Clavibacter michiganensis Downregulates Photosynthesis and Modifies Monolignols Metabolism Revealing a Crosstalk with Tomato Immune Responses

Abstract

The gram-positive pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial canker disease in tomato, affecting crop yield and fruit quality. To understand how tomato plants respond, the dynamic expression profile of host genes was analyzed upon Cmm infection. Symptoms of bacterial canker became evident from the third day. As the disease progressed, the bacterial population increased in planta, reaching the highest level at six days and remained constant till the twelfth day post inoculation. These two time points were selected for transcriptomics. A progressive down-regulation of key genes encoding for components of the photosynthetic apparatus was observed. Two temporally separated defense responses were observed, which were to an extent interdependent. During the primary response, genes of the phenylpropanoid pathway were diverted towards the synthesis of monolignols away from S-lignin. In dicots, lignin polymers mainly consist of G- and S-units, playing an important role in defense. The twist towards G-lignin enrichment is consistent with previous findings, highlighting a response to generate an early protective barrier and to achieve a tight interplay between lignin recomposition and the primary defense response mechanism. Upon progression of Cmm infection, the temporal deactivation of phenylpropanoids coincided with the upregulation of genes that belong in a secondary response mechanism, supporting an elegant reprogramming of the host transcriptome to establish a robust defense apparatus and suppress pathogen invasion. This high-throughput analysis reveals a dynamic reorganization of plant defense mechanisms upon bacterial infection to implement an array of barriers preventing pathogen invasion and spread.

Keywords: RNA-seq; Solanum lycopersicum; defense lignin; gram-positive; monolignols; phenylpropanoids; photosynthesis; plant immunity; plant pathogen interaction; transcriptomics.

Figure 1

Disease symptoms in tomato plants infected with Cmm. (A) Bacterial canker lesions on inflorescence stems at 3, 6, 9, 12, 15 and 18 dpi. The dashed boxes in white indicate the symptoms. (B) Bacterial growth in tomato stem samples, which were harvested at different time points during an 18-day period after inoculation. Data represent the mean ± SD (n = 4). (C) Effect of Cmm infection on total chlorophylls of the true leaves. The relative chlorophyll content was calculated by comparing the concentration of chlorophylls quantified in mg/g of fresh weight between Cmm infected and control plants. Data are shown as means ± SD (n = 3). Asterisks indicate significant differences (t-test) between Cmm infected and control plants (p ≤ 0.05). (D) Trypan blue stained cross-sections at 6 and 12 dpi stems of tomato plants; e: epidermis, ct: cortex, x: xylem, pi: pith. (Scale bar = 500 μm).

Figure 2

Overview of differentially expressed genes (DEGs) upon Cmm infection at 6 and 12 dpi. (A) Comparative analysis of gene expression distribution among the samples of control and Cmm infected plants. Red bars depict the highly expressed genes (FPKM ≥ 10), blue bars the genes with intermediate level of expression (1 < FPKM < 10), while green bars depict genes with low expression levels (FPKM ≤ 1). Values are mean ± standard deviation of three biological replicates of each sample. * p < 0.05 indicates significant difference between control and Cmm infected samples judged by Student’s t-test. (B) The number of genes that were significantly upregulated or downregulated in response to Cmm infection at 6 and 12 dpi. (C) Venn diagram showing a pairwise comparison of the number of upregulated and downregulated genes at 6 and 12 dpi of Cmm infection. Padj < 0.05 and log₂FC ≥ 1 or ≤ −1.

