Molecular Plant Advance Access originally published online on May 9, 2008
Molecular Plant 2008 1(3):471-481; doi:10.1093/mp/ssn014
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Mutation of a Gene in the Fungus Leptosphaeria maculans Allows Increased Frequency of Penetration of Stomatal Apertures of Arabidopsis thaliana
a School of Botany, The University of Melbourne, Melbourne, Vic 3010, Australia
b Current address: Laboratory of Forest Protection, Faculty of Forestry, Gadjah Mada University, Yogyakarta, Indonesia 55281
1 To whom correspondence should be addressed. E-mail bhowlett{at}unimelb.edu.au, fax +61 39347–5460, tel. +61 38344–5062.
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Abstract |
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Leptosphaeria maculans, a pathogen of Brassica napus, is unable to invade most wild-type accessions of Arabidopsis thaliana, although several mutants are susceptible. The infection pathway of L. maculans via a non-invasive inoculation method on A. thaliana lms1 (undefined), pmr4-1 (defective in callose deposition), and pen1-1 and pen2-1 (defective in non-host responses to several pathogens) mutants is described. On wild types Col-0 and Ler-0, hyphae are generally arrested at stomatal apertures. A T-DNA insertional mutant of L. maculans (A22) that penetrates stomatal apertures of Col-0 and Ler-0 five to seven times more often than the wild-type isolate is described. The higher penetration frequency of isolate A22 is associated with an increased hypersensitive response, which includes callose deposition. Complementation analysis showed that the phenotype of this isolate is due to T-DNA insertion in an intronless gene denoted as ipa (increased penetration on Arabidopsis). This gene is predicted to encode a protein of 702 amino acids with best matches to hypothetical proteins in other filamentous ascomycetes. The ipa gene is expressed in the wild-type isolate at low levels in culture and during infection of A. thaliana and B. napus.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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The ascomycete, Leptosphaeria maculans, causes blackleg (or phoma stem canker)—the major disease of Brassica napus worldwide (Fitt et al., 2006). Sexual and asexual spores germinate upon contact with leaves or cotyledons of B. napus, and hyphae penetrate through stomatal apertures and grow asymptomatically in the mesophyll layer before entering vascular tissue via minor veins in the leaf. A trailing necrosis develops in the leaf as the fungus grows asymptomatically through the vascular tissue down to the stem base and even into the root (Hammond and Lewis, 1987; Sprague et al., 2007). This fungus cannot normally infect Arabidopsis thaliana. However, an accession and several mutants that are susceptible have been described. Bohman et al. (2004) classified an accession (An-1) and EMS-generated mutants (lms1 to lms11), as well as mutants (pad3-1, deficient in biosynthesis of camalexin, and pmr4-1, impaired in callose synthase) as susceptible after inoculating wounded leaves. In susceptible plants, the first symptom was chlorosis, followed by a grey or brown discoloration. The centre of the lesion collapsed and pycnidia formed. These plants (including An-1 and lms-1) had wild-type levels of camalexin, indicating that this phytoalexin was not the only determinant of resistance. Also, several susceptible mutants and resistant wild types produced callose after inoculation, suggesting that callose was only partially responsible for resistance to L. maculans (Kaliff et al., 2007; Staal et al., 2006).
Further studies by this research group led to the identification of two resistance loci in A. thaliana. Staal et al. (2006) examined several recombinant inbred lines and found susceptible progeny, regardless of the genotype of L. maculans isolate used in the screening. A locus (RLM1Col = At1g64070) in Col-0 background comprised a complex of seven structurally related toll interleukin receptor (TIR)–nucleotide-binding site (NB)–leucine-rich repeat (LRR) genes. Another locus (RLM2Ler) in Ler-0 contained a paralogue. The NB–LRR class of genes usually encodes cytoplasmic proteins and some are involved in host resistance mechanisms, whilst others are involved in non-host interactions (Jones and Dangl, 2006). These seven genes at the RLM1Col locus are members of the H subgroup of TIR–NB–LRR genes. This subgroup has not as yet been associated with any classical resistance (R) genes (Meyers et al., 2003). In contrast to such classical R genes, which are constantly evolving in concert with their cognate Avr genes in the pathogen, members of the H subgroup do not appear to be under positive selection (Palomino et al., 2002). Indeed, Jones and Dangl (2006) classify A. thaliana as a non-host for L. maculans, on the basis of characteristics of RLM1 and RLM2 and on the type of resistance mediated by these genes.
