR proteins, with some motifs only found in a few HIR proteins, except for BnHIR1 proteins that contain only motif 9. For example, motif 10 was only detected in Group 3, while motif 4 was unique to the HIR subfamily. Furthermore, there is a high degree of similarity between BnHIR and its cognate AtHIR proteins.
To better investigate structural diversity among BnHIR proteins, MEME was used to analyze 15 conserved motifs identified in the full-length BnHIR protein (Figure 2b). Motif 2 or motif 3 was observed in all 50 BnHIR proteins. Multiple motifs were observed in almost all BnHIR proteins, with some motifs only found in a few HIR proteins, except for BnHIR1 proteins that contain only motif 9. For example, motif 10 was only detected in Group 3, while motif 4 was unique to the HIR subfamily. Furthermore, there is a high degree of similarity between BnHIR and its cognate AtHIR proteins.

Cis-Acting Elements in the Promoter Regions of BnHIR Genes
To further explore the transcriptional regulation of the BnHIR genes, the potential cisacting elements were identified in their upstream 2000 bp genomic DNA sequence. To better understand the potential function of the BnHIR genes, cis-acting elements involved

Cis-Acting Elements in the Promoter Regions of BnHIR Genes
To further explore the transcriptional regulation of the BnHIR genes, the potential cis-acting elements were identified in their upstream 2000 bp genomic DNA sequence. To better understand the potential function of the BnHIR genes, cis-acting elements involved in light response, defense, and stress response were preserved. All BnHIR genes contained light responsiveness, such as GT1-motif and G-box, and at least four cis-acting elements were found in the upstream region of each BnHIR gene ( Figure 3). Elements responsive to phytohormones were also found in most promoter regions of HIR genes, including abscisic acid (ABRE) and cis-acting regulatory element (CAT-box). Members in Groups 1/2/4 had more abundant cis-acting elements, but members in Group 3 had fewer cis-acting elements, and BnPHB1.3 contained only four elements, with no phytohormone and stress cis-acting elements. In addition, the promoter regions of BnHIR included those cis-acting elements, such as the protein binding region (HD-Zip 3), enhancer (CAAT-box), and abiotic stress response (MBS) ( Figure S1). Notably, the promoter region of BnPHB2.4 had the most multitudinous cis-acting elements, including 10 phytohormone-responsive elements, 11 light-responsive elements, and six other cis-acting elements. In contrast, BnPHB1.3 had scisic acid (ABRE) and cis-acting regulatory element (CAT-box). Members in Groups 1/2/4 had more abundant cis-acting elements, but members in Group 3 had fewer cis-acting elements, and BnPHB1.3 contained only four elements, with no phytohormone and stress cis-acting elements. In addition, the promoter regions of BnHIR included those cis-acting elements, such as the protein binding region (HD-Zip 3), enhancer (CAAT-box), and abiotic stress response (MBS) ( Figure S1). Notably, the promoter region of BnPHB2.4 had the most multitudinous cis-acting elements, including 10 phytohormone-responsive elements, 11 light-responsive elements, and six other cis-acting elements. In contrast, BnPHB1.3 had the fewest cis-acting elements in the promoter region, indicating that their functions are conserved or degenerated. Figure 3. Cis-acting elements in the promoter region of BnHIR genes. Cis-acting elements in promoter regions were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 March 2022) and plotted using R package tidyverse. All cis-acting elements are grouped into four categories and marked with different colored boxes. The red star represents the target gene for subsequent functional verification.

Expression Patterns of BnHIR Genes at Different Developmental Stages
To better study the biological functions of BnHIRs, we analyzed their expression profiles in numerous tissues during different stages. No expression was detected in BnHIR2.6, BnHIR7.2, BnHIR7.7, BnPHB1.1, or BnPHB1.6 in any examined tissue. In Group 1, BnHIR2.2 and BnHIR2.3 were only highly expressed in leaves, and BnHIR2.7, BnHIR3.2, and BnHIR3.3 were highly expressed in all detected tissues (Figure 4a). However, Figure 3. Cis-acting elements in the promoter region of BnHIR genes. Cis-acting elements in promoter regions were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare /html/, accessed on 17 March 2022) and plotted using R package tidyverse. All cis-acting elements are grouped into four categories and marked with different colored boxes. The red star represents the target gene for subsequent functional verification.

