Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae.

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defense compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

Introduction

The interaction between plants and pathogenic fungi is complex, with the continuous coevolution of plant defense mechanisms and virulence mechanisms of pathogens (Anderson ). Upon invasion by a pathogen, plants defend themselves from infection through various mechanisms, such as the formation of physical barriers, synthesis of antimicrobial proteins, and chemical defense compounds (Jones and Dangl 2006; Ferreira ). Plants synthesize a variety of chemical compounds (terpenes, alkaloids, and different types of phenolics) that are either preformed or produced upon infection (Lattanzio , 2008). Some of these phenolic compounds include known antifungal agents, such as catechin, stilbenes, isoflavonoids, and condensed tannins (Jeandet ; Kocaçalişkan ; Lattanzio ; Liu ; Ullah ).

Phytopathogenic fungi have evolved to overcome host defense responses. For example, some fungi can degrade phenolics that are part of the host defense response via specialized enzymatic pathways (Westrick ). In many instances, the phenolic degradation pathways in fungi are clustered and connected by key classes of enzymes (Gluck-Thaler ; Gluck-Thaler and Slot 2018). This includes the enzyme class catechol dioxygenases (CDO), which can cleave aromatic rings at the catecholic bond through the addition of molecular oxygen (Hayaishi 1966; Broderick 1999). The CDO enzymes are divided into 2 families either catalyzing the first reaction of the ortho-cleavage or meta-cleavage pathways. These enzyme families have evolved independently from one another and are found in both eukaryotic and prokaryotic microorganisms (Harayama and Rekik 1989; Eltis and Bolin 1996; Vaillancourt ).

The intradiol dioxygenases (EC 1.13.11.1), are part of the catechol branch of the 3-oxoadiptae pathway (also known as the ortho-cleavage pathway or the β-ketoadipate pathway) (Harwood and Parales 1996; Martins ). Intradiol dioxygenases cleave the carbon–carbon bond of catecholic aromatic compounds between the 2 adjacent hydroxy substituents (Fig. 1) (Broderick 1999; Vaillancourt ). In this pathway, the catecholic substrate is cleaved to form a muconolactone (Harwood and Parales 1996), which is further broken down via multiple enzymatic reactions to form the products succinyl-CoA and acetyl-CoA which enter the tricarboxylic acid cycle and provide substrates for the production of the high-energy molecules, NADH and ATP (Fig. 1) (Ornston and Stanier 1966; Harwood and Parales 1996; Martins ). In the meta-cleavage pathway, on the other hand, an extradiol dioxygenase cleaves the catecholic substrate adjacent to a hydroxy substituent to form a muconate semialdehyde (Vaillancourt ). This compound is further broken down to form pyruvate and acetaldehyde which are metabolized by the glycolysis pathway (Fig. 1) (Maruyama ).

Fig. 1.

The ortho- and meta-cleavage routes of phenolic degradation by microbial species. Blue bracket over C-1,2-DO represents the step in the ortho-cleavage pathway that CDO1, 2, and 4 of the Ceratocystidaceae are involved in. Green bracket over C-3,4-DO indicates the step in the meta-cleavage pathway -in which CDO3 of the Ceratocystidaceae is involved in. (Enzymes: C-1,2-DO—catechol 1,2-dioxygenase, 4CML—4-carboxymuconate lactonase, 4CMH—4-carboxymuconolactone hydrolase, KSCT—β-ketoadipate:succinyl-CoA transferase, KCT—β-ketoadipate-CoA thiolase; C-3,4-DO—catechol 3,4-dioxygenase, 2H4CSHD—2-hydroxy-4-carboxymuconate-6-semialdehyde dehydrogenase, 2P4,6DH—2-pyrone-4,6-dicarboxylate hydrolase, 4OMH—4-oxalomesaconate hydratase, CHOA—4-carboxy-4-hydroxy-2-oxoadipate aldolase, OD—oxaloacetate β-decarboxylase). Chemical structures and pathways, adapted from Wadke et al. (2016).

Fungi are thus able to utilize phenolic plant defense metabolites as carbon sources through the use of CDOs and their respective catabolic pathways (Camarero ; Bugg ). The action of CDOs during plant infection has been shown to contribute to the pathogenicity and virulence of some phytopathogenic fungi (Shanmugam ; Hammerbacher ; Wadke ). For example, the fungal maize pathogen, Cochliobolus heterostrophus, produces an intradiol CDO in response to phenolic compounds present in its environment (Shanmugam ). Members of the family Ceratocystidaceae utilize CDOs to overcome host responses. For example, the pathogen Endoconidiophora polonica makes use of CDOs in the degradation of flavonoids and stilbenes produced by its conifer host (Wadke ). Previous studies revealed that increased expression of the CDO genes in this fungus increased virulence of the pathogen (Hammerbacher ; Wadke ).