The best characterized plant-fungal system displaying non-host resistance is the interaction between A. thaliana and the powdery mildew fungus, Blumeria graminis f. sp. hordei. Screening of A. thaliana EMS-generated mutants with B. graminis f. sp. hordei led to the discovery of three mutants (pen1-1, pen2-1, and pen3-1) that allowed higher penetration frequencies of this fungus (Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006). Although these mutants allowed a high rate of hyphal penetration, post-invasive growth was prematurely arrested. The three genes responsible for the mutant phenotypes were identified by map-based cloning. PEN1 encodes a syntaxin containing a SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) domain, suggesting involvement in membrane fusion and secretion events (Collins et al., 2003). PEN2 encodes a peroxisomally located glycosyl hydrolase (Lipka et al., 2005), whilst PEN3 encodes the putative ATP-binding cassette (ABC) transporter, pleotropic drug resistance 8 (PDR8) and contributes to defense at the cell wall, and also intracellularly (Stein et al., 2006). In contrast to pen1-1, which only allows growth of a limited range of pathogens, pen2-1 and pen3-1 are susceptible to a wide spectrum of other non-host pathogens, such as Erysiphe pisi, Phytophthora infestans, Plectosphaerella cucumerina, and Pythium irregulare (Adie et al., 2007; Lipka et al., 2005; Stein et al., 2006).
In contrast, little is known about molecules in the pathogen involved in non-host interactions. The availability of a bank of tagged T-DNA insertional transformants of L. maculans offered us the opportunity to screen for fungal mutants with altered infection phenotypes on A. thaliana. In this paper, we describe a L. maculans mutant that has an increased penetration frequency on A. thaliana wild-type accessions Col-0 and Ler-0, as well as on several mutant plant lines. We also describe the gene that is mutated by the T-DNA insertion.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Infection Pathway of Leptosphaeria maculans on Intact Leaves of Arabidopsis thaliana
Inoculation via wounding is used widely to assess the interaction between fungi and their host plants. However, wounding often triggers host defense responses, such as production of callose, pathogenesis-related proteins, phytoalexins, oxidative compounds and defense signaling molecules (for instance, jasmonate, salicylate and ethylene) and these wounding responses complicate the assessment of host responses to infection (Leon et al., 2001). Accordingly, we developed a non-invasive method to inoculate intact leaves of A. thaliana. Initially, pycnidiospores of the wild-type isolate (IBCN18) in water were applied to intact leaves of ecotype Ler-0. Tissue was harvested at various times and stained with lactophenol-trypan-blue. Even after 3 d, no germinating spores were observed, and lesions never developed (Figure 1A). A similar result was seen on Col-0 and on mutants lms1, pad3-1, and pmr4-1 (data not shown). However, when inoculum was prepared in 10% Campbell's V8 juice or 1% glucose, the germination rates on leaves of Ler-0 were 86 and 74%, respectively (data not shown). This increase in germination was not peculiar to isolate IBCN18; five randomly chosen unrelated field isolates behaved similarly (data not shown). For all subsequent inoculations, a drop of pycnidiospores in 1% glucose was placed on each half of each intact leaf of A. thaliana or a cotyledon of B. napus cv. Monty, a moderately susceptible cultivar. At 3 d post-inoculation (dpi), a high proportion of spores had germinated (Figure 1B, 1D, 1I, and 1N). Hyphal invasion was always associated with penetration of the stomatal aperture. By 5 dpi, hyphae had penetrated stomatal apertures of mutants lms1, pmr4-1, and B. napus (Figure 1E, 1J, and 1O) and were colonizing the spaces between mesophyll cells (Figure 1F, 1K, and 1P). In contrast, hyphae seldom penetrated stomatal apertures of Ler-0 (Figure 1C) or Col-0 (data not shown). By 7 dpi, some localized trypan-blue-staining material, indicative of a cell death (hypersensitive) response, was apparent in lms1, pmr4-1, and B. napus, but hyphae were not observed in direct contact with these cells (Figure 1G, 1L, and 1Q). By 14 dpi, pycnidia (arrows) were obvious in these plants (Figure 1H, 1M, and 1R).