Expression Analysis of BnHIRs under S. sclerotiorum Induction
To further investigate the role of BnHIRs under S. sclerotiorum induction, we analyzed the expression profiles of BnHIRs at different time points in response to S. sclerotiorum infection.
HIR genes in Group 1 showed different expression patterns. BnHIR2.4, BnHIR2.5, BnHIR3.1, and BnHIR3.2 were all highly expressed in the susceptible variety and the resistance variety after S. sclerotiorum infection. In particular, BnHIR2.7 was weakly expressed in the susceptible variety. Still, the expression level in resistant materials was higher and up-regulated rapidly 24 h after infection, reaching a maximum at 48 h and gradually decreasing (Figure 5a). Members in Group 2 had relatively weak responses to S. sclerotiorum (Figure 5b). The PHB genes in Group 3 were consistently expressed, with no apparent specificity between the two varieties ( Figure 5c). As with Group 3, each member of Group 4 had high expression, with no obvious species specificity. However, 48 h after S. sclerotiorum infection in the disease-resistant material, all members of Group 4 had

Expression Analysis of BnHIRs under S. sclerotiorum Induction
To further investigate the role of BnHIRs under S. sclerotiorum induction, we analyzed the expression profiles of BnHIRs at different time points in response to S. sclerotiorum infection.
HIR genes in Group 1 showed different expression patterns. BnHIR2.4, BnHIR2.5, BnHIR3.1, and BnHIR3.2 were all highly expressed in the susceptible variety and the resistance variety after S. sclerotiorum infection. In particular, BnHIR2.7 was weakly expressed in the susceptible variety. Still, the expression level in resistant materials was higher and up-regulated rapidly 24 h after infection, reaching a maximum at 48 h and gradually decreasing (Figure 5a). Members in Group 2 had relatively weak responses to S. sclerotiorum (Figure 5b). The PHB genes in Group 3 were consistently expressed, with no apparent specificity between the two varieties ( Figure 5c). As with Group 3, each member of Group 4 had high expression, with no obvious species specificity. However, 48 h after S. sclerotiorum infection in the disease-resistant material, all members of Group 4 had a higher response to S. sclerotiorum except for BnPHB2.2, with the highest expression levels for BnPHB3 and BnPHB 4 (Figure 5d).
The expression levels of SA and MeJA pathway marker genes of Col-0, Athir2.7-Mu, and OV-BnHIR2.7 plants were detected at different times after inoculation with S. sclerotiorum ( Figure S3a). We analyzed the phenotypes of OV-BnHIR2.7 transgenic A. thaliana overexpression lines, Athir2.7-Mu mutants, and wild-type Col-0 plants. At the seedling stage, there was no noticeable difference between Col-0 wild-type, OV-BnHIR2.7, and Athir2.7-Mu ( Figure S3b). At the maturity stage, OV-BnHIR2.7 plants were prominently higher than Athir2.7-Mu plants ( Figure S3c). In addition, the siliques of the Col-0 wildtype were prominently smaller than those of the OV-BnHIR2.7 plants but prominently longer than those of the Athir2.7-Mu ( Figure S3d). These results suggest that HIR2.7 is a critical positive regulator of plant growth and development, especially in promoting silique length. Moreover, there was no extreme difference in BnHIR2.7 expression in the overexpressed lines before and after S. nucleobium inoculation (Figure 6a). Notably, trypan blue staining showed that overexpression of BnHIR2.7 in A. thaliana did not induce programmed cell death (Figure 6b). Col-0, Athir2.7-Mu, and OV-BnHIR2.7 A. thaliana leaves showed different degrees of lesions at 24 h and 36 h after inoculation with S. sclerotiorum The blue-to-red color bar on the right represents the normalized expression value, and expression levels of BnHIRs were normalized by log 2 (FPKM + 1). Expression data were retrieved from the NCBI SRA database (PRJNA274853). The red star represents the target gene for subsequent functional verification.
