Comparative genome and transcriptome analyses reveal adaptations to opportunistic infections in woody plant degrading pathogens of Botryosphaeriaceae

Abstract

Botryosphaeriaceae are an important fungal family that cause woody plant diseases worldwide. Recent studies have established a correlation between environmental factors and disease expression; however, less is known about factors that trigger these diseases. The current study reports on the 43.3 Mb de novo genome of Lasiodiplodia theobromae and five other genomes of Botryosphaeriaceae pathogens. Botryosphaeriaceous genomes showed an expansion of gene families associated with cell wall degradation, nutrient uptake, secondary metabolism and membrane transport, which contribute to adaptations for wood degradation. Transcriptome analysis revealed that genes involved in carbohydrate catabolism, pectin, starch and sucrose metabolism, and pentose and glucuronate interconversion pathways were induced during infection. Furthermore, genes in carbohydrate-binding modules, lysine motif domain and the glycosyl hydrolase gene families were induced by high temperature. Among these genes, overexpression of two selected putative lignocellulase genes led to increased virulence in the transformants. These results demonstrate the importance of high temperatures in opportunistic infections. This study also presents a set of Botryosphaeriaceae-specific effectors responsible for the identification of virulence-related pathogen-associated molecular patterns and demonstrates their active participation in suppressing hypersensitive responses. Together, these findings significantly expand our understanding of the determinants of pathogenicity or virulence in Botryosphaeriaceae and provide new insights for developing management strategies against them.

Keywords: Lasiodiplodia theobromae; de novo assembly; RNASeq; low-depth sequencing; virulence factors.

Figure 1

Differences in the virulence and the phylogenomic analyses of the fungi used in the current study. (A) Differences in the virulence of isolates used for whole genome analyses on grapevine. Results indicate Lasiodiplodia theobromae as the most virulent among the three species. CK indicate the control for which normal PDA plug was used without any fungal inoculum. Figures showed the results of the pathogenicity test conducted for all the isolates used in the study. (B) Phylogenomic analyses for evolutionary relationships among different fungi. Time scale of the neighbour-joining phylogenomic tree was shown by million years ago at each node. Estimates of divergence times (millions of years) calculated from the rate of sequence similarity are indicated at each node. The red dots are the fossil calibrations from the Time Tree database.

Figure 2

Overview of the gene family size comparison between the L. theobromae (CSS-01s) and other fungi. (A and B) Venn diagram of predicted gene families in (A) L. theobromae, B. dothidea and N. parvum and (B) Botryosphaeriaceae family with other fungi. The values explain the union counts of orthologous gene families shared among different fungi groups. Botryosphaeriaceae taxa share the highest number of unique gene families with opportunistic pathogens. (C) Top 20 Pfam domains in L. theobromae CSS-01s genome, compared with those in other fungi used in the comparative analysis. (D) Pair-wise comparison of Pfam gene family sizes between L. theobromae CSS-01s and other fungi used in the comparative analysis.

Figure 3

Difference in the virulence of Lasiodiplodia theobromae and their gene expression at different stages of growth and at different temperatures. (A) Difference in the virulence of L. theobromae at different infection stages on grape shoots. Figure shows the progress of infection of L. theobromae isolate CSS-01s at (i) 8, (ii) 12 and (iii) 24 hpi. (B) Differences in the virulence of L. theobromae isolate CSS-01s at different temperatures on grape shoots and their growth differences on the axenic cultures at respective temperatures: (i) control test with PDA without inoculum, (ii) virulence at 25 °C (iii) virulence at 35 °C, (iv) axenic culture growth at 25 °C and (v) culture growth at 35 °C. (C) Venn diagram showing in planta differentially expressed genes of L. theobromae at different time points after inoculation. Numbers indicate differentially expressed genes that are specific to each time point, or shared by two different time points. Differentially expressed genes at 8 and 12 hpi are represented with circles. (D) Up-regulation of LysM domain genes of L. theobromae during transcriptome analysis. L indicates samples collected from axenic cultures, M indicates samples of fungus and host tissues. Two different temperatures of 25 °C and 35 °C indicated by numbers 25 and 35. Two sample collection time points were indicated by 8 and 12 hpi.

Figure 4

Gene family expansions and contractions of L. theobromae, and up-regulation of gene expressions during infection. (A) Comparison of membrane transporter families in L. theobromae (CSS-01s). The families with red colour mean expansion in the L. theobromae genome, whereas the blue means contraction. (B) Plots of up-regulated CWDE gene expressions in each family during the infections of L. theobromae. Transcriptomic analysis revealed several CWDE belonging to different families up-regulated during infection. These gene families were involved in pathogenicity processes of the fungi. PL indicates pectin lyases.

Figure 5

Putative effectors and their hypersensitive responses in N. benthamiana. Putative Botryosphaeriaceae effectors suppressed B. glumae-induced hyposensitive response in N. benthamiana. Representative cell death symptoms were photographed at 2 days after B. glumae inoculation. The right half leaf sections were injected with B. glumae with the pEDV empty vector and the left half sections were injected with B. glumae with effector gene constructs. Black circle represents pEDV empty vector, red circle represents effectors specific to Botryosphaeriaceae, orange circle represents commons effectors in Botryosphaeriaceae and the purple circle represents leaves injected with 0.9%NaCl.

Figure 6

Phylogentic analyses, gene expression and pathogenicity assays conducted for glycosyl hydrolase family genes. (A) One of the 1000 most parsimonious trees obtained from a heuristic search of glycosyl hydrolase family genes and 36 related sequences from closely related species. Parsimony bootstrap support values ≥ 50% are indicated at the nodes and branches and the sequences from this study are given in bold. (B) Gene expression analysis of selected glycosyl hydrolase family genes. This includes the gene expression at different conditions (on axenic cultures and on grape shoots after inoculation at 25 °C and at 35 °C) and at different infection stages (8 and 12 hpi). (C) Lesions on stems were photographed at 3 days post-inoculation of the above L. theobromae transformants. WT indicates the wild-type strain of L. theobromae CSS-01s and numbers 1–10 represent the replicates of each overexpressing transformants conducted for each gene. First 10 stems were inoculated with the GH 2.1698 overexpressing transformants of L. theobromae, and the second 10 stems were inoculated with the GH 7.188 overexpressing transformants of L. theobromae. (D) Bar chart showing the mean lesion lengths of the overexpressing transformants. WT indicates the wild-type strain of L. theobromae CSS-01s. H2-OV1–H2-OV10 represents the mean lesion lengths of the GH 2.1698 overexpressing transformants of L. theobromae, and H7-OV1–H7-OV10 represents the GH 7.188 overexpressing transformants of L. theobromae.