Mycol. Res. 104 (11) : 1342–1347 (November 2000). Printed in the United Kingdom.
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Comparative analysis of an endopolygalacturonase coding gene in isolates of seven Fusarium species
Martha L. POSADA1, Bele! n PATIN4 O1, Aitor DE LA HERAS2, Salvador MIRETE2, Covadonga VA! ZQUEZ1 and M. Teresa GONZA! LEZ-JAE! N2* " Departamento de MicrobiologıT a III, # Departamento de GeneT tica, Facultad de BiologıT a, Universidad Complutense, 28040-Madrid, Spain. E-mail : tegonja!eucmax.sim.ucm.es Received 29 April 1999 ; accepted 1 February 2000.
Polygalacturonase (PG) synthetized by Fusarium spp. is a polymorphic enzyme with complex isoform patterns influenced by culture conditions and which are variable at the intra- and inter-specific levels. The partial sequence of an endopolygalacturonase coding gene (endopg) has been analyzed in isolates of seven species of Fusarium, most of them associated with Pinus pinea. The genomic fragment analyzed, 740 bp long, included two introns and the exon 4, which contained most of the amino acid motifs conserved in PGs from different origins. The high similarity of the amino acid sequences found among the isolates would indicate that differences in amino acid composition are not probably the main reason of the variability of this enzyme. Therefore, differences in the gene expression and regulation patterns of endopg gene among the isolates should be considered. Both types of sequences, the coding and the two intron sequences contained in the endopg fragment, were compared among the isolates to evaluate their potential use for phylogenetic studies. The different levels of variability of these sequences and the similar results obtained from the phylogenetic analyses when the three sequences (coding, intron and amino acid sequences) were used indicate that this endopg region would be very useful for phylogenetic analysis in the genus Fusarium.
INTRODUCTION Fusarium is a diverse genus which includes species pathogenic on a multitude of economically important plant species, responsible for damping-off, root rot and vascular wilts (Nelson et al. 1981). The process of infection, colonization of host tissues and disease induction by Fusarium spp. involves extensive cell wall degradation of the host tissue, mainly due to the depolymerization of pectin, which is the main component of cell walls and the middle lamella (Durrans & Cooper 1988). Pectin-degrading enzymes (pectinases) are produced by the fungal pathogen throughout the process of infection (Leone 1992, Centis et al. 1997, Tenberge et al. 1996), among which polygalacturonases are the most studied. Polygalacturonases (PGs, poly-α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) are produced by the pathogen when it is cultured on substrates such as pectin, polygalacturonic acid or galacturonic acid (Ferna! ndez et al. 1993). PGs are also important enzymes in saprophytic isolates of Fusarium and other filamentous fungi such Aspergillus (Bussink et al. 1992). The study of PG enzymes has been enhanced by their importance in particular industrial processes such as fruit-juice clarification (Voragen et al. 1986). PG is a polymorphic enzyme which exhibits considerable variation in physicochemical features, such as substrate * Corresponding author.
preferences, optimal pH and mode of action (endo and exo modes of action) (Stratilova! et al. 1993). Both endo- and exoPG enzymes would perform different roles during the course of fungus–host plant interactions (Bateman & Basham 1976, Cooper 1983, Walton 1994). The analysis of PG isoform patterns by IEF (isoelectric focusing) from in vitro cultures of Fusarium isolates have revealed substantial variability at both inter- and intra-specific level (Ferna! ndez et al. 1993, Posada et al. unpubl.). There were also differences in depolymerization efficiency of different pectin-related substrates among isolates from different species (Posada et al. unpubl.). This variability, together with the diversity of hosts colonized by Fusarium spp. (from pine trees to tomato) and the morphological and physiological diversity within the genus could suggest a considerable structural variability of PG enzymes in this genus. However, there is still little information about the genetic control of these enzymes that could help analyze their structural variability. The existence of an endoPG coding gene has been reported in F. moniliforme (Caprari et al. 1993), F. oxysporum f. sp. lycopersici (Di Pietro & Roncero 1998) and F. oxysporum f. sp. radicis lycopersici (Patin4 o et al. 1997). The three of them are very similar in amino acid sequence. This information, although still sparse, points to a structural conservation of the gene in contrast to the observed variation in the PG enzymatic system as mentioned before. Our objective was to study the degree of conservation of the endopg gene in Fusarium. It has been obtained and
M. L. Posada and others
1343 conditions. The mycelia were harvested by filtration through Whatman paper No. 1 and were frozen in liquid nitrogen for DNA isolation and stored at k80 m.