The expression levels of SA and MeJA pathway marker genes of Col-0, Athir2.7-Mu, and OV-BnHIR2.7 plants were detected at different times after inoculation with S. sclerotiorum ( Figure S3a). We analyzed the phenotypes of OV-BnHIR2.7 transgenic A. thaliana overexpression lines, Athir2.7-Mu mutants, and wild-type Col-0 plants. At the seedling stage, there was no noticeable difference between Col-0 wild-type, OV-BnHIR2.7, and Athir2.7-Mu ( Figure S3b). At the maturity stage, OV-BnHIR2.7 plants were prominently higher than Athir2.7-Mu plants ( Figure S3c). In addition, the siliques of the Col-0 wild-type were prominently smaller than those of the OV-BnHIR2.7 plants but prominently longer than those of the Athir2.7-Mu ( Figure S3d). These results suggest that HIR2.7 is a critical positive regulator of plant growth and development, especially in promoting silique length. Moreover, there was no extreme difference in BnHIR2.7 expression in the overexpressed lines before and after S. nucleobium inoculation (Figure 6a). Notably, trypan blue staining showed that overexpression of BnHIR2.7 in A. thaliana did not induce programmed cell death (Figure 6b). Col-0, Athir2.7-Mu, and OV-BnHIR2.7 A. thaliana leaves showed different degrees of lesions at 24 h and 36 h after inoculation with S. sclerotiorum (Figure 6c). At 48 h after S. sclerotiorum inoculation, the lesions expanded to the entire A. thaliana leaf ( Figure S3e). Correspondingly, the infected area of strain in overexpressed lines was signifi-cantly (p = 0.00007) lower than that of Col-0 at 36 h inoculated, and that of Athir2.7-Mu was significantly (p = 0.0036) higher than that of Col-0 at 24 h inoculated (Figure 6d).  Figure 6c). At 48 h after S. sclerotiorum inoculation, the lesions expanded to the entire A. thaliana leaf ( Figure S3e). Correspondingly, the infected area of strain in overexpressed lines was significantly (p = 0.00007) lower than that of Col-0 at 36 h inoculated, and that of Athir2.7-Mu was significantly (p = 0.0036) higher than that of Col-0 at 24 h inoculated (Figure 6d).

Discussion
HIR family genes play key roles in plant development, metabolism, and stress resistance, and they have been identified in many plant species. In wheat, TaHIR1 and Ta-HIR3 expression were reduced in response to environmental stimuli such as low temperature, drought, and high salt stress. TaHIR1 and TaHIR3 genes' silencing induced by barley streak mosaic virus reduced the resistance of the wheat variety Suwon11 to the avirulent stripe rust type CYR23 and the necrotic cell region near the infection site and altered the expression levels of defense-related genes [10]. In pepper, overexpression of CaHIR1 gene resulted in spontaneous cell death, enhanced disease resistance against P. syringae [14], and enhanced sensitivity to salinity and drought stress during germination [35]. However, the gene function of BnHIR in B. napus has not been studied in depth.
Previous studies have shown that HIR genes have conserved functions and profound evolutionary origins in each kingdom [7]. It has been reported that 19 and 16 HIR genes have been identified in the rice and maize genomes, respectively [7,12]. In this study, we identified 50 BnHIRs, and these genes encode HIR proteins with an SPFH or PHB domain, consistent with canonical HIR proteins in other plants [36]. B. napus has more HIR genes than those plants, probably owing to additional whole-genome triploidy (WGT) during the evolutionary history of Brassica crops [15]. In higher plants, HIR genes can be divided

Discussion
HIR family genes play key roles in plant development, metabolism, and stress resistance, and they have been identified in many plant species. In wheat, TaHIR1 and TaHIR3 expression were reduced in response to environmental stimuli such as low temperature, drought, and high salt stress. TaHIR1 and TaHIR3 genes' silencing induced by barley streak mosaic virus reduced the resistance of the wheat variety Suwon11 to the avirulent stripe rust type CYR23 and the necrotic cell region near the infection site and altered the expression levels of defense-related genes [10]. In pepper, overexpression of CaHIR1 gene resulted in spontaneous cell death, enhanced disease resistance against P. syringae [14], and enhanced sensitivity to salinity and drought stress during germination [35]. However, the gene function of BnHIR in B. napus has not been studied in depth.