In spite of the importance of CDOs in the plant host-fungal pathogen interaction, very little is known about CDOs in the Ceratocystidaceae. This fungal family includes agricultural crop pathogens such as the sweet potato black rot pathogen, Ceratocystis fimbriata, the banana crown rot pathogen, Thielaviopsis musarum, and the causal agent of carrot root rot, Berkeleyomyces basicola (Halsted and Fairchild 1891; Melo ; Nel ). There are also many species which cause disease on important forest trees, including the conifer blue-stain fungi, E.polonica and Endoconidiophoralaricicola, the wattle wilt pathogen, Ce. albifundus, and the causal agent of sapstreak in sugar maple trees, Davidsoniella virescens (Krokene and Solheim 1998; Roux ; Richter 2012). Apart from these pathogenic species there are also a number of saprophytic species in the Ceratocystidaceae that do not cause disease or which are considered weakly pathogenic. These include Huntiella species, particularly Huntiellamoniliformis and Huntiellabhutanensis (Wilson ; Wingfield ), weakly pathogenic fungi such as D. eucalypti, found on Eucalyptus (Kile ), and the ambrosia beetle symbionts, Ambrosiella xylebori and Ambrosiellacleistominuta (von Arx and Hennebert 1965; Mayers ).

There are a large number of sequenced genomes in the Ceratocystidaceae (Wilken ; Van der Nest ; Wingfield , 2016a, 2017) which provide an important resource for determining the presence of these enzymes across different genera. However, to date, only 4 CDOs were identified in E. polonica (Wadke ). It is still unknown whether other members of the Ceratocystidaceae with different ecological lifestyles have similar enzymes. The aim of this study was to identify and characterize the CDOs in pathogenic and nonpathogenic species of the Ceratocystidaceae, as well as understand the evolutionary history of the catechol dioxygenases and their involvement in fungal nutrition.

Materials and methods

Identification and characterization of putative CDOs in the Ceratocystidaceae

To identify putative CDO homologs present in the genomes of the fungal species, we used the sequences identified in E. polonica to search the publicly available genomes. These included the nucleotide sequences of 3 intradiol dioxygenases (EpCDO1, GenBank Accession: KU221039; EpCDO2, GenBank Accession: KU221040; EpCDO4, GenBank Accession: KU221042) and 1 extradiol dioxygenase (EpCDO3, GenBank Accession: KU221041) (Wadke ).

Putative CDO homologs were identified using local BLAST searches (tBLASTn, expect [E]-values > 105) using CLC Genomics Workbench v 8.1 (https://www.qiagenbioinformatics.com/). The corresponding scaffold or contig on which there was a hit was extracted in CLC Main Workbench and open reading frames were predicted using Web AUGUSTUS (http://bioinf.uni-greifswald.de/augustus/) (Hoff ; Hoff and Stanke 2013). The genes identified from the annotations were confirmed with a reciprocal BLASTp on NCBI. The functional domains of the CDOs were annotated using InterProScan (Jones ), the conserved domains were identified using the conserved domain (CD) function on NCBI (http://www.ncbi.nlm.-nih.gov/Structure/cdd/wrpsb.cgi). The predicted proteins were also assessed for transmembrane domains using TMHMM (Krogh ) and for signal peptides using SignalP (Petersen ). Orthologous proteins were identified using OrthoMCL (Li ).

Phylogenetic analysis

A phylogenetic approach was taken to determine if the genes could be considered orthologous and to determine the evolutionary history and relationships of the respective genes. The genome sequence information for 30 species of the Ceratocystidaceae was used, in addition the genome sequences from Fusarium circinatum and Fusariumfujikuroi were used to provide outgroup gene sequences (Table 1). They were obtained from the GenBank database of the National Centre for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/).

Table 1.

Presence of the CDO 1-4 gene transcripts across the Ceratocystidaceae with the genome information and ecological lifestyle of each species used in this study.

Species	Genome ID	CDO1	CDO2	CDO3	CDO4	Lifestyle
A. cleistominuta	JABFIG000000000.1	—	—	√	√	Beetle-vectored, weakly pathogenic
A. xylebori	PCDO00000000.1	—	—	√	√	Beetle-vectored, weakly pathogenic
Be. basicola	PJAC00000000.1	√	√	√	√	Necrotroph
Br. fagacearum	MKGJ00000000.1	—	√	√	√	Necrotroph
Ca. adiposa	LXGU00000000.1	—	—	√	√	Weakly pathogenic
Ce. albifundus	JSSU000000000.1	√	√	√	√	Necrotroph
Ce. eucalypticola	LJOA00000000.1	√	√	√	√	Necrotroph
Ce. cacaofunesta	PEJQ00000000.1	√	√	√	√	Necrotroph
Ce. fimbriata	APWK00000000	√	√	√	√	Necrotroph
Ce. harringtonii	MKGM00000000.1	√	√	√	√	Necrotroph
Ce. manginecans	JJRZ01000000	√	√	√	√	Necrotroph
Ce. platani	LBBL00000000.1	√	√	√	√	Necrotroph
Ce. smalleyi	NETT00000000.1	√	√	√	√	Necrotroph
Ch. thielavioides	BCGU00000000.1	—	√	√	√	Necrotroph
Davidsoniella australis	RHLR00000000.1	√	√	√	√	Necrotroph
D. eucalypti	RMBW00000000.1	√	√	√	√	Weakly pathogenic
D. neocaledoniae	RHDR00000000.1	√	√	√	√	Necrotroph
D. virescens	LJZU00000000.1	√	√	√	√	Necrotroph
E. laricicola	LXGT00000000.1	√	√	√	√√	Necrotroph
E. polonica	LXKZ00000000.1	√	√	√	√√	Necrotroph
H. bhutanensis	MJMS00000000.1	—	—	√	—	Saprotroph
H. decipiens	NETU00000000.1	—	—	√	—	Saprotroph
H. moniliformis	JMSH00000000.1	—	—	√	—	Saprotroph
H. omanensis	JSUI00000000.1	—	—	√	—	Saprotroph
H. savannae	LCZG00000000.1	—	—	√	—	Saprotroph
Thielaviopsis cerberus	JACYXV00000000.1	√	√	√	√	Necrotroph
T. ethacetica	BCFY00000000.1	√	√	√	√	Necrotroph
T. euricoi	BCHJ00000000.1	√	√	√	√	Necrotroph
T. musarum	LKBB00000000.1	√	√	√	√	Necrotroph
T. punctulata	LAEV00000000.1	√	√	√	√	Necrotroph
F. circinatum	AYJV02000000	√	√	√	√	Necrotroph
F. fujikuroi	GCA_900079805.1	√	√	√	√	Necrotroph