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Responses to inoculation by isolate IBCN18 were assessed on A. thaliana mutants pen1-1 and pen2-1. At 3 dpi, pycnidiospores had germinated on leaves of the mutants and Col-0 (Figure 2A, 2G, and 2K). Although, at 5 dpi, hyphae penetrated leaves of pen1-1 and pen2-1 (but not Col-0) via the stomatal aperture (Figure 2B, 2H, and 2L), only in pen1-1 did hyphae grow into intercellular spaces of one or two layers of the palisade mesophyll (data not shown). Trypan-blue staining indicated an intense hypersensitive response in pen1-1 and pen2-1, but not in Col-0 at 5 dpi (Figure 2C, 2I, and 2M). In addition, aniline-blue staining revealed increased fluorescence in pen1-1 and pen2-1 compared with Col-0, indicative of callose accumulation in these mutants (Figure 2D, 2J, and 2N). Intercellular hyphae were colonizing palisade and spongy mesophyll layers in pen1-1 by 7 dpi (Figure 2E) and sporulation occurred at 14 dpi (Figure 2F). In contrast, after 5 dpi, fungal growth was arrested in pen2-1.
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Identification of a T-DNA Insertional Mutant (A22) of L. maculans with an Increased Penetration Frequency on Arabidopsis thaliana
Individual wild-type L. maculans (IBCN18) isolates that had been transformed with an Agrobacterium tumefaciens T-DNA construct were inoculated onto intact leaves of A. thaliana lines Col-0, Ler-0, and mutants pen1-1 and pen2-1. At 5 dpi, tissue was harvested, cleared, and stained with lactophenol-trypan-blue. Potential infection sites were identified by counting the frequency of stomatal apertures with associated hyphae (with or without penetration). When hyphae were associated with, but not penetrating, the stomatal aperture, this was classified as an unsuccessful penetration event (Figure 3A). When hyphae had entered through the stomatal aperture, this was scored as successful (Figure 3B and 3C). Of 142 L. maculans transformants tested, one (A22) showed an increased frequency of penetration (42% on Col-0; 40% on Ler-0) compared with the wild-type isolate (6% on Col-0; 10% on Ler-0) (Figure 3D). Hyphae of isolate A22 were arrested in the palisade mesophyll layer on these lines and, consequently, this isolate was not able to complete its vegetative lifecycle (data not shown). The penetration frequencies of the wild-type isolate on pen1-1 and pen2-1 were 15 and 18%, respectively, and those of isolate A22 were 37 and 40%, respectively. However, both these isolates had similar penetration frequencies (42%) on B. napus (Figure 3D). This fungal mutant elicited a stronger hypersensitive response than the wild-type isolate on A. thaliana Col-0 or Ler-0, as seen by increased deposition of trypan-blue-staining material (Figure 4A–4D) and aniline-blue-staining material (Figure 4E–4H). The increase in HR appeared to be associated with an increase in fungal biomass, even though the staining material was seldom in direct contact with hyphae. Aniline-blue-staining material did not accumulate in uninfected leaves of any of the A. thaliana plants tested (data not shown). The A22 isolate was also inoculated onto intact leaves of mutants pad3-1, pmr4-1, and lms1, previously shown to be susceptible to the wild-type L. maculans isolate. No differences in infection phenotype between isolate A22 and the wild-type isolate were observed (data not shown).
