Global transcriptional analysis suggests Lasiodiplodia theobromae pathogenicity factors involved in modulation of grapevine defensive response

Abstract

Background: Lasiodiplodia theobromae is a fungus of the Botryosphaeriaceae that causes grapevine vascular disease, especially in regions with hot climates. Fungi in this group often remain latent within their host and become virulent under abiotic stress. Transcriptional regulation analysis of L. theobromae exposed to heat stress (HS) was first carried out in vitro in the presence of grapevine wood (GW) to identify potential pathogenicity genes that were later evaluated for in planta expression.

Results: A total of 19,860 de novo assembled transcripts were obtained, forty-nine per cent of which showed homology to the Botryosphaeriaceae fungi, Neofusicoccum parvum or Macrophomina phaseolina. Three hundred ninety-nine have homology with genes involved in pathogenic processes and several belonged to expanded gene families in others fungal grapevine vascular pathogens. Gene expression analysis showed changes in fungal metabolism of phenolic compounds; where genes encoding for enzymes, with the ability to degrade salicylic acid (SA) and plant phenylpropanoid precursors, were up-regulated during in vitro HS response, in the presence of GW. These results suggest that the fungal L-tyrosine catabolism pathway could help the fungus to remove phenylpropanoid precursors thereby evading the host defense response. The in planta up-regulation of salicylate hydroxylase, intradiol ring cleavage dioxygenase and fumarylacetoacetase encoding genes, further supported this hypothesis. Those genes were even more up-regulated in HS-stressed plants, suggesting that fungus takes advantage of the increased phenylpropanoid precursors produced under stress. Pectate lyase was up-regulated while a putative amylase was down-regulated in planta, this could be associated with an intercellular growth strategy during the first stages of colonization.

Conclusions: L. theobromae transcriptome was established and validated. Its usefulness was demonstrated through the identification of genes expressed during the infection process. Our results support the hypothesis that heat stress facilitates fungal colonization, because of the fungus ability to use the phenylpropanoid precursors and SA, both compounds known to control host defense.

Keywords: Botryosphaeria dieback; Fungal gene expression; Graevine vascular diseases; Melanin; RNA-seq; RT-qPCR; Transcriptome; Vitis vinifera.

Fig. 1

Contrasting conditions in RNAseq evaluated in general linear model statistics to identify differentially expressed genes. The conditions evaluated were from fungus grown in Vogel’s salts without heat stress (F) or under heat stress (FS), Vogel’s salts with 1 % of grapevine wood (GW), without heat stress (FW) or under heat stress (FWS). To evaluate the gene expression under the effect of heat stress (HS), the expression of FS was compared to F (FS/F). To test only the effect of HS when GW was present, FWS/FW ratio was measured, while to test both effects at the same time the ratio FWS/F was considered. Finally, fungal gene expression only as effect of GW, was measured evaluating FW/F ratio

Fig. 2

Pie chart describing the main Gene Ontology (GO) of molecular functions categories of transcripts with putative pathogenicity role. The predicted open reading frames encoded by transcripts were used in a local Blastp to find hits in the experimentally verified Pathogen-Host Interaction database (PHI-base). The PHI-base hits were classified based on its GO information

Fig. 3

Heatmap showing clusters of co-regulated genes with their expression profiles. The dendrogram indicates the relationship between gene expression profiles as determined by hierarchical clustering (Pearson correlation). The differentially colored boxes at the left indicate different clusters of genes with similar expression profiles. The color key indicates logFC values ranging from bright red for most up-regulated to bright green for the most down-regulated genes, considering a FDR < 0.01. The expression pattern for clusters with functional enrichment (clusters 1, 3 and 7) are shown in a graph with gray lines for each gene while the average for all the genes in the cluster is shown in red line. The contrasting conditions evaluated are those indicated in Fig. 1. Briefly, FS/F: effect of heat stress (HS); FWS/FW: effect of HS but in the presence of grapevine wood; FWS/F: effect of HS in the presence of grapevine wood and FW/F: effect of only GW

Fig. 4

Gene Ontology (GO) of molecular functions categories with enrichment in hierarchical clusters of similar expression profiles. The GO term differential expression is considered through z-score ($\mathrm{zscore}=\frac{\left(\mathrm{upregulated}‐\mathrm{downregulated}\right)}{\sqrt{\left(\mathrm{upregulated}+\mathrm{downregulated}\right)}}$, described in [53]). Red color indicates higher proportion of up-regulated genes and blue color corresponds to higher proportion of down-regulated genes. The color key indicates logFC values ranging from golden for up-regulated genes, to cyan for down-regulated genes. The effects of only grapevine wood (FW/F) and heat stress in the presence of grapevine wood (FWS/FW) were evaluated

Fig. 5

Functional enrichment of Gene Ontology (GO) categories based on the differentially expressed genes in each analyzed contrast. The color key indicate from pale yellow to blue, the increasing percentage of genes representing each functional category. To eliminate general and excessively specific categories, only the functional categories that have at least 5 and maximum 500 genes were considered in the analysis. The contrasting conditions evaluated are those indicated in Fig. 1. Briefly, FS/F: effect of heat stress (HS); FWS/FW: effect of HS but in the presence of grapevine wood; FWS/F: effect of HS in the presence of grapevine wood and FW/F: effect of only GW. * FDR < 0.2 and ** FDR < 0.05