The amino acid sequences for each of the individual genes (CDO1-4) were aligned using MAFFT (Multiple sequence alignment based on fast Fourier transform) v 7 (https://mafft.cbrc.jp/alignment/server/) using the L-INS-i option (Katoh and Toh 2008) to generate a single alignment per gene. The alignments were trimmed and in the case of CDO4, the N-terminal signal peptide was excluded. The alignments were then analyzed using a Maximum Likelihood (ML) method with RAxML v8 (Stamatakis 2014). The predicted best-fit substitution model for amino acid data as indicated by ProtTest 3 (Darriba ) was incorporated in the ML analyses. The evolutionary models were the Whelan and Goldman (2001) with gamma distribution (WAG+G) for CDO1 and CDO2 data, and the Le-Gascuel model (Le and Gascuel 2008) with gamma distribution (LG+G) for CDO3 and CDO4. The branch support was determined using 1000 bootstrap replicates. The phylogenetic trees were viewed using MEGA v.7 (Kumar ) and FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).

A multi locus species tree was generated for all the species used in this study. The nucleotide sequences for the Minichromosome Maintenance Complex Component 7 (MCM7), DNA-directed RNA polymerase II subunit RPB1 and RPB2 (RPB1, RPB2), and elongation factor 2 (EF2) genes were identified in the genomes using the BLASTn function in CLC Main Workbench v 8.1. The nucleotide sequences were aligned using MAFFT v 7 and concatenated using FASconCAT-G v 1.04 (Kück and Longo 2014). The alignments were converted to Phylip files using PAUP v 4.0a (Swofford 2002). An ML analysis was conducted on the data using the predicted evolutionary model of a General Time Reversible with gamma distribution (GTR+G) as determined with jModelTest (Darriba ). RAxML v8 was used to perform the ML analysis, with the data being partitioned into the respective loci, and the parameters for each partition allowed to vary independently. Bootstrap support was determined with 1000 rapid bootstrap replicates. The tree was viewed using MEGA v.7 and FigTree v1.3.1.

Gene synteny flanking CDOs

The gene synteny surrounding the CDO genes was investigated to determine whether the CDOs are found in specialized enzymatic clusters. For each of the CDOs, the 5 genes flanking either side of the genes, where available, were identified using BLASTx searches on NCBI. These flanking regions were assessed for synteny using EasyFIG 2.2.3 (Sullivan ). This was done to identify any possible gene clusters involved in the degradation of catechol derivatives, and to determine whether there were regions in the surrounding genes that were indicative of gene expansions, gains or losses. The programme uses a BLASTn function to compare the sequences of the input fasta files using a similarity threshold with a maximum E-value of 0.001.

Analysis of CDO gene family evolution

The gene family contractions and expansions across the Ceratocystidaceae were inferred using the Count software package (http://www.iro.umontreal.ca/~csuros/gene_content/count.html) through inferring the character’s history given the phylogeny (Csűrös and Miklós 2006; Csűrös 2010). The analysis was performed using a Wagner parsimony method that penalizes individual family members for their loss (loss penalty = 1) and gains, inferring the history with a minimum penalty on the previously described multilocus species tree generated. To further analyze the data, the ancestral character states were constructed for each individual gene using Mesquite v 3.0.4 (Maddison and Maddison 2019). The analyses were based on Parsimony reconstruction and used the tree generated from the combined data (see above) as the phylogenetic hypothesis.

Identification of the ortho- and meta-cleavage pathways in the Ceratocystidaceae

To identify the putative homologs of the ortho- and meta-cleavage pathways, the characterized genes of the pathways from closely related species were used in a tBLASTn search against the 30 genomes of the Ceratocystidaceae (Table 1) using CLC Genomics Workbench v 8.1. The reference genes used as queries include carboxy-cis, cis-muconate cyclase (Ce. fimbriata PHH5432.1); 3-oxoadipate enol-lactonase (Ce. platani KKF95260.1); 3-oxoadipate CoA-transferase (Ce. platani KKF92536.1); acetyl-CoA acyltransferase (Ce. fimbriata PHH52801.1). The corresponding scaffold or contig on which a gene was identified was extracted in CLC Main Workbench and open reading frames were predicted using web AUGUSTUS. The genes identified from the annotations were confirmed with a reciprocal BLASTp on NCBI.

