Department of Environmental Biology, University of Guelph, Guelph, Ontario, , Canada N1G 2W11
Author for correspondence: Paul H. Goodwin. Tel: +1 519 824 4120 ext. 2754. Fax: +1 519 837 0442. e-mail: pgoodwin{at}uoguelph.ca
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ABSTRACT |
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Keywords: anthracnose, biotrophy, hemibiotrophy, mallow, necrotrophy
Abbreviations: CBD, cellulose-binding domain; CWDE, cell-wall-degrading enzyme; Cgm, Colletotrichum gloeosporioides f. sp. malvae; LPH, large primary hyphae; MCWE, mallow-cell-wall extract; PDA, potato dextrose agar; PEL, pectate lyase; PNL, pectin lyase; RACE, rapid amplification of cDNA ends; TSH, thin secondary hyphae
b The GenBank accession numbers for the sequences reported in this paper are AF158256 and AF156984.
a These authors contributed equally to this work.
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INTRODUCTION |
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The interaction between Cgm and mallow indicates that cell-wall-degrading enzymes (CWDEs) produced by the fungus may be involved in disease development. Among fungal CWDEs, pectinases have been the most widely studied because pectic compounds are important to the structure of the plant cell wall and middle lamella, and pectinases alone can directly kill plant cells (Keon et al., 1987 ). A pectate lyase (PEL) gene of C. gloeosporioides has been shown to be important for the virulence of the fungus in avocado fruit (Yakoby et al., 2001
), indicating that at least some pectinases are involved in the virulence of Colletotrichum. For Cgm, two PEL genes (pel-1 and pel-2) have been described thus far (Shih et al., 2000
). Only pel-2 expression could be detected during infection or in pure culture with glucose as the sole carbon source, but both pel-1 and pel-2 transcripts could be detected in pure cultures with either mallow-cell-wall extract (MCWE) or purified apple pectin as sole carbon source. Pectic lyase enzyme activity, which includes both PEL and pectin lyase (PNL) activities, was detected in infected mallow leaves at the onset of the necrotrophic phase of infection, which is also the period when pel-2 transcripts were detected (Shih et al., 2000
).
Multiple pectinase isozymes are relatively common in phytopathogenic fungi and these isozymes typically differ in their regulation (Annis & Goodwin, 1997 ). Different pectinase isozymes may be active in a stage-specific manner during the saprotrophic, biotrophic or necrotrophic phases of infection, synergistically, at the same time or during several phases of infection. For example, Fusarium solani f. sp. pisi possesses at least four PEL genes: pelA is induced by oligomers produced by pelB; pelB is constitutively expressed; pelD is only expressed in planta; and pelC may encode an intracellular PEL that degrades oligomers generated by other pectinases (González-Candelas & Kolattukudy, 1992
; Guo et al., 1995a
, b
, 1996
). These four genes are believed to act synergistically to provide rapid degradation of the pectin, and although disruption of pelA or pelD alone did not affect virulence, a loss in virulence was observed when both pelA and pelD were disrupted (Rogers et al., 2000
).
A further examination of Cgm has revealed that the fungus contains additional pectinase genes. The work presented here describes the characterization of two PNL genes, pnl-1 and pnl-2, from Cgm. These encode extracellular PNLs that differ considerably in their expression in pure culture and in their expression during infection. In addition, pnl-1 has a novel structure that has not previously been described for any pectinase gene.
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METHODS |
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Fungal in vitro culture.
Conidia from 8-day-old cultures, grown on PDA, were transferred to liquid medium to give a final concentration of 1x107 conidia ml-1 and were grown at 22 °C with shaking at 120 r.p.m. The medium was a minimal salts broth (0·3 g NaNO3 l-1; 0·5 g KCl l-1; 0·5 g MgSO4.7H2O l-1; 0·01 g FeSO4.7H2O l-1; 1·0 g K2HPO4 l-1) with a sole carbon source of either 2% glucose, 0·5% apple pectin (galacturonic acid content, 77·5%; methoxy content, 7·8%; Sigma), 0·5% mallow-leaf cell wall extracted according to Mankarios & Friend (1980) or 0·5% apple pectin plus 0·5% MCWE. The mycelium of a 3-day-old culture of Cgm was harvested by filtration through two layers of Miracloth (Calbiochem), ground to a fine powder in liquid nitrogen and stored at -70 °C for RNA extraction.