+1
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
DNA isolation DNA was obtained from 1 g of mycelium of each isolate according to Dellaporta et al. (1983). PCR amplifications of endopg gene
Intron 3 50-52 bp Exon 3 74 bp
Intron 4 45-55 bp Exon 4 456 bp
Exon 5 105 bp
Fig. 1. Representation of endopg gene showing exons and introns after Caprari et al. (1993), and the partial genomic endopg sequence analyzed showing the length of the different regions included.
compared with a partial endopg sequence of six isolates belonging to different Fusarium sections. These were isolated from seeds of close cones of Pinus pinea which have previously been shown to have differences in several aspects related with PG activity (Posada et al. unpubl.). Sequence data collected from Fusarium isolates from other origins obtained in our laboratory or reported by other authors were also included in the comparison. Both the coding and the two intron sequences included in the endopg fragment were individually analyzed to evaluate the possibility of using them in phylogenetic analyses. MATERIALS AND METHODS Fungal isolates Isolates of Fusarium equiseti, F. episphaeria, F. roseum, F. moniliforme, F. solani and F. oxysporum were isolated from seeds from closed cones collected in forests of P. pinea in Madrid. They were obtained and identified in the Department of Zoology, Plant Pests and Diseases, Escuela Universitaria de Ingenierı! a Te! cnica Forestal of the Universidad Polite! cnica, Madrid. The isolates r2, r6 of F. oxysporum f. sp. radicis lycopersici and 1a of F. oxysporum f. sp. lycopersici were initially obtained from infected tomato plants by Dr Tello (Instituto Nacional de Investigaciones Agrarias, Madrid). Fungi were maintained as stock cultures on potato-dextrose agar slopes at 4 mC and k80 m in 15 % glycerol. Culture conditions The isolates were cultured in 100 ml Erlenmeyer flasks containing 20 ml liquid medium (Va! zquez et al. 1986) with glucose (1n5 g 100 ml−") (Merck) as the carbon source. The cultures were inoculated with mycelial disks of 8 mm cut from the margins of 7 d colonies and incubated at 25 m under static
Genomic DNA (0n5 µg) of the F. equiseti, F. episphaeria, F. roseum, F. moniliforme, F. solani and F. oxysporum isolates was amplified by the polymerase chain reaction (PCR) using as primers the following oligonucleotides : upper 5h-ATCTGGCCATGTCATTGA-3h and lower 5h-GGTCGGCTTTCCAGTAGG-3h based on sequences of fungal pg genes already published (Caprari et al. 1993). PCR reactions were conducted in a Peltier PTC-100 Thermal Cycler (M. J. Research). For a 30 µl reaction mixture 1 unit of Taq polymerase (Promega) was used. Oligonucleotide primers were used at a final concentration of 1 µ. The amplification protocol for F. equiseti, F. episphaeria. F. roseum and F. solani isolates was performed as follows : a first step of 5 min at 94 m followed by 29 cycles (1 min at 94 m, denaturation ; 1 min at 54 m, annealing ; 5 min at 72m, extension) and a final extension of 5 min at 72 m. The amplification protocol for F. moniliforme and F. oxysporum was : 5 min at 94 m followed by 34 cycles (1 min at 94 m, denaturation ; 1 min at 52 m, annealing ; 2 min at 72 m, extension) and a final extension of 5 min at 72 m. Cloning and sequencing of endopg fragments The amplification products were cloned into the pMOS Blue T-vector and subsequently transformed following the manufacturers’ instructions (Amersham Life Science). Both strands of each cloned fragment were sequenced by the ALF DNA Sequencer (Pharmacia LKB Biotechnology) according to the manufacturers’ instructions in the Sequencing Service Unit of the Universidad Complutense de Madrid and by the ABI PRISM 377 DNA Sequencer (Perkin–Elmer) in the Sequencing Unit of the Centro de Investigaciones Biolo! gicas (Consejo Superior de Investigaciones Cientı! ficas, Madrid). In several isolates more than one clone was sequenced and no differences were observed. Aminoacid and nucleotide sequence analysis The cloned sequences were analyzed by using the Lasergene Biocomputing Software for Windows (DNASTAR, Madison). The sequences were compared at both DNA and amino acid levels with PG sequences from isolates r2 and r6 of F. oxysporum f. sp. radicis lycopersici (Patin4 o et al. 1997, Posada 1999), isolates 880621a-1 (Arie et al. 1998), 42-87 (Di Pietro & Roncero 1998) and 1a (Posada 1999) of F. oxysporum f. sp. lycopersici, Aspergillus oryzae (Kitamoto et al. 1993) and Cochliobolus carbonum (Scott-Craig et al. 1990). Phylogenetic analyses were performed with the PHYLIP 3.5 package (Felsenstein 1993) using the method of Kimura for amino acid comparisons (Program PROTDIST) and the method