Statistical analysis

The data were separated into pathogens and nonpathogens (as seen in Table 1), and the number of species in each category were further assigned to a category of (1) having all 4 CDOs present or (2) not having all 4 CDOs present. This count data were subjected to a Fisher’s Exact Test using a 95% confidence interval (2-tailed), with an alternate hypothesis that the true odds ratio is not equal to 1. All analyses were conducted using R (www.r-project.org).

Results

From the 32 genomes used in this study (Table 1), putative CDOs (CDO1—CDO4) were identified based on their sequence identity to previously identified genes from E. polonica (Wadke ). The genes identified were consistent between species within the respective genera (Table 1). Only one of the genes, CDO3, was identified in all the species included in this study (Table 1). Ceratocystis spp., Davidsoniella spp., 1 Berkeleyomyces sp., and Thielaviopsis spp. had single copies of all 4 of the previously described CDO genes. Bretziella fagacearum and Chalaropsis thielavioides genomes contained gene sequences for CDO2, 3, and 4. Ambrosiella spp. and Catunicaadiposa contained gene sequences for CDO3 and CDO4. Species of Huntiella had only 1 extradiol dioxygenase, CDO3. We also identified a gene duplication of CDO4 in E. polonica and E. laricicola. The genomes of the outgroup species F. circinatum and F. fujikuroi contained 1 copy of each of the CDOs (Table 1).

The genome architecture of CDO1 using InterProScan, CD and SignalP identified a conserved intradiol ring-cleavage dioxygenase core domain (IPR015889) adjacent to a conserved intradiol ring-cleavage dioxygenase C-terminal (IPR000627). The sequences contained the conserved residues of the active site and dimer interface, including the residues involved in binding the iron(III) cofactor.

The predicted conserved domains identified in the CDO2 genes included an N-terminal domain (IPR007535) characteristic of an intradiol cleaving dioxygenase and an intradiol ring-cleavage dioxygenase core domain (IPR015889). A third intradiol dioxygenase was identified, CDO4, was identified. All the CDO4 homologs contained an N-terminal signal peptide as predicted by InterProScan and SignalP, which was consistent with findings by Wadke . They were well conserved and spanned 20 amino acids into the gene. Thus, the CDO4 protein is predicted to be secreted to the extracellular space. The analyses identified the ASA-HP cleavage site between amino acids 20 and 21. This is a standard secretory peptide transported by the Sec translocon protein and cleaved by Signal Peptidase I (Auclair ). This was consistent in each CDO4 gene included in this study. The intradiol ring-cleaving dioxygenase core C-terminal domain (IPR000627) was present in each of the CDO4 transcripts.

One extradiol cleaving dioxygenase, CDO3, was identified. This extradiol ring-cleavage dioxygenase was similar to a class III enzyme, with a conserved subunit B domain (IPR004183), with similarity to a 4,5-DOPA extradiol dioxygenase (EC 1.13.11.29). A gene duplication of CDO4 was identified in the E. polonica and E. laricicola genomes. The genes were located on different scaffolds, and were surrounded by different flanking genes. The sequence similarity of the coding regions of the 2 genes was 92.73% in E. polonica and 93.9% in E. laricicola (Supplementary Fig. 1). When the amino acid sequences of the homologous CDO4 genes at the same genomic location were compared to one another, they were 99.13% similar in percentage identity to each other (Supplementary Fig. 1). The duplicate gene is located on scaffolds that are 24,364 and 24,420 bp in length in E. polonica and E. laricicola, respectively (Wingfield ). Each scaffold had 3 genes with a predicted ORF present. The other genes present on the scaffold are predicted to be an isoamyl alcohol oxidase/dehydrogenase and a hypothetical protein. The cleavage site for the secretory signal was identified in the Endoconidiophora CDO4 gene duplicates.

The ML phylogenies of the individual genes showed close congruency to the species tree, and each gene formed a clade that included members of the same genus (Figs. 2–5). The duplicated CDO4 genes identified in the Endoconidiophora species grouped with the CDO4 genes identified previously (Fig. 5), but formed their own clade, suggesting that the gene expansion occurred in an ancestral Endoconidiophora lineage.

Fig. 5.

Maximum likelihood gene tree (left) for CDO4 amino acid sequences (1000 bootstrap replicates) with outgroup species F. circinatum and F. fujikuroi. Synteny map (right) for representative species of each of the genera present in the gene tree representing the sequence similarity of the genomic regions flanking CDO4. *CDO4 (6, orange) including 5 genes upstream and downstream of the CDO. Genes which are numbered and colored are shown to be syntenic and conserved through the majority of the species, with some unique genes also included. The genes without numbers or colored gray were not found to be syntenic across genera. From the left: 1—catabolic 3-dehydroquinase; 2–3-dehydroshikimate dehydratase; 3—quinate dehydrogenase; 4—quinate permease; 5—hypothetical protein; 6—CDO4; 7—Hypothetical protein part of fungal TF MHR superfamily, containing a GAL4 zinc finger DNA-binding domain; 8—phosphopantetheine adenylyltransferase; 9—subtilase family protein; 10—DNA polymerase kappa; 11—hypothetical protein part of beta elim lyase superfamily; 12—subtilase/peptidase family; 13—subtilase/peptidase family; 14—histone-lysine N-methyltransferase SET9; 15—FK506-binding protein 2.

