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Eukaryot Cell. Oct 2002; 1(5): 719–724.
PMCID: PMC126752

Isocitrate Lyase Is Essential for Pathogenicity of the Fungus Leptosphaeria maculans to Canola (Brassica napus)

Abstract

A pathogenicity gene has been identified in Leptosphaeria maculans, the ascomycetous fungus that causes blackleg disease of canola (Brassica napus). This gene encodes isocitrate lyase, a component of the glyoxylate cycle, and is essential for the successful colonization of B. napus. It was identified by a reverse genetics approach whereby a plasmid conferring hygromycin resistance was inserted randomly into the L. maculans genome. Twelve of 516 transformants tested had reduced pathogenicity on cotyledons of B. juncea and B. napus, and 1 of these 12 had a deletion of the isocitrate lyase gene, as well as an insertion of the hygromycin resistance gene. This mutant was unable to grow on fatty acids, including monolaurate, and the isocitrate lyase transcript was not detected. When the wild-type gene was reintroduced into the mutant, growth on monolaurate was restored and pathogenicity was partially restored. L. maculans isocitrate lyase is produced during infection of B. napus cotyledons, while the plant homologue is not. When 2.5% glucose was added to the inoculum of the isocitrate lyase mutant, lesions of sizes similar to those caused by wild-type isolate M1 developed on B. napus cotyledons. These findings suggest that the glyoxylate pathway is essential for disease development by this plant-pathogenic fungus, as has been shown recently for a fungal and bacterial pathogen of animals and a bacterial pathogen of plants. Involvement of the glyoxylate pathway in pathogenesis in animals and plants presents potential drug targets for control of diseases.

Fungal plant pathogens employ diverse strategies to infect their hosts. Mechanisms underlying these strategies are being revealed because of the increasing amenity of many fungi to molecular genetic analysis. In particular, techniques including insertional mutagenesis have identified fungal pathogenicity genes crucial to disease. Such genes are defined on the basis of mutagenesis techniques; disruption of a pathogenicity gene results in significant or complete loss of disease symptoms when mutants are inoculated onto hosts that are susceptible to the wild-type pathogen. The types of pathogenicity genes expressed depend on the infection process of a particular fungus, for instance, whether hydrolytic enzymes, specialized infection structures, or toxins are required, as well as the nutritional status of the pathogen (for reviews, see references 8 and 22). Pathogenicity genes may be valuable as fungicide targets for disease control. If such genes are specific to the fungus, inhibitors may be designed to bind to their products and block the growth of the fungus without affecting the plant.

Leptosphaeria maculans (anamorph = Phoma lingam) causes blackleg disease of canola (Brassica napus) worldwide. Its infection pathway is well characterized (for a review, see reference 7). Hyphae enter the host through stomata without the aid of specialized infection structures such as appressoria. The fungus initially grows in planta as a biotroph and later becomes necrotrophic. The fungus grows down the stem and produces a dark canker girdling the base of the plant, hence the name blackleg. In spite of the economic importance of blackleg disease, L. maculans has not been studied in great detail at a molecular genetic level. This paper describes an insertional mutagenesis approach to identifying pathogenicity genes of L. maculans and the characterization of one such gene encoding isocitrate lyase.

MATERIALS AND METHODS

Fungal culturing and transformation.

L. maculans isolate M1, which has well-defined host specificity, was mutated. This isolate causes cotyledonary and stem lesions on all of the B. napus and B. juncea (Indian mustard) cultivars tested, and its ability to attack B. juncea is controlled by a single locus (3, 15). Isolate M1 was transformed to hygromycin resistance by using plasmid pUCATPH (11) and restriction enzyme-mediated integration in the presence of 10 to 40 U of HindIII, KpnI, or SacI as previously described (17). Transformed protoplasts were plated onto regeneration medium (1.2 M sucrose, 10% Campbell's V8 juice, 1.5% agar) containing hygromycin (50 μg/ml; Sigma). Hyphae were visible after 2 to 3 weeks and were subjected to two rounds of hyphal-tip culturing onto 10% V8 juice agar plates containing hygromycin before transfer to nonselective medium. For Northern blot analysis, mycelia were grown in complete medium (17.5 g of Czapek-Dox per liter, 0.005% yeast extract) in still culture (50 ml) in petri dishes (15-cm diameter) for 15 to 25 days, washed in sterile water, and transferred to complete medium or minimal medium (14) containing no carbon or nitrogen source, ammonium acetate (0.5%) only, or ammonium acetate (0.5%) and glucose (0.25%). Mycelia were harvested 7 h later.