DNA and RNA isolation.
Genomic DNA was isolated from the mycelia of 7-day-old cultures of Cgm, Colletotrichum graminicola, Colletotrichum gloeosporioides f. sp. hyperici and Colletotrichum lindemuthianum grown on PDA covered with cellophane (Flexel), as described by Goodwin et al. (2000) . Total RNA from fungus, host and infected host tissue was extracted according to the method of Chen et al. (2000)
.
Amplification of partial pnl fragments.
Genomic DNA from Cgm was used as a template for PCR with primers PL5 (5'-CAGCGGTGTCATCAAGGG-3') and PL3 (5'-AACAGTGGTCAGATCCAGAC-3'; bold primer is degenerate, A or G), corresponding to the peptide sequences SGVIKG and VWIDHV, respectively, which are highly conserved among several known pnl and pel genes (Templeton et al., 1994 ). The PCR conditions used were one cycle of 5 min at 94 °C, 2 min at 65 °C and 3 min at 72 °C, followed by 39 cycles of 1 min at 94 °C, 1 min at 65 °C and 1·5 min at 72 °C, with a final extension for 10 min at 72 °C. PCR products from this amplification were made blunt-ended using DNA polymerase I Klenow fragment (Amersham Pharmacia). The PCR products were then cloned into pBluescript KS- (Stratagene).
cDNA-library screening.
A cDNA library was constructed in ZAPII (Stratagene), according to the manufacturers instructions, using poly(A)+ RNA isolated from the infected area of mallow leaves at 7296 h after inoculation with Cgm (the transition period from LPH to TSH). The cloned pel-1 and pel-2 PCR products, obtained using primers PL5 and PL3 with genomic DNA from Cgm, were labelled using the Random Primed DNA Labelling Kit (Roche Diagnostics) and were used to screen the cDNA library. Plaque-lifts were done with a MagnaGraph nylon transfer membrane (MSI Micron Separations) according to Sambrook et al. (1989)
. Hybridizations of plaque-lifts were performed according to the manufacturers protocols (MSI Micron Separations). The blots were washed in 1xSSC at 42 °C (Sambrook et al., 1989
). Selected cDNA clones in the
ZAPII library were transformed into pBluescriptII SK- by the in vivo excision procedure (Stratagene).
Rapid amplification of cDNA ends (RACE).
This procedure was performed as described by Shih et al. (2000) , with the following modifications. Primers AP1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') and PL5 were used in the amplification of the 3' RACE product of pnl-2; primers AP1 and PNL2 (5'-CCGAGGACGATGTGCTGGC-3') were used for amplification of the 5' RACE product. Primer PNL2 was designed based on the sequence of the 3' RACE product of pnl-2. PCR conditions for the amplification of the 5' RACE product were 1 min at 94 °C, followed by 30 cycles of 15 s at 94 °C, 30 s at 66 °C and 45 s at 72 °C, with a final extension of 3 min at 72 °C. The PCR conditions for amplification of the 3' RACE product consisted of 1 min at 94 °C, followed by 25 cycles of 45 s at 94 °C and 2 min at 68 °C. The primers PNL2F (5'-CACACCGACCTCTACATTC-3') and PNL2R (5'-GTCCTCGACGGTAAGTAAG-3'), which were used for the amplification of the full-length pnl-2 cDNA, were designed based on sequences of the overlapping 5' and 3' RACE products. The PCR conditions for cloning the full-length pnl-2 cDNA were the same as those described for 5' RACE, except that the annealing temperature was 58 °C. PCR products were cloned according to Shih et al. (2000)
.
Sequence analyses.
Both strands were sequenced by the dye-terminator method on an automated DNA sequencer (model 377; Applied Biosystems). Computer analysis and phenogram construction based on the nucleotide and deduced amino acid sequences was performed according to Shih et al. (2000) .
Northern and Southern hybridizations.