The genes flanking the CDOs differed between genera. Within-genus comparisons revealed high conservation of gene order and orientation. When compared across genera, there were certain genera that had conserved gene order and orientation, such as Ceratocystis and Davidsoniella (Figs. 2–5). However, there were some outliers, such as Br. fagacearum and the genus Huntiella, showing low synteny compared to the other genera included in the dataset (Figs. 2–5).

Fig. 2.

Maximum likelihood gene tree (left) for CDO1 amino acid sequences (1000 bootstrap replicates) with outgroup species F. circinatum and F. fujikuroi. Synteny map (right) for representative species of each of the genera present in the gene tree representing the sequence similarity of the genomic regions flanking CDO1. *CDO1 (6, orange) including 5 genes upstream and downstream of the CDO. Genes which are numbered and colored are shown to be syntenic and conserved through the majority of the species, with some unique genes also included. The genes without numbers or colored gray were not found to be syntenic across genera. From the left: 1—hypothetical protein; 2—putative phosphatidylinositol N-acetylglucosaminyltransferase subunit C; 3—mediator of RNA polymerase II transcription subunit 31; 4—DNA-directed RNA polymerase III subunit RPC10; 5—hypothetical protein containing CFEM domain; 6—CDO1; 7—tRNA (uracil-O(2)-)-methyltransferase; 8—eukaryotic translation initiation factor eIF-1; 9—N-glycosylation protein; 10—hypothetical protein; 11—sodium/pantothenate symporter; 12—Ser/Thr protein phosphatase family protein; 13—reverse transcriptase.

The gene orientation and order for CDO1 showed gene synteny between the Ceratocystis species and the Berkeleyomyces species (Fig. 2). There are many similarities between the genera, with only a few genes that differed between them. A similar pattern is observed for CDO2, with genes downstream of the CDO2 gene showing high synteny between the genera, whereas, upstream of the gene, the gene order is less conserved in Thielaviopsis (Fig. 3). One of the genes upstream of CDO2 included a carboxy-cis, cis-muconate cyclase (see gene 5 in Fig. 3) which is one of the enzymes used in the catechol branch of the ortho-cleavage pathway. The genes upstream of CDO3 were more conserved than those downstream in some of the species. There were striking differences between the flanking genes of CDO3 of H.moniliformis compared to the rest of the species in the other genera (Fig. 4).

Fig. 3.

Maximum likelihood gene tree (left) for CDO2 amino acid sequences (1000 bootstrap replicates) with outgroup species F. circinatum and F. fujikuroi. Synteny map (right) for representative species of each of the genera present in the gene tree representing the sequence similarity of the genomic regions flanking CDO2. *CDO2 (6, orange) including 5 genes upstream and downstream of the CDO. Genes which are numbered and colored are shown to be syntenic and conserved through the majority of the species, with some unique genes also included. The genes without numbers or colored gray were not found to be syntenic across genera. From the left: 1—neutral ceramidase; 2—ATP-dependent DNA helicase srs2; 3—hypothetical protein containing DUF 4591 domain; 4—sterol regulatory element-binding protein 1; 5—carboxy-cis, cis-muconate cyclase; 6—CDO2; 7—nitronate monooxygenase; 8—endonuclease/exonuclease/phosphatase, COG 2374; 9—double-stranded RNA binding motif; 10–37S ribosomal protein S7 mitochondrial; 11—hypothetical protein.

Fig. 4.

Maximum likelihood gene tree (left) for CDO3 amino acid sequences (1000 bootstrap replicates) with outgroup species F. circinatum and F. fujikuroi. Synteny map (right) for representative species of each of the genera present in the gene tree representing the sequence similarity of the genomic regions flanking CDO3. *CDO3 (6, orange) including 5 genes upstream and downstream of the CDO. Genes which are numbered and colored are shown to be syntenic and conserved through the majority of the species, with some unique genes also included. The genes without numbers or colored gray were not found to be syntenic across genera. From the left: 1–40S ribosomal protein S19; 2—hypothetical protein; 3—fluoride export protein 1; 4—hypothetical protein containing DUFF 4449 domain; 5—DNA ligase 4; 6—CDO3; 7—hypothetical protein; 8—COP9 signalosome complex subunit 5; 9—hypothetical protein; 10—putative RNA-binding protein sce3; 11—hypothetical protein; 12—universal stress protein A family protein C25B2.10; 13—GPCR-type G protein 2; 14—putative RNA-binding protein sce3; 15—hypothetical protein; 16—putative vacuolar membrane protein; 17—glycerol kinase; 18—mannose-1-phosphate guanyltransferase; 19—hypothetical protein; 20—protein farnesyltransferase subunit beta.

