Identification and analysis of major latex protein (MLP) family genes in Rosa chinensis responsive to Botrytis cinerea infection by RNA-seq approaches

Abstract

Roses (Rosa chinensis) are among the most cherished ornamental plants globally, yet they are highly susceptible to infections by Botrytis cinerea, the causative agent of gray mold disease. Here we inoculated the resistant rose variety 'Yellow Leisure Liness' with B. cinerea to investigate its resistance mechanisms against gray mold disease. Through transcriptome sequencing, we identified 578 differentially expressed genes (DEGs) that were significantly upregulated at 24, 48, and 72 hours post-inoculation, with these genes significantly enriched for three defense response-related GO terms. Further domain analysis of the genes in these GO terms reveal that 21 DEGs contain the Bet v 1 family domain, belonging to the major latex protein (MLP) gene family, suggesting their potential key role in rose disease resistance. Furthermore, we systematically identified 46 RcMLP genes in roses and phylogenetically categorized them into two distinct subfamilies: group I and II. Genomic duplication analysis indicates that tandem duplication is the main driver for the expansion of the RcMLP family, and these genes have undergone by purifying selection. Additionally, detailed analyses of gene structure, motif composition, and promoter regions reveal that RcMLP genes contain numerous stress-responsive elements, with 32 RcMLP genes harboring fungal elicitor/wound-responsive elements. The constructed potential transcription factor regulatory network showed significant enrichment of the ERF transcription factor family in the regulation of RcMLP genes. Gene expression analysis reveal that DEGs are mainly distributed in subfamily II, where four highly expressed genes (RcMLP13, RcMLP28, RcMLP14, and RcMLP27) are identified in a small branch, with their fold change exceeding ten folds and verified by qRT-PCR. In summary, our research results underscore the potential importance of the RcMLP gene family in response to B. cinerea infection and provide comprehensive basis for further function exploration of the MLP gene family in rose resistance to fungal infections.

Keywords: Botrytis cinerea; RNA-Seq; major latex protein; rose; tandem duplication.

Figure 1

Development of B. cinerea on rose petal and analysis of DEGs in roses across control and time-based infections. (A) Variably severe disease lesions observed in two rose cultivars post-inoculation under different time of treatment. The volcano plots show DEGs of ‘Yellow Leisure Liness’ cultivar between uninfected rose petals (0 hour) and infected by B. cinerea at 24 hours (B), 48 hours (D), and 72 hours (F), based on the criteria of log2 fold change (FC) > 1 and FDR value < 0.01. Heatmaps show the expression levels of DEGs in different samples at 24 hours (C), 48 hours (E), and 72 hours (G) post-infection with B. cinerea. Data were homogenized by Z-score.

Figure 2

Identification of candidate B. cinerea infection-regulated rose genes. (A) Venn diagram depicting number and overlap among upregulated DEGs from each time point. (B) The bar plot illustrates the top 10 Biological Process (BP) GO terms enriched with commonly upregulated DEGs in (A), highlighting the three most significant infection-related terms in red, and the genes enriched within these terms are considered potential candidates. (C) Heatmap depicting the expression patterns and protein domain annotations of the candidate genes identified in (B). Data normalized using Z-score method. FC represents fold change between control and different infection time points.

Figure 3

Evolution analysis of RcMLPs in rose. (A) The multiple sequence alignment of RcMLPs Bet v1 domain sequences. Sequence logo of Bet v1 domain, generated by WebLogo. We have marked the positions of the 3-D structure predicted in (B) atop the aligned 1D sequence diagrams. (B) The 3D structure model of Bet v1 domain predicted by AlphaFold3. (C) The ML phylogenetic tree of 117 MLPs from four species.

Figure 4

Collinearity analysis of RcMLP gene family. (A) Chromosomal locations and their synteny of RcMLP genes in rose. Gray lines indicate all synteny blocks in the rose genome and red line indicates segmental duplication of RcMLP genes. (B) Synteny analysis RcMLP genes between rose and R. roxburghii, A. thaliana, and O. sativa, respectively. The syntenic MLP gene pairs were highlighted in red lines.

Figure 5

The phylogenetic tree, gene structures, protein motifs, and cis-regulatory elements analysis among RcMLPs. (A) The phylogenetic tree of RcMLPs was built with full-length protein sequences by the maximum likelihood (ML) method. (B) The gene structures of RcMLP gene family genes. Yellow boxes, green boxes and black horizontal lines displayed the CDS regions, UTR regions and introns, respectively. (C) The 9 conserved motifs of RcMLP gene family were represented by boxes with different colors. (D) Cis-regulatory elements (CREs) distribution of each RcMLP was shown.

Figure 6

The putative TFs regulatory network analysis of RcMLPs. (A) All putative transcription factors (TFs) were represented by diamond nodes, RcMLPs were represented by rectangle nodes. The putative TFs and related mediated RcMLPs were linked by grey lines. (B) Wordcloud for TFs. Font size is positively correlated with the gene number of corresponding TFs. (C) Top 10 highly enriched and targeted RcMLPs were shown and the darker the color shows highly enriched. (D) The number of top 10 enriched TFs distributions across the targeted RcMLP genes.

Figure 7

Expression patterns of RcMLP family at different time points during B. cinerea infection of rose. (A) The heatmap shows the expression levels of 46 genes encoding MLP gene family in rose petals at 0 hours (uninfected control) and at 24, 48, and 72 hours post-B. cinerea infection. (B) qRT-PCR results of eight selected RcMLPs genes under B. cinerea infection stress. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.