The Characterization of the Phloem Protein 2 Gene Family Associated with Resistance to Sclerotinia sclerotiorum in Brassica napus

Abstract

In plants, phloem is not only a vital structure that is used for nutrient transportation, but it is also the location of a response that defends against various stresses, named phloem-based defense (PBD). Phloem proteins (PP2s) are among the predominant proteins in phloem, indicating their potential functional role in PBD. Sclerotinia disease (SD), which is caused by the necrotrophic fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum), is a devastating disease that affects oil crops, especially Brassica napus (B. napus), mainly by blocking nutrition and water transportation through xylem and phloem. Presently, the role of PP2s in SD resistance is still largely estimated. Therefore, in this study, we identified 62 members of the PP2 gene family in the B. napus genome with an uneven distribution across the 19 chromosomes. A phylogenetic analysis classified the BnPP2s into four clusters (I-IV), with cluster I containing the most members (28 genes) as a consequence of its frequent genome segmental duplication. A comparison of the gene structures and conserved motifs suggested that BnPP2 genes were well conserved in clusters II to IV, but were variable in cluster I. Interestingly, the motifs in different clusters displayed unique features, such as motif 6 specifically existing in cluster III and motif 1 being excluded from cluster IV. These results indicated the possible functional specification of BnPP2s. A transcriptome data analysis showed that the genes in clusters II to IV exhibited dynamic expression alternation in tissues and the stimulation of S. sclerotiorum, suggesting that they could participate in SD resistance. A GWAS analysis of a rapeseed population comprising 324 accessions identified four BnPP2 genes that were potentially responsible for SD resistance and a transgenic study that was conducted by transiently expressing BnPP2-6 in tobacco (Nicotiana tabacum) leaves validated their positive role in regulating SD resistance in terms of reduced lesion size after inoculation with S. sclerotiorum hyphal plugs. This study provides useful information on PP2 gene functions in B. napus and could aid elaborated functional studies on the PP2 gene family.

Keywords: Brassica napus; Sclerotinia disease resistance; phloem protein 2; phylogenetic analysis.

Figure 1

The phylogenetic analysis of PP2 proteins in A. thaliana, O. sativa and B. napus. All PP2 proteins were clustered into four subfamilies (I–IV) with differently colored branches (I, green; II, orange; III, purple; IV, red). The gene IDs for B. napus are black, the gene IDs for A. thaliana are bright yellow and those for O. sativa are blue.

Figure 2

The gene structures and conserved motifs analysis of BnPP2 genes: (a) gene ID; (b) conserved motif distribution in BnPP2 proteins. Numbers 1–8 are displayed in differently colored boxes. Motif 1 is the F-box domain and motifs 2–7 are part of the PP2 domain, located at 16–44 aa,1–15 aa, 126–146 aa, 45–64 aa, 56–78 aa and 98–116 aa, respectively. Motif 3 and motif 2 are labeled as motif A, motif 4 is motif D, motif 7 is motif C and motifs 5 and 6 is motif B. Table S7 contains more detailed information. (c) The gene structures of BnPP2 genes. The dark brown boxes represent untranslated transcript regions (UTRs) and the blue boxes represent coding sequences (CDSs).

Figure 3

The expression patterns of the BnPP2 genes in clusters II–IV: (a) the expression heatmap of BnPP2 genes in various tissues from the root, stem, leaf, bud and silique; (b) the expression heatmap of BnPP2 genes in Westar and ZY821 cultivars at 0 and 24 h after S. sclerotiorum inoculation. The expression data were processed with the log2 normalization of fragments per kilobase million (FPKM).

Figure 4

The expression validation of 12 candidate BnPP2 genes in response to S. sclerotiorum by qRT-PCR. The time points 0 h, 12 h, 24 h, 36 h and 48 h represent hours after inoculation with S. sclerotiorum. The error bars show the standard error of three replicates.

Figure 5

(a) The hormone-related and stress-related cis-acting regulatory elements in the promoters of the BnPP2 genes in clusters II–IV. The bar graph indicates the total number of each cis-acting element found in the BnPP2 promoters (royal blue box), as well as the number of BnPP2 promoters that included a specific cis-regulatory element (red circle). (b) The gene ontology enrichment analysis of proteins that interacted with BnPP2 proteins. Table S4 contains more detailed information.

Figure 6

The genome-wide association analysis (GWAS) for SD resistance in a B. napus population comprising 324 accessions and Manhattan plots of SD resistance from association analyses: (a) Manhattan plots of the disease after 48–24 h; (b) Manhattan plots of the disease after 36–24 h; (c) Manhattan plots of the disease after 48 h; (d) Manhattan plots of the disease after 24 h. The red dashed line shows the GWAS threshold (1/SNP number).

Figure 7

The functional validation of BnPP2-6 for SD resistance using transient expression in tobacco: (a) the disease lesion sizes on leaves at 24, 36 and 48 h post inoculation with S. sclerotiorum, transiently injected through agrobacterium containing BnPP2-6-ox recombinant vectors and empty vectors (negative control) into tobacco leaves; (b) the disease lesion sizes were analyzed statistically by comparing the overexpressed BnPP2-6 to the control. The data represent the means ± 2 SDs from three independent experiments, each containing 20 leaves. Significant differences in lesion sizes between BnPP2-6- Ox and the control are indicated (Student’s t-test) as follows: *** p < 0.001.