Previous studies have shown that HIR genes have conserved functions and profound evolutionary origins in each kingdom [7]. It has been reported that 19 and 16 HIR genes have been identified in the rice and maize genomes, respectively [7,12]. In this study, we identified 50 BnHIRs, and these genes encode HIR proteins with an SPFH or PHB domain, consistent with canonical HIR proteins in other plants [36]. B. napus has more HIR genes than those plants, probably owing to additional whole-genome triploidy (WGT) during the evolutionary history of Brassica crops [15]. In higher plants, HIR genes can be divided into four classes, including I (HIR7 clade), II (HIR1/2/3 clade), III (PHB1/2/3/4 clade), and IV (PHB5/6 clade), some of which can be subdivided into smaller subclasses [7,12]. Phylogenetic analysis of the HIR genes of A. thaliana and B. napus was performed. They were divided into four groups according to the bootstrap value, structural and functional characteristics, and named as HIR subfamily (Group 1/2) and PHB (Group 3/4) subfamily, respectively. They were not completely the same as previous studies [11,37]. The BnHIR family genes have relatively diverse gene structure changes (Figure 2a). In addition, the homologous genes in each subfamily have similar characteristics, including gene structure, conserved motifs, and cis-acting elements, suggesting their functional conservation ( Figure 1). It is predicted that most BnHIR proteins were localized in mitochondria. Furthermore, several cis-acting elements have multiple copies in the BnHIR promoter region. This is particularly true for cis-acting elements that confer light and hormone responsiveness, which would enhance the response of the BnHIR gene to stress and developmental signals, and suggests that the BnHIR gene plays an important role in these processes. Furthermore, in B. napus, there are more members in Groups 1 and 4 than in Groups 2 and 3, suggesting that the ancestral evolutionary branch of Group 1/4 has undergone more expansion than the other two evolutionary branches. BnPHB1.3, BnPHB1.5, and BnPHB1.7 corresponded to AtPHB1.2 and independently became Group 3 in the phylogenetic tree, and they were not divided into the same group with other BnPHBs (Figure 1). This may indicate that the HIR genes in B. napus have differentiated and performed different functions.
The expression pattern of HIR genes has been studied in several species. In maize, the expression pattern was classified into three groups, A, B, and C, based on the results of cluster analysis. The expression of HIR genes in Group A was higher in the embryo and stem. HIR genes in Group B may play an important role in seed development, while HIR genes in Group C may alter the development and senescence of maize [12]. In B. napus, BnPHB3.2 and BnPHB3.3 in Group 4 were also highly expressed in seeds and embryos, suggesting that HIR genes play a considerable role in embryo and seed development. In soybean, GmHIR1 is highly expressed in flower buds, while GmHIR2 is mainly expressed in globular embryos. The majority of the BnHIR genes are not active in buds, which may be due to the function degradation in B. napus. In the GmHIR15 and GmHIR16 gene pair, the expression of GmHIR15 in leaves increased to a higher level than that of GmHIR16. In the GmHIR14 and GmHIR17 gene pair, GmHIR14 was expressed in seeds, but GmHIR17 was not [38]. This is basically consistent with the function of the BnHIR genes. Furthermore, in tomato (Solanum lycopersicum L.), the expression pattern of tomato SlHIR genes is spatially specific; for example, SlPHB4 and SlPHB10 were expressed in root and flower tissues, respectively. SlPHB8 and SlPHB9 were highly expressed in 2 cm fruit, while the expression patterns of SlPHB5, SlPHB14, and SlPHB15 increased with the fruit development stages [35]. In our study, most members of the BnHIR2/3 and BnPHB2/3 subfamilies were highly expressed in seeds, seed coats, and embryos during seed development and siliques during maturation. HIR genes were highly expressed in seeds and siliques in several species, suggesting that these genes have important roles in seed development and maturation. Compared with other species, however, the HIR gene in B. napus appears to have lost its function in promoting root development and promoting flowering. After inoculation with S. sclerotiorum, the comparison of gene expression between the two materials found that the expression levels of 31 HIR genes were up-regulated at 48 h and 96 h ( Figure S4c), while that of nine HIR genes were down-regulated ( Figure S4d), suggesting that most of the HIR genes had a positive response to the induction of S. sclerotiorum.