Pathogenicity tests.

Transformants were screened for the ability to infect cotyledons of 14-day-old seedlings of B. juncea cv. Stoke wounded with a 26-gauge needle as previously described (15). B. juncea was chosen for the initial screen so that target pathogenicity genes in L. maculans would include the host specificity gene (3). Lesion development was assessed between 10 and 14 days postinoculation and compared visually to lesions caused by isolate M1. Transformants with reduced pathogenicity were rescreened on B. juncea cv. Stoke and B. napus cv. Westar, and those with confirmed reduced pathogenicity were characterized further.

Nucleic acid manipulations and analysis of a nonpathogenic mutant.

Chromosomal and genomic DNA or total RNA from L. maculans was isolated, resolved by gel electrophoresis, blotted onto nylon membranes, and hybridized as previously described (17). Regions of L. maculans DNA flanking the pUCATPH plasmid were obtained by inverse PCR, and the resultant products were cloned into pGEM T-Easy (Promega). The inverse PCR product derived from a nonpathogenic mutant, which has a single insertion of pUCATPH, was radiolabeled and used to probe a cosmid library (pWEB vector; EpiCentre Technologies) containing 30- to 40-kb inserts of DNA from isolate M1 to obtain the wild-type copy of the mutated L. maculans gene. Insert DNA from a hybridizing cosmid was subcloned, and resultant inserts were sequenced by using ABI dye-primer cycle reactions and an automated sequencer (Applied Biosystems model 373A). The DNA sequence obtained was compared to those in the GenBank database by using BLAST (1), and genes were predicted by using FGENESH software (http://www.softberry.com) and GENSCAN (www.bionavigator.com). One subclone contained an open reading frame with a high degree of sequence similarity to that of isocitrate lyase (icl1). A 6-kb XbaI fragment containing icl1 was ligated into the XbaI site of pAN8-1, a plasmid that confers phleomycin resistance (12). Protoplasts of the nonpathogenic mutant (Δicl1) were transformed with this construct or pAN8-1 as described above, and resultant transformants were selected for growth on phleomycin at 20 μg/ml (Cayla).

Putative complemented strains were inoculated onto B. napus cv. Westar cotyledons and pycnidiospores germinated on water agar plates. Strains were also grown on minimal medium agar supplemented with monolaurate (0.25% Tween 20, polyoxyethylene sorbitan monolaurate) or glucose (0.25%) as the carbon source. Additionally, growth on this carbon source was tested in the presence of inhibitors of isocitrate lyase, i.e., 0.5 or 1 mM 3-nitropropionate (an analogue of succinate) and 0.5 or 1 mM 3-bromopyruvate (an analogue of glyoxylate) (19). After 9 days, colony diameters were compared to those of the wild type (M1) and the Δicl1 mutant. These inhibitors were also added to inoculum and tested for the ability to affect disease development on B. napus cotyledons. The infection patterns of the wild-type and Δicl1 strains were investigated by light microscopy. Fourteen days after inoculation, cotyledon tissue was placed in boiling 95% ethanol for 15 min, cleared in saturated chloral hydrate for 5 days, mounted in Hoyer's medium, and examined with an Olympus light microscope (BH-2) as previously described (18).

To investigate transcription of isocitrate lyase during infection, cotyledons were harvested over the course of infection with isolate M1 and total RNA was examined by Northern blot analysis. The L. maculans icl1 gene and the B. napus isocitrate lyase gene which was cloned by PCR with primers 5′-GAAGGGAGATTCGAGGCG-3′ and 5′-GCCACACTTCTTGGTGAC-3′ (4) were radiolabeled and hybridized to the RNA blot. Control RNA samples were purified from germinated seed, uninfected cotyledons and leaves, and L. maculans mycelia.