These were done with MagnaGraph nylon transfer membranes, as described above for the plaque-lifts. Hybridization probes were prepared from full-length cDNA clones of pnl-1 and pnl-2, and were labelled as previously described. The blots were washed in 1xSSPE (for Northern) or 1xSSC (for Southern) at 42 °C for medium stringency and in 0·1xSSPE or 0·1xSSC at 65 °C for high stringency (Sambrook et al., 1989 ).
RT-PCR analyses.
DNA-free total RNA was extracted from inoculated and un-inoculated mallow leaves, treated with RNase-free DNase (Life Technologies), and reverse transcribed into cDNA with SUPERSCRIPT II (Life Technologies) using oligo(dT) primers. For RT-PCR of pnl-1, the cDNA was used with the pnl-1-specific primers PNL1F (5'-ATGAAGTCGGCTTCAGCC-3') and PNL1R (5'-ATGGGTGGTGACTTAGAC-3'); the PCR procedure was the same as previously described for the PL5 and PL3 primers. Amplification of actA from the same samples was performed according to Goodwin et al. (2000) . Relative RT-PCR for co-amplification of pnl-2 and actA (Jin et al., 1999
) was performed according to Goodwin et al. (2000)
, with the following modifications. Primers PNL2F and PNL2 were used to amplify a portion of pnl-2; the PCR conditions used were 3 min at 94 °C, followed by 35 cycles of 50 s at 94 °C, 50 s at 56 °C, and 60 s at 72 °C, and a final extension of 7 min at 72 °C. All experiments were repeated at least twice.
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RESULTS |
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Sequence analyses
pnl-1 (GenBank accession no. AF158256) encoded a putative protein of 479 aa. The predicted amino acid sequence from pnl-1 had a signal-peptide-cleavage site between Ala20 and Gln21 that conformed with the (-3, -1) rule (von Heijine, 1991 ). The mature protein from pnl-1 would have a molecular mass of 47·3 kDa, consisting of 459 aa, and a pI of 5·5. Two potential N-glycosylation sites were found at Asn39 and Asn124. Codon usage was highly biased, with 61% of all codons having a C in the third position (C, 61%; G, 21%; T, 12%; A, 5%).
pnl-2 (GenBank accession no. AF156984) encoded a putative protein of 379 aa with a signal-peptide-cleavage site between Ala19 and Ala20 that conformed with the (-3, -1) rule (von Heijine, 1991 ). The mature protein would have a molecular mass of 37·5 kDa, consisting of 360 aa, one potential N-glycosylation site at Asn129 and a pI of 8·3. Codon usage was also highly biased, with 72% of all codons having a C in the third position (C, 72%; G, 17%; T, 9%; A, 2%).
The amino acid sequences from pnl-1 and pnl-2 were compared with those of other fungal and bacterial PNLs (Fig. 1). The deduced protein sequence from pnl-1 had 4146% similarity (identity and conserved substitutions) to PNLs of other fungi. pnl-2 was closely related to the pnlA gene of Glomerella cingulata (93% similarity). pelA, pelB and pelD all clustered together, which is not surprising since these three PNL genes are all from Aspergillus niger.
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Southern-hybridization analysis
pnl-1 and pnl-2 are likely to be single-copy genes in the Cgm genome, as indicated by single bands in each of the digests in Southern hybridizations (Fig. 3). Hybridization with pnl-1 under low-stringency conditions revealed only one additional weakly hybridizing band, indicating that Cgm may have only two PNL genes (data not shown). Because pnl-1 is unique in having a CBD, an examination for homologues of pnl-1 was made in several related fungi; a single major hybridization band of about the same size as the one seen for Cgm was observed (Fig. 4
). However, one extra, faint band (of approx. 9·4 kb) in C. graminicola and two extra, faint bands (of approx. 8·9 and 5·3 kb) in C. gloeosporioides f. sp. hyperici and C. lindemuthianum were also observed, which were more pronounced under medium-stringency conditions.