Fig. 6

Gene Ontology (GO) of biological process categories with enrichment and showing differentially expressed genes in all the contrasting conditions evaluated (FDR < 0.05). The color key indicates logFC values ranging from golden for up-regulated genes, to cyan for down-regulated genes. The dendrogram indicates the relationship through the hierarchical clustering of gene expression (the middle rings show from the center out: effect on L. theobromae gene expression of heat stress (HS) and grapevine wood (FWS/F), only grapevine wood (FW/F), only HS (FS/F), and effect of HS in the presence of grapevine wood (FWS/FW). The Biological Process GOterms are indicated in different colors on the outermost circle

Fig. 7

L. theobromae gene expression during grapevine infection. Fungal gene expression was evaluated at 7, 11 and 15 days post-infection in unstressed or heat-stressed grapevine (HS). Normalization was done using β-tubulin and the relative gene expression is indicated as logFC (in planta/in vitro) using HTqPCR package in R. One asterisk indicates significant differences with general linear model statistics (p-values < 0.05) for in planta/in vitro and quotation marks for heat-stressed/non stressed contrasting conditions. The dendrogram indicates the relationship between gene expression profiles through calculates of Canberra distances and Pearson correlation. The color key indicates logFC values ranging from golden for up-regulated genes, to cyan for down-regulated genes. IRCD: intradiol-ring cleavage dioxygenase (comp4276_c0_seq1); SH: salicylate hydroxylase (comp12473_c0_seq1); FMH: fumarylacetoacetate hydrolase (comp14342_c0_seq1); SIT:sugar inositol transporter (comp8181_c0_seq1), MCO: multicopper oxidase (comp7300_c0_seq1), PL: pectate lyase (comp16237_c0_seq1), AML: amylase (comp7101_c0_seq1), HGD: homogentisate dioxygenase (comp8784_c0_seq1), CHD: choline dehydrogenase (comp5526_c0_seq1), GH3: glycoside hydrolase family 3 (comp13725_c0_seq1), XGH: xylosidase glycoside hydrolase (comp5761_c0_seq2), HPPD: 4- Hydroxyphenylpyruvate dioxygenase (comp18638_c0_seq1)

Fig. 8

Hypothetical model of in vitro L. theobromae heat stress (HS) response, in the presence of grapevine wood (GW). The model of whole cell response was constructed using genes from clusters 1, 3 and 7 (in Fig. 2) showing both co-regulation and GO term enrichment. Additional file 13 gives the gene name with its expression pattern. The illustration shows the main functions and processes being carried out by the fungus when growing on GW (a), and the changes that are proposed to be occurring after HS (b). The putative cellular location (intra or extracellular) is indicated in the scheme. A continuous arrow line indicates an enzymatic reaction that is supported on the differentially expression of its encoding gene (shown in red). A dashed arrow line indicates a suggested enzymatic reaction. The substrate/product of reaction is indicated on boxes. TYR: tyrosinase, MCO: multicopper oxidase, DOPA: L-3,4-dihydroxyphenylalanine, HPP: 4-hydroxyphenylpyruvate, HPPD: 4-hydroxyphenylpyruvate dioxygenase, HGD: homogentisate dioxygenase, HGA: homogentisic acid, 4-MAA: 4-maleylacetoacetate, 4-FMAA: 4-fumarylacetoacetate, FM: fumarate, AA: acetoacetate, FMH:fumarylacetoacetate hydrolase, SH: salicylate hydroxylase, IRCD: intradiol ring cleavage dioxygenase, DDP: domon domain containing protein, PCWDEs: Plant cell wall degrading enzymes; PCW: Plant cell wall

Fig. 9

The effect of heat stress (HS) on grapevines uninfected or infected with L. theobromae. Stereomicroscope images were taken at 15 days post-infection, corresponding to 10 days of HS treatment (day-night cycles, with temperatures ranging from 42 to 20 °C). Uninfected and unstressed grapevine show healthy green tissue (a). Uninfected and heat-stressed grapevine show a change to a white color of the pith and phloem impairment (b). Infected but unstressed grapevine show black, yellow and brown color tissues close to the site of inoculation (c). Infected and stressed grapevine show white pith and brown/yellow color in primary xylem (d)

Fig. 10

Proposed model of fungal-plant interaction under heat stress (HS). The model was done using gathered information of in vitro L. theobromae HS global transcriptional response, gene expression in planta (Fig. 7) and documented HS response in plant. a When fungus infects unstressed plant, selectively degrades pectin (up-regulation of PL compared with the other PCWDEs), this allows it to growth in intercellular spaces without causing major damage to PCW. The presence of the pathogen could trigger the host defensive mechanism mediated through SA, or by the activation of the phenylpropanoid pathway. However, the host defense could be impaired for the activity of SH and IRCD (degrade SA), or by HPPD (phenylpropanoid precursors). b Heat stress activates the same metabolic pathways increasing SA and the levels of phenylpropanoid precursors, thus the fungus could benefit from the higher availability of compounds using them as carbon sources. The main differential change on stressed plant, compared with unstressed, is the up-regulation of SH, FMH and XGH, whose activities could facilitate fungal colonization. Tyr: tyrosine. Phe: phenylalanine. SA: salicylic acid, IRCD: intradiol-ring cleavage dioxygenase (comp4276_c0_seq1); SH: salicylate hydroxylase (comp12473_c0_seq1); FMH: fumarylacetoacetate hydrolase (comp14342_c0_seq1); SIT:sugar inositol transporter (comp8181_c0_seq1); PL: pectate lyase (comp16237_c0_seq1). XGH: xylosidase glycoside hydrolase (comp5761_c0_seq2), HPPD: 4- Hydroxyphenylpyruvate dioxygenase (comp18638_c0_seq1). FCW: fungal cell wall. PCW: plant cell wall; PCWDEs: plant cell wall degrading enzymes