In many genera, the genes flanking CDO4 are predicted to be involved in the shikimate pathway and the catabolism of quinate, a product of the shikimate pathway (Fig. 5). Quinate is degraded by various enzymes to a form that can enter the β-ketoadipate pathway. The genes identified as flanking CDO4 include a catabolic 3-dehydroquinase (EC 1.1.5.8), 3-dehydroshikimate dehydratase (EC 4.2.1.10), quinate dehydrogenase (EC 1.1.1.282), and a quinate permease (Pf00083) which collectively are involved in the transformation of quinic acid to protocatechuate. This gene cluster was not present in Br. fagacearum and the Ambrosiella species (Fig. 5). There were also large differences in the gene orientation and order between the different species for the genes flanking CDO4 (Fig. 5), as the syntenic genes found in the Ambrosiella species were dispersed in a different order, with some of the genes appearing upstream of CDO4 on the contig. In Br. fagacearum and the genus Ambrosiella only 1 syntenic gene was found on the scaffold (Histone-lysine N-methyltransferase SET9), where the rest of the genes lacked synteny to the other genera (Fig. 5).

Parsimony analysis of the CDOs showed a simple evolutionary history of the genes (Fig. 6). Based on the results of the parsimony analysis, a loss of the CDO1 gene occurred in the common ancestor of Bretziella, Ambrosiella, and Huntiella. This gene was also independently lost in the genus Chalaropsis when it split from the Ceratocystis. Huntiella incurred a simultaneous loss of CDO2 and CDO4 after it split from its closest relatives, the genera Ambrosiella and Bretziella. CDO2 was also lost in Ambrosiella after diverging from Bretziella. A duplication of CDO4 occurred in the common ancestor of E. polonica and E. laricicola, suggesting that they are paralogs which have continued to diverge over time (Fig. 6).

Fig. 6.

Ancestral history of the CDO 1-4 genes (left image) based on a multi-locus species tree. The presence of a gene is indicated by the colored line (CDO1—orange line, CDO2—gray line, CDO3—yellow line, CDO4—light blue line, CDO4 duplication—dark blue line), and the loss of a gene is indicated by the absence of that colored line from the node. Numbers on graph represent gene loss/gain events (1—loss of CDO1; 2—gain of CD04; 3—loss of CDO1; 4—loss of CDO2; 5—loss of CDO4). Graphical representation (right image) of ortho-cleavage pathway genes present in each species.

An alternative evolutionary history was determined using a Count analysis, which determines the distribution of homolog family sizes across the different genera using a Wagner parsimony approach (Supplementary Fig. 2). The predicted gains and losses on the tree (Supplementary Fig. 2) showed that many were lineage specific. Consistent with the previous results, the ancestral state of the genes predicted that the most recent common ancestor of the Ceratocystidaceae contained a single copy of each of the CDOs. A single gene loss for CDO1 was predicted for the Ch. thielavioides (Supplementary Fig. 2). A gene loss event of both CDO1 and CDO2 was predicted to have occurred in the lineage leading to the Huntiella, Bretziella, and Ambrosiella lineages, which included Ca. adiposa. In contrast to the ancestral state reconstruction in Mesquite using parsimony, a gene gain of CDO2 was predicted to have occurred for Br. fagacearum. The gene expansion predicted for the Endoconidiophora species was consistent with that of the method used in Mesquite.

When observed as a whole, the gene loss events were found to have occurred in the clades containing the saprophytic fungi and weakly pathogenic fungi, while the pathogenic fungi contained all 4 CDO copies (Table 1 and Fig. 6). The Fisher’s Exact Test showed an association between CDO copy number and lifestyle of the Ceratocystidaceae (P < 0.01).

The genes for the entire ortho-cleavage pathway in the Ceratocystidaceae were identified in all of the genera except for those of Huntiella, Ambrosiella, Catunica, and Bretziella (Table 2). There was one exception, the acetyl-CoA acyltransferase gene (EC 2.3.1.16) was present in all Ceratocystidaceae represented genomes (Table 2). The genes involved in the entire meta-cleavage pathway could not be identified in any of the fungal species, apart from 1 gene, a putative aldehyde dehydrogenase (EC 1.2.1.85).

Table 2.

Identification of ortho-cleavage genes involved in the degradation of catecholic substrates in the genomes of the Ceratocystidaceae.

Species	Ortho-cleavage pathway genes
	Carboxy-cis, cis-muconate cyclase	3-oxoadipate enol-lactonase	3-oxoadipate CoA-transferase	Acetyl-CoA acyltransferase
A. cleistominuta	—	—	—	√
A. xylebori	—	—	—	√
Be. basicola	√	√	√	√
Br. fagacearum	√	—	—	√
Ca. adiposa	—	—	—	√
Ceratocystis albifundus	√	√	√	√
Ce. eucalypticola	√	√	√	√
Ce. cacaofunesta	√	√	√	√
Ce. fimbriata	√	√	√	√
Ce. harringtonii	√	√	√	√
Ce. manginecans	√	√	√	√
Ce. platani	√	√	√	√
Ce. smalleyi	√	√	√	√
Ch. thielavioides	√	√	√	√
Davidsoniella australis	√	√	√	√
D. eucalypti	√	√	√	√
D. neocaledoniae	√	√	√	√
D. virescens	√	√	√	√
E. laricicola	√	√	√	√
E. polonica	√	√	√	√
H. bhutanensis	—	—	—	√
H. decipiens	—	—	—	√
H. moniliformis	—	—	—	√
H. omanensis	—	—	—	√
H. savannae	—	—	—	√
Thielaviopsis cerberus	√	√	√	√
T. ethacetica	√	√	√	√
T. euricoi	√	√	√	√
T. musarum	√	√	√	√
T. punctulata	√	√	√	√

Reference genes used in BLAST query: Carboxy-cis, cis-muconate cyclase (Ce. fimbriata PHH5432.1); 3-oxoadipate enol-lactonase (Ce. platani KKF95260.1); 3-oxoadipate CoA-transferase (Ce. platani KKF92536.1); acetyl-CoA acyltransferase (Ce. fimbriata PHH52801.1).