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The expression of pnl-1 and pnl-2 during infection was also investigated by RT-PCR, using primers that spanned introns. Amplification with pnl-1-specific primers detected a band of the expected size at 72 h after inoculation, and at subsequent time points, but no pnl-1 transcripts were detected in the biotrophic phase or from spores grown on PDA (Fig. 8). These results confirm the Northern hybridization results. The lack of pnl-1 transcripts in spores and during the biotrophic phase was not due to a lack of cDNA, as amplification of actA was possible whenever the fungus was present (Fig. 8
). For pnl-2, relative RT-PCR (Goodwin et al., 2000
) was performed, where pnl-2 was co-amplified with the constitutively expressed actA gene as an internal control so that expression levels could be compared between the two genes. Although pnl-2 expression could not be detected by Northern hybridization, it could be detected by relative RT-PCR, with detection beginning in the biotrophic phase at 48 h after inoculation (Fig. 9
). Transcripts of pnl-2 were also detectable by RT-PCR from spores grown on PDA. During infection, expression of pnl-2 was first detected at 48 h after inoculation and thereafter appeared to be expressed at a relatively constant level compared to actA (Fig. 9
).
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DISCUSSION |
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Since pnl-2 transcripts were not detected in the cDNA library or by Northern hybridizations, but were detected by RT-PCR from infected tissue, it appears that pnl-2 had a much lower level of expression during infection than pnl-1. pnl-2 is also the first pectinase of Cgm identified thus far that is expressed in the biotrophic phase of infection. Biotrophic and hemibiotrophic fungal pathogens may need to regulate their CWDEs to avoid tissue maceration and the production of oligogalacturonate products that may elicit plant-defence responses (Keon et al., 1987 ). One way to achieve this is by having very low levels of expression; in general, biotrophic fungi are reported to have much lower levels of CWDEs compared to those of necrotrophs (Keon et al., 1987
). Although pnl-2 is also expressed during the necrotrophic phase, its level of expression remains relatively low (i.e. only detectable by RT-PCR).
It is possible that pnl-2 expression was relatively low during infection because it may be more sensitive to catabolite repression. Expression of pnl-2 was relatively high (i.e. detectable by Northern hybridization) in cultures grown with MCWE, but was not detectable by Northern hybridization in cultures grown with glucose, which is well known to repress pectinase genes. Expression was also not detected in cultures grown with purified apple pectin, which is more soluble than MCWE and would not only provide more inducers for pectinases but would also yield catabolites more rapidly than MCWE.
Keon et al. (1987) compared the features of the CWDEs of biotrophs, necrotrophs and hemibiotophs. For biotrophs and the biotrophic phase of hemibiotrophs host-cell-wall penetration is highly localized, with minimal wall degradation, but for many necrotrophs and the necrotrophic phase of hemibiotrophs, widespread cell-wall degradation and maceration occurs. Catabolite repression provides the main means of regulating fungal CWDEs and can be caused by even very low levels of sugars. Keon et al. (1987)
hypothesized that for hemibiotrophs, such as certain Colletotrichum spp., the switch from biotrophy to necrotrophy may be related to the fungal CWDEs ceasing to be affected by host sugars. One way that a hemibiotrophic fungus could do this is by having different CWDEs at different stages of disease development, through the production of multiple isozymes that are regulated in different ways.
pnl-1 of Cgm was regulated quite differently to pnl-2. In culture, pnl-1 transcripts were detected when pectin was the sole carbon source and were only partially repressed by the presence of glucose, indicating that regulation by catabolite repression was limited. This is similar to pel-2 of Cgm, which was also expressed in cultures with pectin and glucose (Shih et al., 2000 ). During infection, pnl-1 appeared to be expressed at a considerably higher level (i.e. detectable by Northern hybridization) than pnl-2, and expression during infection was associated with development of necrotic symptoms. This is also similar to the expression of pel-2 in infected tissue (Shih et al., 2000
). Therefore, pnl-1 and pel-2 could be genes that are not greatly affected by host sugars and that are involved in the widespread cell-wall degradation associated with necrotrophy.