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Identification of the Disrupted Gene in L. maculans Mutant Isolate A22
Sequences flanking the T-DNA insertion in L. maculans mutant isolate A22 were identified by TAIL–PCR and a total of 665 bp of L. maculans DNA was cloned. Southern analysis showed that this is a single copy sequence and that one copy of T-DNA had inserted into the genome of mutant A22 (data not shown). The 665-bp fragment hybridized to a cosmid sized 48 kb from a library of isolate IBCN18. Sequence analysis revealed that the T-DNA had inserted in the open reading frame of a gene, which we denoted as ipa (increased penetration on Arabidopsis), and caused a 16-bp deletion (GCAGAAGCTGACCGAC) 1243 bp downstream of the initiation codon after tryptophan 414. A diagram of the predicted gene including the site of T-DNA insertion and location of primers used for subsequent RT–PCR analysis is presented in Figure 5A. This gene (GenBank EU266496 [GenBank] ) is predicted to be intronless and to encode a protein of 702 amino acids, whose best matches are to hypothetical proteins from other filamentous ascomycetes including the closely related dothideomycete, Phaeosphaeria nodorum (GenBank EAT82059 [GenBank] ; E value 0), Magnaporthe grisea (GenBank XP_360976 [GenBank] ; E value 2e–140), Sclerotinia sclerotiorum (GenBank XP_001584918; E value 5e–131), Aspergillus oryzae (GenBank BAE65568 [GenBank] ; E value 1e–156), Neosartorya fischeri (GenBank XP_001267654; E value 1e–123), and A. terreus (Genbank XP_001215794; E value 9e–53).
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Further sequencing revealed the presence of hypothetical genes flanking ipa. Best matches of these genes were to genes from P. nodorum, one encoding a protein with a sulfatase domain (GenBank EAT81589 [GenBank] ; E value 7e–134) and the other encoding a protein containing an aspartyl protease domain (GenBank EAT86827 [GenBank] ; E value 5e–84). In P. nodorum, these two genes do not flank the ipa homologue, but are located 57 and 13 kb, respectively, from it (data not shown). A suite of software programs was used to seek conserved domains in the predicted IPA protein, but none gave predictions with high probability values. For instance, no domains were revealed by searches of the Conserved Domain Database (CDD) or the Conserved Domain Architecture Retrieval Tool (CDART) at NCBI, nor were any domains, such as secretion signals detected by PROSITE. However, PSORTII predicted two dileucine motifs at amino acid numbers 465 and 466, and 589 and 590. This domain is often involved in protein–protein interactions. This program also predicted a transmembrane domain type 1b with a cytoplasmic tail at amino acids 408–667. A hydropathy analysis was performed using TopPred (Claros and von Heijne, 1994). A transmembrane region, similar to that predicted by PSORTII, was identified with a hydrophobicity value just exceeding the prediction threshold. However, more stringent analyses conducted with the DAS–TMfilter server, containing a filter for false-positive predictions (Cserzo et al., 2002) and two other algorithms, TMHMM2.0 (Krogh et al., 2001) and SOSUI (Hirokawa and Boon-Chieng, 1998), did not predict transmembrane regions within IPA.
Transcriptional Analysis
Conditions under which ipa was expressed were sought. Northern blots of poly A+ RNA of the wild-type and A22 isolates grown in 10% Campbells V8 juice (complete media) were probed with fragments from either the 5' or 3' part of ipa, but no signals were detected. Reverse transcriptase RT–PCR experiments were then performed using primers predicted to amplify a 364-bp fragment (Table 1; Figure 5A). Transcripts were detected when IBCN18 was grown under several conditions, including complete media, minimal media containing 1% glucose, B. napus leaf-extract media, and tissue of B. napus or A. thaliana, 14 and 10 dpi, respectively (data not shown). A fragment of actin was amplified as a control. RT–PCR experiments with a range of primers designed at various positions across ipa were carried out to determine the length and relative abundance of the transcript in the wild-type and mutant isolates. Although a band was not amplified from the wild type with primers spanning the complete gene (P18 and P16), overlapping fragments were amplified with other primer sets. The size of the transcript was calculated as 2109 bp. Similar experiments using sets of primers 5' of the T-DNA insertion revealed the size of the transcript in mutant A22 as at least 1055 bp. To estimate the expression level of ipa, RT–PCR was performed using a five-fold dilution series of cDNA of isolate IBCN18 grown in complete media. While a 141-bp band of actin was amplified from all dilutions after only 25 cycles, 35 cycles were required to detect amplification of a 364-bp band of ipa in all but the 1/625 dilution (Figure 5B). The intensity of the actin band after 25 cycles was higher than that of ipa with 35 cycles, indicating a low level of expression of ipa.
