Genome-wide identification and functional exploration of the legume lectin genes in Brassica napus and their roles in Sclerotinia disease resistance

Abstract

As one of the largest classes of lectins, legume lectins have a variety of desirable features such as antibacterial and insecticidal activities as well as anti-abiotic stress ability. The Sclerotinia disease (SD) caused by the soil-borne fungus Sclerotinia sclerotiorum is a devastating disease affecting most oil crops such as Brassica napus. Here, we identified 130 legume lectin (LegLu) genes in B. napus, which could be phylogenetically classified into seven clusters. The BnLegLu gene family has been significantly expanded since the whole-genome duplication (WGD) or segmental duplication. Gene structure and conserved motif analysis suggested that the BnLegLu genes were well conserved in each cluster. Moreover, relative to those genes only containing the legume lectin domain in cluster VI-VII, the genes in cluster I-V harbored a transmembrane domain and a kinase domain linked to the legume lectin domain in the C terminus. The expression of most BnLegLu genes was relatively low in various tissues. Thirty-five BnLegLu genes were responsive to abiotic stress, and 40 BnLegLu genes were strongly induced by S. sclerotiorum, with a most significant up-regulation of 715-fold, indicating their functional roles in SD resistance. Four BnLegLu genes were located in the candidate regions of genome-wide association analysis (GWAS) results which resulted from a worldwide rapeseed population consisting of 324 accessions associated with SD. Among them, the positive role of BnLegLus-16 in SD resistance was validated by transient expression in tobacco leaves. This study provides important information on BnLegLu genes, particularly about their roles in SD resistance, which may help targeted functional research and genetic improvement in the breeding of B. napus.

Keywords: Brassica napus; Sclerotinia sclerotiorum; genome-wide association study; legume lectin; phylogenetic analysis.

Figure 1

Chromosomal locations and duplicated gene analysis of BnLegLu genes in Brassica napus. The chromosomal locations of all BnLegLu genes are represented on different chromosomes, and different colors represent different BnLegLu subfamily genes. Subfamilies I to VII are indicated by black, purple, medium blue, dark turquoise, dark green, chocolate and slate blue color, respectively. Rose red lines are used to highlight the duplicated BnLegLu gene pairs.

Figure 2

Phylogenetic analysis of BnLegLu proteins in A. thaliana and B. napus. All legume lectin proteins were clustered into seven subfamilies (I–VII) with differently colored branches (I, green; II, blue; III, purple; IV, yellow; V, dark red; VI, rose red; VII, red). The gene IDs for B. napus are black, the gene IDs for A. thaliana are bright blue.

Figure 3

Schematic overview of the domain architecture in BnlegLus.

Figure 4

Analysis of the network for the interaction of BnLegLu proteins in B. napus. (A) Protein–protein interaction network of BnLegLu. The BnLegLu proteins are indicated by medium blue circles, and the green circles represent the proteins that interact with BnLegLu proteins. The red lines indicate the interaction between BnLegLu proteins, and the gray lines represent the interaction between BnLegLus and other proteins. (B) GO analysis of proteins interacting with BnLegLu proteins.

Figure 5

Expression patterns of the BnLegLu genes in Westar and ZY821 cultivars at 0 and 24 h after Sclerotinia sclerotiorum inoculation. The heatmap was generated by taking log2 fold of FPKM values. The color bar shows the relative expression from low (blue) to high (red).

Figure 6

Expression validation of 16 candidate BnLegLu genes in response to S. sclerotiorum determined by qRT-PCR. The time points 0, 12, 24, 36 and 48 h represent hours after inoculation with S. sclerotiorum. The error bars show the standard error of three replicates.

Figure 7

Functional validation of BnLegLu-16 for SD resistance. (A) Genome-wide association analysis (GWAS) for SD resistance in a B. napus population comprising 324 accessions and Manhattan plots of SD resistance from GWAS; Manhattan plots of the disease after 36 h; the red dashed line shows GWAS threshold (1/SNP number). (B) Haplotype analysis of BnLegLu-16. (C) Subcellular localization of BnLegLu-16-ox in tobacco, BnLegLu-16-GFP fusion protein was transiently co-expressed with the mCherry–H⁺-ATPase, which was used as the plasma membrane marker in tobacco leaves. GFP and mCherry were transiently co-expressed as the control. After 48 h post-incubation, the tobacco leaves were observed with confocal microscope (ZEISS LSM710, Germany). (D) Disease lesion sizes on leaves at 24, 36 and 48 hpi with S. sclerotiorum, which was transiently injected through agrobacterium containing BnLegLu-16-ox and the control into tobacco leaves. (E) Disease lesion sizes statistically analyzed by comparing the overexpression BnLegLu-16-ox to the control. The data represent the means ± 2 SD from three independent experiments, with each containing 20 leaves. Significant differences in lesion size between BnLegLu-16-ox and the control are indicated (Student’s t-test) as follows: ***p < 0.001). The error bars show the standard error of three replicates.