Discussion

Throughout the fungal kingdom, closely related species often differ significantly in their lifestyles (Soanes ). This is also observed in the Ceratocystidaceae, a family which includes fungi that are associated with plants in different ways, ranging from saprophytic to highly pathogenic. Different lifestyles in closely related fungi are often determined by different gene inventories and not by evolutionary distances (Schäfer ; Yoder and Turgeon 2001).

Plants synthesize a wide range of phenolic compounds, usually in high concentrations (Levin 1971). These compounds have antifungal activity, through binding to and precipitating extracellular proteins (Charlton ; Ndhlala ), interfering with membrane potentials (Rao ), disrupting membrane structure (Sang Sung and Gun Lee 2010) and the oxidative respiratory electron chain (Shalaby and Horwitz 2015). In order to survive in association with plants, as epiphytes, pathogens or endophytes, fungi have adapted to these compounds, and intricate mechanisms to circumvent their toxicity have evolved (Anderson ; Frantzeskakis ).

An adaptation to phenolic plant defenses are catabolic degradation pathways, where the phenolic ring is cleaved by a catechol dioxygenase enzyme and the linearized carbon chain is subsequently used as an energy source (Broderick 1999). In previous studies, CDO enzymes were identified as pathogenicity and virulence factors in the bark-beetle associated blue-stain fungus, E. polonica, a member of the Ceratocystidaceae (Hammerbacher ; Wadke ). To expand on previous research, CDO genes from different genera of the Ceratocystidaceae with widely different ecological lifestyles were investigated in this study with a focus on genomic differences using a comparative approach.

CDO genes in the Ceratocystidaceae are more abundant in pathogenic species

In previous studies, 4 CDOs were described in E. polonica, (CDO1-CDO4) (Wadke ). In this study, all 4 homologous CDOs were identified in the genera Ceratocystis, Davidsoniella, Berkeleyomyces, Endoconidiophora, and Thielaviopsis. Species in these genera are considered highly pathogenic on diverse hosts. For example, Ce. fimbriata causes black rot of sweet potatoes, Ce. manginecans and Ce. albifundus cause wilt in Acacia trees and Ce. platani causes canker stains in plane trees (Halsted and Fairchild 1891; Johnson ; Ocasio-morales ; Roux ; Tarigan ; Adawi ). Thielaviopsis species cause disease on many species of palm trees around the world (Paulin-Mahady ; Melo ; Saeed ) and Be. basicola causes root rot in many hosts such as cotton, chicory, carrots, groundnuts and tobacco plants (Punja and Sun 1999; Nel ). Endoconidiophora species are vectored by bark beetles to coniferous hosts such as Picea abies and Larix decidua causing blue-stain of the bark and wood, and kill the trees under high inoculation densities (Krokene and Solheim 1997, 1998). The genus Davidsoniella mostly contains pathogenic species. Davidsoniella virescens is a virulent pathogen, causing sap streak in maple trees, but is also weakly pathogenic on other hardwood hosts (Shigo 1962; Richter 2012; Bal ). In contrast, D. eucalypti is weakly pathogenic to certain species of Eucalyptus (Kile ; Richter 2012; Wingfield ), however, as it was only isolated from artificial stem wounds its primary host is not known.

In contrast to the pathogenic genera included in this study, only 1 CDO was identified in our study from the genus Huntiella. Species of this genus are saprophytic and colonize fresh wounds in tree bark, without causing disease (Roux ; Van Wyk ). This dramatic gene loss in the genus Huntiella supports previous findings, that the degradation of phenolic compounds is more common in pathogenic fungi compared to their saprophytic counterparts (Gluck-Thaler and Slot 2018). This pattern of gene loss in the saprophytic species of the Ceratocystidaceae has also been observed in the genes of the glycoside hydrolase protein family, which are involved in the catabolism of sucrose (Van der Nest ).

Ambrosiella species, which are the mycangial symbionts of ambrosia beetles (von Arx and Hennebert 1965; Mayers , 2020) only retained 2 of the 4 CDOs found in the Ceratocystidaceae. As ambrosia beetle symbionts, Ambrosiella species are also only weakly pathogenic, but are required to assimilate sufficient nutrients from the host to sustain the beetles’ development (Hulcr and Dunn 2011; Jankowiak 2011). CDO1 and CDO2, therefore, do not seem to play an important role in nutrient acquisition for these fungi. Furthermore, the genome of Ca. adiposa, formerly Ceratocystis adiposa (Mayers ), also contained only 2 CDOs (CDO3 and CDO4) and is an opportunistic pathogen, which causes root rot in sugarcane cuttings but is considered a weak pathogen and does not attack established plants (Sartoris 1927). Furthermore, CDO1 was lost from the genome of Ch. thielavioides. This species has a similar lifestyle to Ca. adiposa and is an opportunistic pathogen causing black mold in weakened plants, such as rose grafts (Longree 1940).

