Transcriptome and VcCADs gene family analyses reveal mechanisms of blight resistance in rabbiteye blueberry

Abstract

Introduction: Blueberry branch blight is a severe plant disease primarily caused by Neofusicoccum parvum.

Methods: In this study, one-year-old branches of two rabbiteye blueberries (Vaccinium virgatum): resistant Tifblue ('TF') and susceptible Climax ('DF') were selected as experimental materials, the physiological parameters of two cultivars pre- and post- N. parvum inoculation. Transcriptomic analysis and comprehensive gene family characterization were conducted to examine the differential gene expression.

Results: The results indicated that 'TF' exhibited higher lignin content and activity levels of peroxidase enzymes (POD), phenylalanine ammonia-lyase (PAL), and cinnamyl alcohol dehydrogenase (CAD) compared to 'DF'. RNA-sequence analysis revealed that at 24 hours post-inoculation, there were 136 and 121 differentially expressed genes related to lignin synthesis in 'TF' and 'DF', respectively. The analysis of these DEGs revealed that numerous of key enzyme genes in lignin synthesis pathways, including PAL, 4CL, CCR, CAD, HCT, COMT and POD were annotated. Additionally, we characterized the CAD gene family, identifying 93 CAD coding genes at the blueberry genome level, which were classified into four subgroups. Most CAD gene promoters contained response elements associated with plant stress resistance pathways. QRT- PCR experiments demonstrated that the expression levels of VcCAD8 and VcCAD62 genes were significantly upregulated at 24 hours post-inoculation.

Discussion: These findings suggest that VcCADs enhance lignin synthesis, improve the resistance of blueberries to shoot blight, and provide novel insights into the defense mechanisms of blueberries against N. parvum.

Keywords: Neofusicoccum parvum; cinnamyl alcohol dehydrogenase; lignin; rabbiteye blueberry; shoot blight.

Figure 1

Symptoms of one-year-old branches of different blueberry varieties after N. parvum infection.

Figure 2

Effect of inoculation with N. parvum on chlorophyll content, soluble protein content, and lignin content in blueberries. (A) chlorophyll a content, (B) chlorophyll b content, (C) soluble protein content, (D) lignin content. Values are means ± SD (n = 4). Differences in comparisons between data from the same variety of blueberry inoculated at different times were tested using Duncan's method of one-way ANOVA (one-way ANOVA) with a significance level of P < 0.05. The same lowercase letters indicate no significant difference as determined by Duncan's test (P ≥ 0.05), while different lowercase letters indicate significant differences (P < 0.05).Data from different varieties of blueberries with the same inoculation time were processed using t-test, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. ns indicates no significant difference. 'TF', 'Tifblue' blueberry; 'DF', 'Climax' blueberry. Two-years-old plants were used as sample. The experiment was repeated three biological replicates.

Figure 3

Effect of inoculation with N. parvum on POD, PAL, and CAD enzyme activities of blueberry. (A) POD, (B) PAL, (C) CAD. The note was the same to Figure 2 . Differences in comparisons between data from the same variety of blueberry inoculated at different times were tested using Duncan's method of one-way ANOVA (one-way ANOVA) with a significance level of P < 0.05. The same lowercase letters indicate no significant difference as determined by Duncan's test (P ≥ 0.05), while different lowercase letters indicate significant differences (P < 0.05). Data from different varieties of blueberries with the same inoculation time were processed using t-test, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. ns indicates no significant difference. 'TF', 'Tifblue' blueberry; 'DF', 'Climax' blueberry. Two-years-old plants were used as sample. The experiment was repeated three biological replicates.

Figure 4

Analysis of DEGs of blueberry inoculated with N. parvum. (A) gene ontology (GO) functional classification of top 30 differently expressed genes in the comparison of in 'TF'24 vs 'DF'24, (B) the first 20 ways of KEGG enrichment analysis of differential genes in the comparison of in 'TF'24 vs 'DF'24, (C) number of differentially expressed genes in phenylpropanoid biosynthesis pathway ('TF'24 vs 'TF'0, 'DF'24 vs 'DF'0 and 'TF'24 vs 'DF'24).

Figure 5

Differentially expressed genes related to lignin biosynthetic pathway in blueberry inoculated with N. parvum. (A) 'TF'24 vs 'TF'0, (B) 'DF'24 vs 'DF'0, (C) 'TF'24 vs 'DF'24.

Figure 6

Analysis of DEGs of lignin synthesis pathway in blueberry. (A) the Wayne diagram, (B) the heat map of DEGs between 'TF'24 vs 'TF'0 and 'DF'24 vs 'DF'0, (C) the heat map of DEGs between 'TF'24 vs 'TF'0 and 'TF'24 vs'DF'24, (D) the heat map of DEGs among 'TF'24 vs 'TF'0, 'DF'24 vs 'DF'0 and 'TF'24 vs 'TF'24.

Figure 7

Identification of blueberry CAD gene family and physicochemical characterization of encoded proteins. (A) the phylogenetic tree, (B) collinear analysis, (C) chromosome mapping of members.

Figure 8

The heatmap showing expression analysis of VcCADs family genes of blueberry inoculated with N. parvum.

Figure 9

Expression analysis of the VcCAD genes were determined by qRT-PCR of blueberry inoculated with N. parvum. (A) VcCAD8, (B) VcCAD19, (C) VcCAD21, (D) VcCAD58, (E) VcCAD62, (F) VcCAD68, (G) VcCAD72, (H) VcCAD93. Data from different varieties of blueberries with the same inoculation time were processed using t-test, **P < 0.01, ***P < 0.001.

Figure 10

The pathway diagram of CAD genes regulating lignin biosynthesis for blueberry inoculated with N. parvum.