RESULTS AND DISCUSSION

A total of 516 hygromycin-resistant transformants were screened on B. juncea and B. napus, and 12 caused either no lesions or lesions less than 30% of the diameter of those caused by isolate M1. The host specificity of these 12 mutants indicates that none of them had an insertion of pUCATPH in the locus conferring host specificity on B. juncea (3). Nine of these mutants had multiple insertions of pUCATPH, while three had single copies; one of the latter mutants was characterized further. Flanking DNA obtained by inverse PCR was used to screen a cosmid library to obtain the wild-type gene. A sequence including the isocitrate lyase (EC 4.1.3.1) gene (icl1) was isolated (GenBank AY118108), and the intron positions in this gene were determined by sequencing fragments of cDNA of L. maculans amplified by PCR with appropriate primers (Fig. (Fig.1).1). A predicted gene is located downstream of icl1 and transcribed in the opposite direction. This gene has high sequence similarity to that which encodes ribose-phosphate pyrophosphokinase (EC 2.7.6.1; prs5) of Neurospora crassa and Saccharomyces cerevisiae and is predicted to be involved in nucleotide metabolism (6). Pulsed-field gel electrophoresis showed that the L. maculans icl1 gene is located on one member of a chromosome doublet (6/7; size, 2.15 Mb) of isolate M1 (data not shown). Southern blot analysis of isolates M1 and Δicl1 showed that deletion of the icl1 gene and an unknown amount of DNA occurred during integration of pUCATPH (Fig. (Fig.11 and data not shown).

FIG. 1.
Alignment of the L. maculans isocitrate lyase gene (icl1) with homologues. (A) Structure of a 5.8-kb region of L. maculans DNA including icl1 (exons are black) and an open reading frame with high sequence similarity to ribose-phosphate pyrophosphokinase ...

Isocitrate lyase is one of two enzymes in the glyoxylate pathway that are involved in the metabolism of two-carbon compounds such as acetate and fatty acids. Accordingly, growth of the Δicl1 mutant was tested on such carbon sources. This mutant was unable to grow on a C12 fatty acid (monolaurate; 0.25% Tween 20) or ammonium acetate (0.5%) as the sole carbon source, in contrast to the wild-type isolate (Fig. (Fig.2).2). Both the mutant and the wild type grew on 0.25% glucose. An analogue of glyoxylate, 3-bromopyruvate, at a concentration of 0.5 or 1 mM did not inhibit growth of isolate M1 specifically on 0.25% Tween 20 (data not shown). However, 3-nitropropionate (an analogue of succinate) at a concentration of 0.5 or 1 mM prevented growth on Tween 20 when it was the sole carbon source (Fig. (Fig.2).2). Both molecules were toxic to L. maculans at concentrations of greater than 5 mM.

FIG. 2.
Growth of L. maculans isolates on complete medium supplemented with hygromycin or phleomycin and on minimal medium supplemented with glucose or monolaurate (Tween 20). A wild-type isolate (M1), an isocitrate lyase mutant (Δicl1), a Δicl1 ...

No transcript of icl1 was observed in the Δicl1 mutant, while in isolate M1, the gene was induced by starvation conditions and by 0.5% ammonium acetate. Glucose (0.25%) did not affect the induction of transcription by acetate (Fig. (Fig.3).3). Glucose causes catabolite repression of isocitrate lyase in fungal species. However, in Aspergillus nidulans, addition of glucose to acetate-containing medium at concentrations similar to those used here for L. maculans did not result in reduced transcription of isocitrate lyase (2). Transcription of icl1 in isolate M1 was detected in cotyledons of B. napus 14 days after infection (data not shown). Transcription of β-tubulin, a constitutively expressed gene that is an indicator of fungal biomass during infection, was also detected at this stage. A wild-type copy of the icl1 gene was reintroduced into the Δicl1 mutant, and eight transformants (Δicl1+icl1) that grew on 0.25% Tween 20 were tested on B. napus (Fig. (Fig.3).3). The resultant lesions for all strains developed more slowly than those caused by the wild-type isolate; however, they were larger than those produced by the Δicl1 mutant (Fig. (Fig.44).

FIG. 3.
Transcriptional regulation of the isocitrate lyase (icl1) gene of L. maculans. Northern analysis of RNA from isolate M1 or an isocitrate lyase mutant (Δicl1) where mycelia were grown in complete medium and then transferred to minimal medium with ...
FIG. 4.
Pathogenicity of L. maculans isolates, including wild-type isolate M1, an isocitrate lyase mutant (Δicl1), and a strain where Δicl1 was transformed with a wild-type copy of icl1icl1+icl1), to cotyledons of B. napus cv. ...

Pycnidiospores of the Δicl1 mutant had an extremely low germination rate, compared to that of the wild type or complemented strains, on water agar plates. Microscopic examination of the wounded infection site revealed limited hyphal growth of the Δicl1 mutant in planta (Fig. (Fig.5).5). This suggests that, rather than a defect in spore germination leading to reduced pathogenicity, a lack of carbon utilization in planta or on the plant surface (in the form of fatty acids) may be responsible for limiting growth; this hypothesis is supported by the finding that when 2.5% glucose was added to the inoculum of the Δicl1 mutant, lesions of sizes similar to those caused by isolate M1 developed (Fig. (Fig.44).