CDO1 was also lost from the genome of Br.fagacearum, a highly virulent pathogen, causing red oak wilt (Juzwik ; de Beer ). However, this fungus, which enters the tree through wounds and root grafts, restricts itself to the xylem vessels of the outer layers of the sapwood and avoids contact with phenolic-producing parenchyma cells (Juzwik and French 1983; Appel ). Therefore, CDO copy number and type in the Ceratocystidaceae is not only determined by pathogenicity, but also by the pathogen’s mode of infection, its preferred niche within the host and the resilience of the host tissue.

Although there does appear to be a trend in CDO copy number and ecological lifestyle, it is clear that the gene loss is largely restricted to a single clade with fewer pathogens (Fig. 6). As such the variation of the distribution of CDOs observed could be due to phylogenetic relatedness. To further investigate this, future studies will need to include more species, providing multiple independent pathogen and nonpathogen clades that could be compared for CDO copy number distribution.

The loss of the ortho-cleavage pathway in the Ceratocystidaceae

Another contributing factor to the differences in mode of infection among the Ceratocystidaceae representatives may be attributed to the complete absence of the ortho-cleavage pathway in the genera that have lost some of their intradiol CDOs. The species which have lost this pathway are all grouped in one clade suggesting that the loss of this pathway is in some way related to their evolution over time. The clade includes the species of Huntiella, Bretziella, Ambrosiella, and Catunica.

The absence of the ortho-cleavage pathway in Huntiella, Catunica, Ambrosiella, and Bretziella will require further research to understand if the gene loss in these genera provides a fitness disadvantage for these fungi and limits them to their specific ecological niches. The ortho-cleavage pathway is predominantly used by fungi to degrade phenolic compounds (Leatham ; Camarero ; Fountoulakis ; Shanmugam ). As the ortho-cleavage pathway produces intermediates which enter the β-ketoadipate pathway, it also provides a nutritional advantage to the fungus (Hammerbacher ; Wadke ).

The single CDO present in Huntiella provides interesting insight into some differences between the pathogenic and saprophytic species. The lack of synteny observed between the flanking regions surrounding CDO3 of Huntiella and other genera of the Ceratocystidaceae provides further evidence of differences in the evolutionary histories of the gene in the different species. Discordance in local synteny has been found to indicate nonorthologous gene relationships (Jun ) and may contribute to the lifestyle adaptations of fungi (Yoder and Turgeon 2001). A lack of local synteny surrounding genes involved in pathogenicity in the Ceratocystidaceae has also been observed for genes involved in the utilization of plant-derived sucrose (Van der Nest ), where the gene loss and lack of synteny were attributed to the action of transposable elements (TEs) found in the Ceratocystidaceae genomes. TEs have been shown to have an important impact on fungal lifestyles (Grandaubert ; Van der Nest ; Muszewska ).

Although the meta-cleavage pathway has not been characterized in fungi, the presence of the extradiol dioxygenase (CDO3) in the Huntiella genomes and the increase in expression of CDO3 in H. moniliformis when grown in the presence of caffeic acid indicates that it does utilize the enzyme (data not shown). In E. polonica expression of CDO3, which was shown to have ring-cleaving activity in vitro, also increased during spruce infection (Wadke ). As the complete meta-degradation pathway has not been resolved in fungi, there are 2 possible scenarios. The first is that all the fungi in this study can fully metabolize the phenolics using an as yet uncharacterized pathway involving extradiol cleavage. The second scenario is that the extradiol enzyme detoxifies the plant secondary metabolites through cleavage, promoting the survival of the fungus, but does not fully metabolize them (Yu and Keller 2005). Such a scenario was observed in the saprophyte Ophiostoma piceae which was able to initially degrade, but not metabolize, monoterpenoids to ensure its continued survival (Yu and Keller 2005; Haridas ). Further investigation into the importance of extradiol dioxygenases in fungal plant infection should be undertaken to determine how they are utilized, as well as their role during plant infection. Due to their presence across all the species of the Ceratocystidaceae, it is likely that the extradiol dioxygenases are important for the metabolism of both the pathogenic and saprophytic species.

Conclusions

In this study, we considered the evolution of CDOs in a subset of species of the Ceratocystidaceae and their potential links with fungal lifestyle. Publicly available genomes were used to identify the full complement of genes encoding catechol dioxygenase enzymes in multiple species of the Ceratocystidaceae. While the genomes of the necrotrophic pathogens, with the exception of Be. fagacearum, contained 4 different genes encoding CDOs, mildly pathogenic species only contained 2 to 3 genes and saprophytic species only contained a single gene. The loss of the genes and the associated metabolic pathway appears to have occurred in a lineage-specific manner.

Further investigation into the ecological importance of the CDOs in a wider range of species from the Ceratocystidaceae needs to be conducted to determine which CDOs are favored by species that occupy different ecological niches. Future studies should also include the analysis of CDO gene expression while the fungi are occupying their specific niches and enzyme activity assays. This information will further aid in understanding the importance of CDOs in plant-pathogen fitness and evolution.

Data availability

The datasets used and/or analyzed in this study are available in the NCBI Database (https://www.ncbi.nlm.nih.gov/nucleotide/). Accession numbers are detailed in Table 1.

Supplementary material is available at G3 online.

Click here for additional data file.