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Searches of collections of ESTs at NCBI using TBLASTN revealed ESTs from other filamentous ascomycetes, two from Gibberella moniliformis grown in rich media and on maize tissue (GenBank DR640899; E value 2e–58 and DR630230; E value 8e37) and one from Geomyces pannorum (GenBank DY993070; E value 3e–55) grown in rich media. The sequences of these ESTs overlapped such that the 5' end of the Ge. pannorum EST matched that from the ATG of ipa, while the 3' end of the longest Gi. moniliformis sequences ended 1347 bp downstream of the initiation codon of ipa (data not shown).
Complementation of Leptosphaeria maculans Mutant A22
To confirm that the T-DNA insertion in isolate A22 was responsible for increased penetration of A. thaliana, this fungal mutant was transformed with a plasmid containing the complete coding sequence of ipa and 2 kb each of 5' and 3' flanking sequence, as well as a gene conferring nourseothricin resistance. PCR-amplification and Southern analysis showed that each of five transformants had an intact copy of ipa integrated into their genomes. Two transformants, A22c6 and A22c8, caused a similar hypersensitive response on A. thaliana Col-0 to that caused by isolate IBCN18 (Figure 6A, 6C, and 6D), while the other three caused a similar response to that of isolate A22 (Figure 6B and data not shown). These findings are consistent with frequencies of penetration of the stomatal aperture by these isolates. Transformants A22c6 and A22c8 showed penetration frequencies similar to those of IBCN18 and significantly less than that of mutant A22 (Figure 6E). Thus, these two transformants had a similar phenotype to the wild-type isolate and, accordingly, had been complemented by ipa. Transformants that induced a strong hypersensitive response (isolates A22c11, A22c12, and A22c18) penetrated intact leaves at frequencies of 19, 27, and 35%, respectively (data not shown), and thus had a similar phenotype to that of mutant A22.
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DISCUSSION |
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To be successful as plant pathogens, microbes must first recognize a surface as an appropriate place on which to initiate germination and growth programs, and then must successfully penetrate the plant surface, all the while avoiding recognition and defense responses of the plant (Thordal-Christensen, 2003). Leptosphaeria maculans has developed a varied arsenal to attack and colonize its natural host, as it has a hemibiotrophic lifestyle with alternating periods of biotrophy and necrotrophy (Rouxel and Balesdent, 2005). Despite this arsenal, L. maculans fails to invade A. thaliana; indeed, pycnidiospores do not germinate on intact leaves. This finding was unexpected, since such spores germinate on intact cotyledons of B. napus at frequencies upwards of 50% (Li et al., 2004). The reason for the lack of germination on A. thaliana is unknown, but this barrier was overcome by addition of a carbon source.
In contrast to the frequent penetration by hyphae on particular A. thaliana mutants, penetration of stomatal apertures was rare on wild types, suggesting that the plant is preventing entry by the fungus, or that the fungus cannot recognize appropriate sites of entry. Fungi such as Puccinia striiformis f. sp. tritici follow surface contours on wheat to find stomatal openings (Moldenhauer et al., 2006). Cytological studies of the Brassica juncea–L. maculans interaction showed no evidence of surface recognition, although, after hyphae began to penetrate the stomatal aperture, the adjacent guard cell often collapsed as a resistance response (Chen and Howlett, 1996). Guard cell collapse was never observed in this study.
