System-wide hypersensitive response-associated transcriptome and metabolome reprogramming in tomato

Abstract

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR.

Figure 1.

Transcriptome profiles of the Cf-4/Avr4 DS and their PLS before (0 h) and 1, 3, and 5 h after the temperature shift that initiates the HR in the DS. A, HCA of the transcriptome profile. For each time point, the columns labeled “DS” or “PLS” represent gene expression data obtained in three independent replicate experiments. Marked changes in the gene expression profiles of the DS and the PLS at 0 h and at 3 and 5 h after the temperature shift are indicated by the yellow-boxed gene sets. At 0 h, an additional gene set selection was performed to group genes based on their expression profiles. 0_U, Up-regulated at 0 h; 0_D, down-regulated at 0 h; 35_D, down-regulated at 3 and 5 h; 35_U, up-regulated at 3 and 5 h. The bar at bottom shows the color code for the relative change in gene expression of log₂-transformed and autoscaled data. B, Venn diagram showing the temporal distribution of unique and overlapping genes differentially regulated upon comparison of the averages between the three biological replicates of the DS and those of the PLS for the different time points. The top panel shows genes up-regulated (U) in the DS compared with the PLS, and the bottom panel shows genes down-regulated (D) in the DS compared with the PLS. Numbers in parentheses represent the number of differentially regulated genes that are unique or overlapping at time points 0, 1, 3, and/or 5 h after the temperature shift.

Figure 2.

Visualization of the transcriptional reprogramming occurring in the DS as compared with their PLS at 0, 1, 3, and 5 h after the temperature shift that induces the HR in the DS. Each panel represents an individual time point and depicts the expression profile of genes (represented by the colored squares) involved in the indicated biological processes. The bar at bottom shows the color codes for the FC in gene expression in the DS compared with the PLS. The map does not represent all differentially regulated genes, as only genes that can be related to specific biological processes are displayed. ABA, Abscisic acid; BA, benzyladenine; IAA, indole-3-acetic acid; JA, jasmonic acid; SA, salicylic acid.

Figure 3.

ORA of the differentially regulated genes in the DS when compared with the PLS at 3 and 5 h after the temperature shift that induces the HR in the DS. A similarity matrix forms a rectangle that is a mirror image of two identical triangles; therefore, only the right triangle is shown. The red triangles along the long sides of the triangles represent clusters of highly similar GO subcategories, whereas the blue blocks represent clusters of unrelated subcategories. A, Similarity matrices (GO subcategories × GO subcategories) of the overlapping genes differentially expressed at time points 3 and 5 h. B, Up-regulated genes unique for time point 5 h. C, Down-regulated genes unique for time point 5 h. ABA, Abscisic acid; JA, jasmonic acid; NA, nucleic acid; NB, nucleobase; NS, nucleoside; NT, nucleotide; ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired resistance. The strength of the correlation is indicated by the color bar on the left: red, strong correlation; blue, not correlated. For details, see Supplemental Materials and Methods S1.

Figure 4.

Coexpression analysis of genes differentially regulated during the HR and identification of central regulators in the regulatory network. A, Coexpression networks, representing all differentially regulated genes that have a correlation coefficient of 0.92 or greater, at 0, 1, 3, and 5 h after the temperature shift that induces the HR in the DS. Shown is an output of Cytoscape software that was used to build a coexpression network of all 1,152 genes that are significantly differentially regulated between the DS and the PLS under at least one of the conditions analyzed. The dots represent individual genes (the “nodes”), and the length of the connecting lines (the “edges”) between them is a measure of their coexpression. Short edges indicate strong coexpression, whereas long edges indicate weak coexpression. Four clusters of strongly coregulated genes were identified. The bar at bottom shows the color code for the FC in gene expression. B, MapMan representation of genes involved in the proteasome pathway, depicting differentially regulated genes at 5 h after the temperature shift involved in ubiquitin activation (E1), ubiquitin conjugation (E2), and ligation of ubiquitin to proteins that are targeted for degradation (E3). SKP, Cullin, and FBOX together constitute the SCF complex. RBX (for ring box protein), APC (for anaphase-promoting complex), HECT (for homology to E6-AP C terminus), and RING (for really interesting new gene) are different protein complexes in the E3 ubiquitin ligase family. DUB, Deubiquitinating enzyme. Red ovals represent individual ubiquitin proteins. C, POBO output interface indicating a “good” motif with spread sample distribution. In this case, the test set (all cluster 1 genes) and the background (the promoter sequences of all genes of Arabidopsis) as well as the cluster 4 gene set (genes with an opposite expression pattern to the test set) are well separated. This indicates a significant difference in the occurrence of the motif searched for among these clusters (in this case, TTTGAC, the W-box motif). “Bad” motifs are characterized by uniform sample distribution, indicating an absence of variation in the occurrence of the motif (cluster 4 and background).

Figure 5.

Ethylene emission and ACS2 gene expression of the seedlings and C. fulvum-inoculated tomato, respectively. A, Ethylene emission patterns of the DS and their PLS upon rescue at 33°C and 100% RH and after a shift to 20°C and 100% RH. B, Relative expression levels of the ACS2 gene in resistant (R; MM-Cf0:Cf-4) and susceptible (S; MM-Cf0:Avr4) parental lines at 6 and 10 d post inoculation (dpi) with C. fulvum. Bars represent expression of the gene (FC) in inoculated plants relative to the mock-inoculated resistant and susceptible PLS.

Figure 6.

Metabolome reprogramming in the DS as compared with their PLS. Shown are the metabolome profiles, comprising all differentially regulated PPM and SPSM, of the DS as compared with the PLS at 0, 1, 3, and 5 h after the temperature shift. A, Principal component analysis depicting metabolome reprogramming in the DS as compared with the PLS. PC1 and PC2 represent principal components 1 and 2, respectively. B, HCA of the PPM and SPSM profiles. The bar at bottom shows the color code for the relative change in metabolite accumulation of log₂-transformed and autoscaled data. The enlarged part of the heat map at right indicates the aromatic amino acid-derived metabolites that differentially accumulate in the DS at time points 3 and 5 h after the temperature shift. OA, Clade containing most of the organic acids that are differentially down-regulated in the DS.

Figure 7.

Alternative energy and carbon skeleton generation strategies of tomato DS undergoing massive transcriptome and metabolome reprogramming. A, Cell wall invertase-mediated generation of Glc from monosaccharides, disaccharides, and polysaccharides to maintain a continuous supply for the tricarboxylic acid cycle and thereby for amino acid biosynthesis. The gray and black rectangles represent biosynthesis and degradation processes, respectively. B, Expression levels of alcohol dehydrogenase (ADH) and LDH genes in the DS as compared with their PLS at 5 h. C, Redirection of common precursors, such as Phe and Tyr, toward targeted metabolite pathways (red and green shapes represent up-regulation and down-regulation of a gene [circles] or a metabolite [rectangles], respectively). CHS, Chalcone synthase; PAL, Phe ammonia lyase; PK, pyruvate kinase; TyrDC, Tyr decarboxylase.