Comparative analysis of a endopolygalacturonase coding gene in isolates of seven Fusarium species of Kimura two parameter distance (program DNADIST) for nucleotide sequences to calculate the distance matrix. Dendograms were obtained using UPGMA in the program NEIGHBOUR and bootstrap analyses were performed using programs SEQBOOT and CONSENSE. RESULTS A partial region of endopg gene has been amplified by PCR in isolates belonging to six Fusarium species by PCR using two 18mer oligonucleotide primers. In all the cases an amplification product of similar length (740 bp) was obtained which showed high amino acid sequence similarity with the endoPG
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coding gene of F. moniliforme (Caprari et al. 1993). The amplification product included the full sequence of exon 4 and introns 3 and 4 and part of exons 3 and 5 (Fig. 1). Exon 4 contained the amino acid residues conserved in endopg genes from different origins, some of them seem to be involved in catalysis or substrate interactions (Annis & Goodwin 1997). The amino acid sequences of the putative pg gene region were deduced and compared among the six isolates (Fig. 2). The amino acid sequences of the same region of F. oxysporum f. sp. radicis lycopersici r6 (Patin4 o et al. 1997) and of two other filamentous fungi, Aspergillus oryzae and Cochliobolus carbonum, were also included. The results indicated a high similarity, about 90 %, among the Fusarium isolates while the similarity
Fig. 2. Comparison of the partial endoPG amino acid sequences among the Fusarium isolates from this study and those of F. oxysporum f. sp. radicis lycopersici r6, Aspergillus oryzae and Cochliobolus carbonum (see text). Amino acid motifs common to PGs are shown in bold (Annis & Goodwind 1997). A dot represents the same amino acid residue as in the sequence of F. oxysporum f. sp. radicis lycopersici and a dash indicates that the aminoacid in this position is not present.
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3
4
Figs 3–4. Fig. 3. Intron 3 sequence alignment of Fusarium isolates. Fig. 4. Intron 4 sequence alignment of Fusarium isolates. 5h and 3h junction sequences and the conserved internal sequence CTA\GAC are underlined.
with the sequences belonging to the other two species of filamentous fungi was about 50 %. Changes in the conserved amino acid motifs were not found. All the changes were point mutations (substitutions) being transitions more frequent than transversions (data not shown). The phylogenetic analysis included one endopg sequence of isolate r2 of F. oxysporum f. sp. radicis lycopersici (Posada 1999) and three corresponding to isolates 880621a-1 (Arie et al. 1998), 42-87 (Di Pietro & Roncero 1998) and 1a (Posada 1999) of F. oxysporum f. sp. lycopersici. The dendogram was obtained by UPGMA. Two groups of Fusarium isolates were observed. One group contained the isolates of F. oxysporum, F. episphaeria and F. solani, while the other group contained the isolates of F. equiseti, F. roseum and F. moniliforme. The similarity among isolates from the first group is higher than among the three isolates from the second group. In the first group several bootstrap values were lower than 50 %, indicating that the endopg sequence did not separate these isolates. The various isolates of F. oxysporum did not form an homogenous group, since the F. oxysporum isolate obtained from pine seeds was clearly different to all the other Fusarium isolates (99 %). In the second group, F. equiseti appeared in a different branch than both F. roseum and F. moniliforme (94 %). The bootstrap value obtained at the node separating F. roseum from F. moniliforme was 80 %. Separation of both A. oryzae and C. carbonum, from Fusarium isolates was supported by a bootstrap value of 100 %. 100 % was also obtained for the node separating A. oryzae from C. carbonum. The nucleotide sequences of the seven isolates of Fusarium, including F. oxysporum f. sp. radicis lycopersici, r6 corresponding to introns 3 and 4 are shown in Figs 3–4. In all isolates, both introns showed the consensus sequences at 5h and 3h ends and the internal sequence involved in splicing described for filamentous fungi (Gurr et al. 1987, Ballance 1991). Introns had a lower GjC content (36 % to 49 %) than exon 4 (48n3 % to 49n3 %). Deletion\insertion was the most frequent change observed in these sequences, in contrast with the endoPG coding sequence where substitution was the only type of change. The seven isolates showed quite similar intron