FIG. 5.
Phenotypes of isocitrate lyase mutant (Δicl1) and wild-type (M1) L. maculans strains in planta. Tissue of B. napus was cleared 14 days after inoculation. The plane of focus is just below the cotyledon surface (s, plant stoma; arrow, fungal hypha). ...

The finding that isocitrate lyase is involved in pathogenesis of L. maculans is consistent with recent findings that this gene is necessary for the full virulence of Mycobacterium tuberculosis and Candida albicans in mice (10, 13). L. maculans icl1 is transcribed during infection of B. napus, which is consistent with the induction of transcription of this gene in C. albicans cells grown in the presence of mouse macrophages (10). Vereecke et al. have reported that, in the plant-pathogenic bacterium Rhodococcus fascians, the second enzyme in the glyoxylate pathway, malate synthase, is essential for pathogenicity on tobacco (25). These authors proposed that glyoxylate buildup in the bacterium inhibits further growth after colonization of the plant.

Unlike animals, plants utilize the glyoxylate pathway, so inhibitors of fungal isocitrate lyase and malate synthase would only be effective for disease control if they did not bind the plant homologues (which is unlikely, given the high degree of amino acid sequence similarity between isocitrate lyase genes; Fig. Fig.1)1) or if the plant homologues were not induced during infection. We cloned a fragment of the B. napus isocitrate lyase gene to investigate its transcription during infection. This gene was transcribed at high levels in germinating seeds, as seen previously (4). However, transcription was not detected at 3, 6, 9, and 12 days postinoculation or in uninfected leaves or cotyledons, although a control plant gene (that which encodes actin) was detected (data not shown). Although inhibitors of icl1 might be expected to lead to a decrease in lesion size caused by the wild type on B. napus cotyledons, this did not occur when 1 mM 3-nitropropionate was added to inoculum (data not shown). Higher concentrations of 3-nitropropionate (>5 mM) caused necrosis, suggesting that this compound is phytotoxic at high concentrations.

The glyoxylate pathway occurs in the peroxisome, an organelle involved in various other functions, including β-oxidation of fatty acids and metabolism of reactive oxygen species. The role of peroxisomal proteins in plant-pathogenic fungi for the formation of infection structures such as appressoria has been demonstrated by disruption of the ClaPEX6 gene in Colletotrichum lagenarium, whereby the mutant does not make appressoria and only colonizes wounded tissue (9). Appressorial turgor in the rice blast fungus Magnaporthe grisea depends on lipid reserves to make glycerol (24), an activity probably reliant on peroxisomal function and the glyoxylate pathway. Recently, an isocitrate lyase mutant of M. grisea has been shown to have reduced pathogenicity (Nick Talbot, personal communication). An additional pathway for utilization of acetyl coenzyme A derived from β-oxidation of fatty acids involves carnitine acetyltransferases (23). A carnitine acetyltransferase gene, probably located in the peroxisomes and mitochondria, is required by M. grisea for full pathogenicity (21). Curiously, the transcription of another carnitine acetyltransferase gene, probably located in the cytoplasm, was induced even more highly than the glyoxylate pathway genes in S. cerevisiae during phagocytosis by macrophages (10). Unlike M. grisea, L. maculans does not make appressoria, so the role of fatty acid utilization in the latter fungus is not to provide molecules to promote turgor pressure for infection structures. These results indicate that the glyoxylate pathway functions in pathogenicity in two pathogenic plant fungi with very different modes of infection.

Although the glyoxylate pathway genes are involved in pathogenicity in several animal and plant pathogens, they are not universal pathogenicity factors. Isocitrate lyase is dispensable for disease development in an isolate of S. cerevisiae that is pathogenic to mammals (5), as well as in Cryptococcus neoformans, an opportunistic pathogen of animals (16). Glyoxylate pathway genes are absent from the plant-pathogenic bacterium Xylella fastidiosa (20).

Acknowledgments

We thank Olen Yoder and Gillian Turgeon for plasmid pUCATPH, Peter Punt for plasmid pAN8-1, Richard Oliver for phleomycin, Peter Dunn for protoplasting enzyme, Leanne Wilson for RNA from B. napus infected with L. maculans, and Nick Talbot for permission to cite unpublished observations. Also, we thank Alice Churchill for critical appraisal of the manuscript.

We thank the Australian Grains Research and Development Corporation for funding.

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