Some studies have been conducted on the function and mechanism of the HIR gene. AtPHB3 and AtPHB4 are mainly expressed in shoot and proliferative root tissues, and overexpressed AtPHB3 and AtPHB4 exhibit an irregular leaf shape and extensive branching phenotype [39,40]. It has been reported that NbPHB1 and NbPHB2 are prominently reduced during plant senescence. This may be because HIR proteins interact directly or indirectly with mitochondrial DNA (mtDNA) at the molecular level to regulate reactive oxygen species' (ROS) formation and oxidative phosphorylation (OXPHOS) [41][42][43][44]. HIR proteins may also be involved in the mitochondrial organization [45,46]. Athir2-1 and Athir3-1 mutants allow normal growth of Pto DC3000 AvrRpt2 but not Pto DC3000, and overexpression of AtHIR1 and AtHIR2 reduces Pto DC3000 growth [8]. In rice, overexpression of OsHIR1 could make plants more susceptible to hypersensitivity, resulting in a faster response to restrict the development of invading pathogens from the site of infection to otherwise healthy tissue [9]. Overexpression of CaHIR1 in pepper enhances basal resistance to infection by hemi-biotrophic bacteria or biotrophic oomycete pathogens [33]. Compared with Col-0, the lesion area of OV-BnHIR2.7 A. thaliana leaves was smaller, while Athir2.7-Mu A. thaliana leaves lesions expanded. These results indicate that when the expression level of BnHIR2.7 was low or silenced, plants could not effectively defend against S. sclerotiorum infection (Figure 6c). In addition, qRT-PCR results show that Col-0, OV-BnHIR2.7, and Athir2.7-Mu A. thaliana were prominently up-regulated after inoculation with S. sclerotiorum, suggesting that BnHIR2.7 responds to S. sclerotiorum. In addition to resistance to S. sclerotiorum, the A. thaliana Athir2.7-Mu and OV-BnHIR2.7 lines showed differences in biomass ( Figure S3c) and silique size ( Figure S3d) compared with Col-0. This may imply that BnHIR2.7 may be a potential gene with disease resistance and improved crop yield. In recent years, a series of investigations have been performed to study SD resistance in B. napus. Several transcription factors and kinases have been revealed to be associated with SD resistance signaling pathways, such as BnWRKY33 [47,48], BNWRKY 70 [49], and BnMPK3 [50]. In addition, rapid alkalinization factors (RALF), such as BnRALF10, induce multiple immune responses to exhibit resistance to S. sclerotiorum, including ROS accumulation, cytoplasmic Ca 2+ promotion, and defense-related gene expression induction [51]. It has also been speculated that Phloem proteins (PP2s) can cross-link together through disulfide bonds to form high molecular weight polymers, which block the sieve to form a physical barrier to slow mycelial colonization [1]. As a potential gene family for disease resistance, HIR may be located upstream of SA-and MeJA-related pathways and inhibit the spread of S. sclerotiorum by promoting the accumulation of SA and H 2 O 2 in the plant [14]. To clarify the possible mechanism of BnHIR2.7 disease resistance, we quantitatively analyzed the key genes of SA and MeJA pathways before and after S. sclerotiorum inoculation. qRT-PCR results showed that marker gene expressions in the SA and MeJA pathways were up-regulated, respectively, after heterologous overexpression of BnHIR2.7 Arabidopsis was inoculated with S. sclerotiorum ( Figure S3a), suggesting that HIR responds to biotic stress through, but not dependent on, SA or JA pathways. Next, further experiments will reveal the mechanisms of BnHIR2.7 to S. sclerotiorum resistance.

Conclusions
In this study, 50 BnHIR genes were identified in the B. napus genome based on publicly available genomic data. The identified BnHIR genes were phylogenetically divided into four groups, and their evolutionary relationships were analyzed. Under normal growth conditions, the majority of the BnHIR genes in Group 1 and Group 4 were highly expressed in leaves, siliques, embryos, and seeds. The HIR gene in Group 2 was low or not expressed in the detected tissues. Except for BnPHB1.7, other genes of Group 3 had high expression levels in the detected tissues. Following inoculation with S. sclerotiorum, the majority of the HIR genes in Group 1 and Group 4 were up-regulated. BnHIR2.7 was barely expressed in the susceptible cultivars but highly expressed in resistant materials. We experimentally verified that overexpression of BnHIR2.7 could inhibit the growth of S. sclerotiorum. In addition, our study found the role of BnHIR2.7 in promoting silique, which also provides clues for further research. These results may suggest further functional characterization of the BnHIR gene family of great value, especially when considering genetic improvements for agronomic traits and S. sclerotiorum resistance in B. napus.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/horticulturae8100874/s1, Figure S1: Statistics of cis-acting elements in the BnHIR promoter region. Figure S2: Relative expression level of Arabidopsis transgenic lines. Figure S3: Phenotype comparison of wild type, Athir2.7-Mu, and OV-BnHIR2.7 lines. Figure S4: Venn diagram of changes in HIR gene expression under Sclerotinia sclerotiorum infection; Table S1: Primers used in this study.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.