5
F. oxysporum 100%
FOL 1a 100%
FOL 880621a-1 100%
FOL 42-87 100%
FORL r2 100% FORL r6
100%
100% F. episphaeria F. solani F. equiseti
100% 100%
F. roseum F. moniliforme
6
F. oxysporum
90%
FOL 880621a-1 87% FOL 1a 100%
FOL 42-87
100%
FORL r2
100%
FORL r6
100% 100%
F. episphaeria F. solani
99%
F. equiseti 100%
F. roseum F. moniliforme
Figs 5–6. Fig. 5. Dendograms corresponding to Fusarium intron 3. Fig. 6. Dendogram corresponding to Fusarium intron 4. Based on Fusarium isolates in this study and of F. oxysporum f. sp. radicis lycopersici r2 (FORL r2) and r6 (FORL r6) and f. sp. lycopersici 880621a-1 (FOL 880621a-1), 42–87 (FOL 42-87) and 1a (FOL 1a).
sequences, especially in the case of intron 3. The percentage of similarity among isolates was estimated and, in the case of intron 3, the lowest value observed was 55n1 % (F. equiseti with F. oxysporum f. sp. radicis lycopersici r2 and F. oxysporum f. sp.
Comparative analysis of a endopolygalacturonase coding gene in isolates of seven Fusarium species lycopersici 1a). The group of F. oxysporum, F. episphaeria and F. solani isolates showed values about 90 %. In the case of intron 4, the lowest value observed was 36n4 % (F. equiseti with F. oxysporum, F. oxysporum f. sp. lycopersici 880621a-1 and F. oxysporum f. sp. radicis lycopersici r2). The group of F. oxysporum, F. episphaeria and F. solani showed values of similarity higher than 80 %. Phylogenetic analyses were performed using both intron sequences and the dendograms are shown in Figs 5–6. The dendograms were almost identical and similar to the one obtained for the coding fragment of the endopg gene (not shown). The values of bootstrap analyses were higher than 50 % in all the cases. The phylogenetic analyses also detected two groups of isolates. The isolates of F. oxysporum, F. episphaeria and F. solani were in one group, separated from F. roseum, F. moniliforme and F. equiseti. F. roseum and F. moniliforme were more closely related to each other than to F. equiseti. The similarity among isolates in the first group is higher than within the second group. The various isolates identified as F. oxysporum did not group together ; the F. oxysporum isolate associated with pine seeds showed more differences with respect to the rest of the isolates within this group. Both isolates of the special form radicis lycopersici seemed to be closely related. All the isolates associated with tomato were from Spain except isolate 880621a-1 of F. oxysporum f. sp. lycopersici isolated from Imachi (Tochigi, Japan). DISCUSSION Comparison of amino acid sequences corresponding to the endopg gene among isolates from different Fusarium species indicated a high degree of sequence similarity (about 90 %). These isolates had shown differences in the PG isoform pattern, activity and other features (Posada et al. unpubl.). None of the amino acid changes observed affected the conserved or the functionally critical amino acids or motifs (indicated in bold, Fig. 1). Although the possibility cannot be discounted that if differences in structural features of the enzymes exist, these could be caused by a few changes in sequence or occur in regions of the protein sequence not analyzed here. These results do not seem to support the view that variability in PG features observed at inter- and intraspecific levels in the genus Fusarium can be explained by differences in the amino acid composition of the enzyme. On the other hand, differences in copy number of endoPG coding sequences are not a likely explanation of the differences on the pattern of endoPG activities found among the isolates. A single copy of the endoPG coding gene has been reported in F. moniliforme (Caprari et al. 1993, Migueli et al. 1993), F. oxysporum f. sp. lycopersici (Arie et al. 1998), F. oxysporum f. sp. radicis lycopersici (Patin4 o et al. 1997) and isolates of F. episphaeria, F. moniliforme, F. solani and F. oxysporum used in this work (Posada 1999). The copy number of the endopg gene could not be determined for F. equiseti and F. roseum. An alternative hypothesis would explain variability of PG isoform patterns and activity as a result of differences in the regulation of pg expression patterns. We are currently using gene specific probes to determine if there are differences in the expression