Expression of pnl-1 was undetectable when Cgm was grown on media with MCWE as a sole carbon source, indicating that expression of pnl-1 cannot be explained simply by substrate inducibility. The results when a combination of apple pectin and MCWE was used as carbon source showed that component(s) of the host-cell material inhibited pnl-1 expression. However, this inhibitory material apparently does not cause significant inhibition of pnl-1 expression during infection. The extraction method employed to obtain cell-wall extract yields a material that is largely free of pigments, lipids and other cytoplasmic contaminants, but in which denatured proteins remain associated with the cell-wall polysaccharides. One possibility is that the inhibitor of pnl-1 expression is a compound released from a particular polysaccharide that could be attacked by CWDEs in medium with MCWE but was not readily available for enzymic digestion in plant tissue because it was associated with phenols, proteins or ions. It would be interesting to isolate the compound(s) responsible for this inhibition and determine how it differs between MCWE cultures and mallow leaves.
A unique feature of the predicted Pnl1 protein is that it contains a CBD linked by a hinge to a PNL catalytic domain. CBDs are commonly found as ancillary domains in cellulases, hemicellulases, xylanases, mannanases and acetylxylan esterases (Tomme et al., 1995 ). Removal of the CBD, genetically or proteolytically, affects enzyme activity on various cellulosic substrates. It has been hypothesized that CBDs enhance enzyme activity to their substrates either by increasing the effective enzyme concentration on the substrate surface by binding to the substrate, or by making the substrate more accessible to the catalytic domain (Tomme et al., 1995
). Pnl1 is the first example of a CBD in a PNL. However, it remains to be seen whether the CBD participates in positioning Pnl1 or whether it directly affects the enzymic activity.
The presence of CBD in Pnl1 implies that pnl-1 may represent a member of a novel PNL gene family that is common to a number of fungi. Southern hybridization demonstrated that pnl-1 homologues were present in at least several other Colletotrichum species, and this has been confirmed by the cloning and sequencing of a highly similar pnl sequence with a CBD from C. lindemuthianum (Y. Wei & P.H. Goodwin, unpublished data).
One indication of the role of CBDs in virulence is indicated by the fact that CBDs are common among cellulases of saprophytic fungi, whereas cellulases from plant-pathogenic fungi often lack CBDs (Müller et al., 1997 ; Sposato et al., 1995
; Wang & Jones, 1996
; Wang & Nuss, 1995
). Wang & Jones (1996)
showed that a cellulase with a CBD from Trichoderma reesei, a saprophyte, caused tissue necrosis when applied to plant tissue, but equivalent levels of a cellulase without a CBD from Macrophomina phaseolina, a plant-pathogenic fungus, did not result in necrosis. For Phytophthora parasitica var. nicotianae, a cell-wall glycoprotein was found to have both elicitor activity and two repeated domains with a motif resembling the fungal CBD consensus sequence (Mateos et al., 1997
). Infiltration of tobacco leaves with this protein elicited necrosis- and defence-gene expression. Although the role of the CBDs in CWDEs of plant-pathogenic fungi remains to be determined, it is interesting to speculate that CBDs may have some function in eliciting host necrosis. For Cgm, expression of the CBD-containing pnl-1 gene was associated with the appearance of host necrosis, whereas expression of pnl-2, which lacks a CBD, was observed prior to host necrosis.
The cytology of the interaction between Cgm and mallow supports a role for CWDEs during infection (Morin et al., 1996 ; Wei et al., 1997
). During the biotrophic phase LPH is highly constricted as it penetrates host cell walls, but in the necrotrophic phase extensive host-cell maceration and no constriction of TSH occurs during cell-wall penetration. Therefore, it appears that LPH and TSH produce different types of cell-wall degradation during infection. LPH may be highly constricted at the plant cell wall as a result of a relatively low level of localized wall degradation, which may be necessary to permit fungal growth while maintaining biotrophy. However, extensive cell maceration by TSH indicates larger amounts of more-damaging CWDEs. Wubben et al. (2000)
hypothesized that fungal pathogens have large pectinase gene families to allow for complex expression patterns that can be altered for different conditions. In this study, we have shown that in addition to pel-1 and pel-2 (Shih et al., 2000
), Cgm also contains two PNL genes, pnl-1 and pnl-2, both of which are expressed during infection. However, the differences in their expression patterns and in the products that they encode may be related to the need for Cgm to have different types of cell-wall penetration during the biotrophic and necrotrophic phases of growth in host tissue.
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ACKNOWLEDGEMENTS |
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Received 3 August 2001;
revised 20 November 2001;
accepted 23 November 2001.