The appearance of necrotic areas that stained with trypan-blue 5 d after inoculation of Col-0 and Ler-0 suggests that the few successful penetration events through stomatal apertures were stopped due to a defence response in the mesophyll layer. Intriguingly, cells undergoing a HR did not often appear to be in direct contact with hyphae. Similarly, the appearance of localized fluorescence after aniline-blue staining of infected Col-0 and Ler-0 suggests the importance of callose in the resistance response to L. maculans. This finding is consistent with the susceptibility of pmr4-1—a mutant that does not produce the callose synthase enzyme responsible for callose accumulation after biotic, abiotic and chemical stresses (Nishimura et al., 2003). These authors showed that callose or callose synthase negatively regulates the salicylic acid signaling pathway in A. thaliana. However, resistance in the A. thaliana in the L. maculans interaction is independent of salicylic acid, ethylene, ABA, and jasmonic acid signaling (Bohman et al., 2004). The callose response has been reported as depending on the presence of RLM1Col and elevated in the absence of camalexin (in the pad3-1 mutant) (Kaliff et al., 2007; Staal et al., 2006). However, we observed no difference in the pattern of aniline-blue staining of Ler-0 (which does not contain RLM1Col) and Col-0. This could be because we examined leaves at 5 dpi, at which time, pycnidiospores would have germinated and penetrated stomatal openings, whereas previous studies (Kaliff et al., 2007; Staal et al., 2006) were carried out at 2 dpi, well before stomatal apertures are penetrated.
Since wild-type accessions of A. thaliana are resistant to infection by L. maculans, we were unable to investigate further the cytology of infection in these lines. Therefore, we examined A. thaliana lms-1, pad3-1, pmr4-1, and pen1-1 mutants previously shown to be susceptible when wounded (Kaliff et al., 2007), to see if they were susceptible when intact leaves were inoculated by the wild-type isolate. Not surprisingly, these mutants all developed disease. Intriguingly, penetration mutant (pen2-1) previously shown to be susceptible to non-host pathogens was not susceptible to L. maculans. PEN2, a glycosyl transferase, is proposed to activate a toxin that poisons fungal penetration pegs at an early stage of invasion (Lipka et al., 2005). Mutation of such a gene may not be expected to affect invasion by L. maculans, as this process does not involve penetration pegs. However, since Arabidopsis and Brassica spp. produce indole-based secondary metabolites, L. maculans may be either less sensitive to such secondary metabolites or better able to detoxify them than B. graminis. Indeed, L. maculans can detoxify indole phytoalexins including brassinin from Brassica species (Pedras et al., 2007).
The screening of mutant banks of A. thaliana with wild-type host and non-host microbes has allowed the unraveling of mechanisms plants use to defend against microbe attack. In contrast, the mechanisms microbes use to attack plants are only just being discovered. Our screening of a bank of L. maculans mutants for the ability to penetrate stomatal openings of wild-type accessions of A. thaliana revealed an isolate with an insertion in an intronless gene, unfortunately with no clue as to its function. The increased penetration frequency of this mutant was the same on Col-0 and Ler-0, thus indicating that this phenomenon is independent of the resistance genes, RLM1Col and RLM2Ler. Interestingly, the penetration frequency of isolate A22 on all A. thaliana lines and mutants tested was similar to that by the wild type on the natural host B. napus isolate.
The finding that ipa was expressed in the wild-type isolate under all conditions tested in vitro and in planta, albeit at a low level, suggests that the function of ipa may not only be related to in-planta growth. This contention is supported by the finding of ESTs similar to ipa in G. moniliformis also expressed in vitro and in planta. Complementation of the L. maculans mutant isolate with an intact copy of ipa confirmed that the phenotype of the A22 mutant could be solely explained by the insertion of the T-DNA resulting in the expression of a truncated transcript.
Clues to the function of the protein encoded by the ipa gene were not gained using bioinformatic tools or by comparative analysis with other fungi. In the L. maculans A22 mutant, the insertion of the T-DNA introduced a premature stop codon, thus resulting in a truncated, perhaps unstable, protein. Searches for similar fungal genes in databases revealed only hypothetical proteins in a few filamentous ascomycetes, none of which shares a common mode of infection or natural host. Until more clues about domains in this protein, or roles of homologues in other fungi, are revealed, a role for this protein cannot be devised.
Arabidopsis thaliana has proven a valuable model for dissecting the mechanisms of non-host resistance of plants. However, the mechanisms used by non-host microbes, especially fungi, are only just being discovered. An increasing number of fungal genomes have been sequenced recently, thus increasing the potential for gene discovery. However, many genes in these genome sequences are annotated as hypothetical proteins and the annotation and assignation of gene function are dependent upon functional studies. The availability of mutant banks in several plant pathogenic fungi should allow further studies to reveal the hidden strategies of fungi that are key to their success as pathogens.