Figure 3

Differentially expressed genes of Cmm infected tomato stems. (A) Volcano plots of differentially expressed genes (DEGs) identified by RNA-seq analysis of Cmm infected versus control plants at 6 (left panel) and 12 dpi (right panel). Red and blue dots indicate up- and down- regulated genes, respectively. Green dots with numbers indicate genes selected for qRT-PCR validation of the transcriptome analysis results. (B) Comparative analysis of gene transcription results derived from qRT-PCR and RNA-seq transcriptome analysis. Green bars represent the fold change value calculated by qRT-PCR analysis as the log2 ratio between the quantitative median expression of the gene in Cmm infected plants relative to the control (n = 4). Red bars represent the fold change value expressed as the log2 between the ratio of the mean FPKM gene expression in Cmm infected and control plants obtained by RNA-seq analysis (n = 3). Positive values correlate with upregulated gene expression, whereas negative values with downregulation of gene (ACO5) expression.

Figure 4

Sorting of tomato DEGs upon Cmm infection. (A) Hierarchical clustering and heat map of the 5527 DEGs showing the expression pattern of Cmm responsive genes at both time points. The red circles designate the nodes of the three major groups: the first group includes Clusters I–III of the upregulated DEGs, the second group Clusters IV–V of the downregulated DEGs and the third group Clusters VI–VII of DEGs without a distinctive pattern of expression between the two time points. (B) Expression pattern of the 5527 DEGs within the eight clusters. The y-axis represents the level of gene expression, whereas the x-axis represents the type of samples. Each line represents the pattern of expression of an individual gene within each Cluster. Red to gray scaling shows the distance of each gene from the center of the Cluster according to k-means clustering, in terms of gene expression pattern. In parenthesis the gene number of each cluster. CT: control plants and Cmm: Cmm infected plants.

Figure 5

Functional classification of Cmm responsive genes within Clusters II, III and V. (A) Gene ontology (GO) analysis of differentially enriched tomato genes. (B) Classification into functional KEGG pathways of DEGs. The fractions on the right display the number of DEGs identified to the total number of genes of each pathway.

Figure 6

Host genes of the plant-pathogen interaction pathway activated by Cmm. (A) Heat map visualizing the stimulated expression pattern of genes involved in the plant-pathogen interaction pathway of Clusters II and III. (B) Graphical presentation of the expression fold change (Cmm/control) of genes controlling plant defense-immunity pathways at 6 and 12 dpi. HR: hypersensitive response; PCD: programmed cell death.

Figure 7

Tomato genes encoding protein components of the photosynthesis complexes were downregulated by Cmm. (A) Heat map visualizing the downregulation of genes involved in photosynthesis of Cluster V. (B,C) Comparative presentation of Cmm infection on the expression fold change (Cmm/control) of genes encoding components of tomato light harvesting complexes (B) and photosynthetic apparatus (C). In (C), the cells in dark green color represent components of tomato photosynthesis that has not been annotated yet.

Figure 8

Cmm infection differentially regulated the tomato phenylpropanoid biosynthetic pathway genes. (A,B) Heat map of tomato DEGs involved in phenylpropanoid metabolism. (A) Genes within Clusters II, III and V that were constantly up- or down-regulated at both time points of Cmm infection. (B) Additional genes of the pathway without a constant expression pattern enclosed in Clusters I, IV and VIII. (C) Presentation of the relative expression change of genes encoding key enzymes of the monolignol biosynthesis pathway. In phenylpropanoid metabolism, the genes that were differentially expressed upon Cmm infection leading to hydroxycinnamyl alcohols which are polymerized into lignin are: L-Phenylalanine Ammonia-Lyase (PAL), Cinnamic acid 4-Hydroxylase (C4H), 4-hydroxycinnamate CoA Ligase (4CL), Hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl Transferase (HCT), Coumaroyl shikimate 3′-Hydroxylase (C3′H), Caffeoyl CoA 3-O-MethylTransferase (CCoAOMT), Ferulic acid/coniferaldehyde 5-Hydroxylase (F5H), Caffeic acid/5-hydroxyconiferaldehyde 3/5-O-MethylTransferase (COMT) and Cinnamyl Alcohol Dehydrogenase (CAD). The lower branch of the pathway demonstrating the suppressed genes at 6 dpi, modulating the synthesis of syringyl (S) lignin units, is highlighted.