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pattern of different PG encoding genes among Fusarium isolates. The sequence similarity found for the endopg gene also contrasts with the diversity of the isolates analyzed. These isolates belong to species from different sections within the genus Fusarium and were isolated from different host plants (pine and tomato). The sequence conservation observed may be related to the existence of a single copy of this gene in the haploid genome of Fusarium. Although one more endoPG coding sequence probably occurs (GenBank AF078156) and at least two exopg genes have been reported (GenBank AF083075, AF136444), it is possible that mutations affecting the endopg gene analyzed in our work cannot be completely compensated for by other PG enzymes under field conditions. More information about the level of variability in other coding sequences within Fusarium, however, would be needed to tell if the level of variability found in the endopg gene is representative of coding sequences in the genus. In this case, it is possible that the high degree of asexual reproduction, total in cases such as F. oxysporum, plays a role in the low level of variability observed. It is generally assumed that introns are more variable than coding sequences and they are often used for phylogenetic studies (Dowling et al. 1996). The partial endopg sequence analyzed in our work contained two introns which could be studied and compared. Little variation in introns 3 and 4 was found among the isolates compared in our study, especially within the group which contained isolates of F. oxysporum (Figs 3–4). These results support our earlier suggestion that genetic variability in the genus Fusarium is relatively low. In this case, the small size of the introns, typical of fungal genes, could also represent a restriction in the variability allowed in order to be correctly spliced. In spite of the different level of variability observed in both introns as well as in the coding sequence the results of the three phylogenetic analyses were consistent. The isolates of F. oxysporum analyzed here did not form an homogeneous group of closely related isolates as might have been expected, mainly due to the isolate obtained from P. pinea. F. oxysporum is a large and diverse taxonomic unit and the different approaches based on DNA techniques suggest it is a species complex (Kistler 1997, O’Donnell & Cigelnik 1997). In our case, both groups of F. oxysporum isolates appear to belong to different taxonomic units. In this context, the idea that variability among species is expected to be higher than within a species should be cautiously assumed for the morphologically defined species of Fusarium. On the other hand, the results indicate a close relationship of isolates of F. oxysporum associated with tomato, F. episphaeria and F. solani. A more extensive study including a larger number of isolates from each species and using a more variable genomic sequence would be necessary to support the close relationship found among these species. The isolates of F. oxysporum associated with tomato also showed very similar intron sequences, identical in the case of intron 3 (Posada 1999). Therefore, it could be suggested that more variable regions such as the intergenic spacer of ribosomal DNA (IGS) (Appel & Gordon 1996) should be used to define more accurately the phylogenetic relationship among isolates from both special forms.
M. L. Posada and others It is concluded that the use of sequences such as the endopg gene which contains regions with different levels of variability, could be useful for comparing Fusarium species. While it is generally recognized that many species of Fusarium appear highly variable morphologically and in terms of host ranges, the current taxonomic treatments of the genus do not provide useful information on the range of variability within species or between them. It seems likely that phylogenetic comparisons at the intraspecific level, especially between closely related species, may require the use of more variable genomic sequences than explored so far. A C K N O W L E D G E M E N TS This work was supported by the Direccio! n General de Investigacio! n, Ciencia y Tecnologı! a (PB96-0586) and by Universidad Complutense de Madrid (PR156\97-7198), B. Patin4 o by a fellowship of the Universidad Complutense de Madrid, M. L. Posada by a fellowship of the Colciencias-Banco Industrial y de Desarrolo (Colombia) and A. de las Heras by a fellowship from the Comunidad Auto! noma de Madrid.
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