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Fungal Culturing, Plant Lines, and Growth Conditions
Leptosphaeria maculans wild-type isolate IBCN18 and derivatives were grown at 22°C on 10% Campbell's V8 juice agar under white fluorescent light. Arabidopsis thaliana mutants pad3-1, pmr4-1, pen1-1, and pen2-1, as well as their parental line, Col-0, and lms1, and its parental line, Ler-0, were grown in Jiffy-7 peat pellets (Jiffy International AS) under controlled conditions (8 h light at 22°C and 16 h darkness at 18°C). Brassica napus cv. Monty, which is moderately susceptible to all L. maculans isolates tested so far, was grown in controlled conditions with 12 h light and 12 h darkness at 22°C. Pycnidiospores were inoculated onto intact leaves of A. thaliana (five to eight leaf stage) and on cotyledons of 10-d-old B. napus plants by placing a drop (10 µL) on each half of each leaf in the presence of water, 10% Campbell's V8 juice or 1% glucose. The inoculated plants were kept at 100% relative humidity for 3 d. After 3, 5, 7, and 14 d post inoculation (dpi), A. thaliana leaves and B. napus cotyledons were detached and photographed, and subsequently were fixed for microscopy examination. For each treatment, leaves of five A. thaliana plants or cotyledons of 10 B. napus seedlings were inoculated and each experiment was repeated at least twice.
Microscopy
Infected plant issue was fixed and decolorized by boiling in 100% ethanol for 3 min and then cooling at room temperature for 1 h. Subsequently, samples were cleared in saturated chloral hydrate (2.5 g mL–1). Fungal structures and host cell death were visualized using trypan-blue according to the method by Keogh et al. (1980). Stained samples were observed using either an Olympus BH2 microscope or a dissecting microscope Leica MZ FLIII and photographed. Accumulation of callose (β-1,3 glucan) was visualized by aniline-blue staining using a modified method of Chen and Howlett (1996). Cleared samples were incubated in 0.01% aniline-blue (Polyscience Inc., PA, USA) in 0.1 M potassium phosphate buffer pH 9.5 for 1 h then mounted in distilled water. Samples were examined by differential interference contrast optics using a microscope equipped with epifluorescence optics model RFL attached to Olympus BH2 (365 nm excitation filter, 420 nm emission filter).
Insertional Mutagenesis and Screening for Penetration of Stomatal Apertures of Leaves of A. thaliana Col-0
Leptosphaeria maculans isolate IBCN18 was transformed with plasmid pGTII, which contains a T-DNA insert with a promoterless enhanced green fluorescent protein gene (EGFP) and a constitutively expressed hygromycin resistance gene using Agrobacterium-mediated transformation (Elliott and Howlett, 2006; Gardiner and Howlett, 2004). This vector was designed with stop codons in all three frames, and so a transcript in the mutant isolate would be expected to terminate 105 bp into the T-DNA. Although the plasmid pGTII contains EGFP, fluorescence was not observed reliably, probably because this construct contains a splice acceptor and an internal ribosomal entry site originating from a non-fungal host (Ishida and Leder, 1999). Transformants were examined for their ability to penetrate stomatal apertures of A. thaliana Col-0 as follows. Pycnidiospores were inoculated onto intact leaves (five to eight leaf stages) by placing a drop (10 µl of 106 spores mL–1) on each half of each leaf in the presence of 1% glucose. Leaves were harvested after 5 dpi. Whole-leaf mounts were prepared and stained with lactophenol-trypan-blue and examined using light microscopy, as described above. Stomatal apertures with associated hyphae (with or without penetration) were counted as infection sites. Penetration through the stomatal aperture was scored as successful, whilst stomatal apertures without hyphal association were scored as unsuccessful penetration events (Figure 3). Initially, such events arising from 100 infection sites (50 sites on each of two leaves from one plant of A. thaliana Col-0) were scored as a percentage of total number of infection sites. Selected L. maculans insertional mutants were re-examined by assessing 180–300 infection sites (30–50 sites examined on each of six leaves from three different plants). One hundred and forty-two transformants were screened and one (A22) had a higher penetration frequency on Col-0 than isolate IBCN18. This transformant was tested further on intact leaves of A. thaliana Ler-0, pad3-1, pm4-1, lms1, pen1-1, and pen2-1 and also on intact cotyledons of B. napus cv. Monty.
Identification of T-DNA Tagged Gene in Leptosphaeria maculans Mutant A22
In order to characterize the mutated gene, genomic DNA was prepared from mycelia and digested with an enzyme that cuts once within the T-DNA and subjected to Southern analysis to confirm single integration of the T-DNA (data not shown). Thermal asymmetric interlaced (TAIL)–PCR was performed to identify DNA flanking the insertion, as described by Mullins et al. (2001). For the primary PCR reaction, template was prepared from mycelial fragments using Extract-N–AmpTM Plant PCR Kits (Sigma) according to the manufacturer's protocol. In addition, two arbitrary degenerate primers were used (Table 1, P1 and P2) along with two sets of border-specific primers (Table 1; left border: P3 –P5; right border: P6 – P8). The resultant product was cloned into plasmid pCR®2.1–TOPO® (Invitrogen), and sequenced using M13 forward and reverse primer. The resultant plasmid was digested with EcoR1 and Xba1 to obtain a 665-bp fragment of the disrupted gene, named ipa. A cosmid library of isolate IBCN18, as well as genomic DNA, was probed with this fragment and a hybridizing cosmid was selected and sequenced using a primer walking strategy. Genes on the cosmid were predicted using FGENESH software (www.softberry.com). A BLASTp search (www.ncbi.nlm.nih.gov) was used to compare amino acid sequences of predicted genes in the flanking region of the T-DNA to sequences with a known or hypothesized function in other fungi. Predicted amino acids were aligned using the ClustalW program (www.ebi.ac.uk). Predicted domains of IPA were sought using PROSITE (http://au.expasy.org/prosite/) (Hulo et al., 2006), SMART (http://smart.embl-heidelberg.de/) (Letunic et al., 2006), Pfam (www.sanger.ac.uk/Software/Pfam/search.shtml) (Finn et al., 2006), ProDom (http://prodom.prabi.fr/prodom/current/html/form.php) (Bru et al., 2005), and DomPred (http://bioinf.cs.ucl.ac.uk/dompred/DomPredform.html) (Marsden et al., 2002).
The subcellular localization of IPA was predicted using PSORT II (http://psort.hgc.jp/form2.html) (Nakai and Horton, 1999), TopPred (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) (Claros and von Heijne, 1994), the DAS–TMfilter server (http://mendel.imp.ac.at/sat/DAS/DAS.html) (Cserzo et al., 2002), TMHMM2.0 (www.cbs.dtu.dk/services/TMHMM) (Krogh et al., 2001), and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui) (Hirokawa and Boon-Chieng, 1998). Amino-terminal secretion signals of IPA were sought using SignalP 3.0 (www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al., 2004).
Complementation of L. maculans A22 Mutant
A 6.5-kb Hpa1 fragment containing the entire ipa coding region was sub-cloned from the cosmid clone into binary vector PZPnat1 (GenBank AY631958
[GenBank]
), which contains the nourseothricin resistance gene from Streptomyces noursei under the control of Aspergillus nidulans Trp C regulatory sequences. The resultant plasmid is denoted as pNat–ipac and was introduced back into L. maculans mutant A22 via Agrobacterium-mediated transformation. Transformants were selected on 50 µL–1 nourseothricin, then screened for the presence of ipa by PCR with primers P9 and P10 (Table 1). Putative complemented isolates had an amplified band sized 364 bp, and five of these were inoculated onto intact leaves of A. thaliana Col-0 in the presence of 1% glucose to see if the phenotype of IBCN18 was restored. Samples were harvested at 5 dpi and whole-leaf mounts were prepared and stained with lactophenol-trypan-blue or aniline-blue. Penetration frequencies and callose accumulation, respectively, were determined. The presence of an intact copy of ipa was confirmed by Southern analysis using standard molecular biology techniques.
Transcriptional Analysis
Isolate IBCN18 was grown on 10% Campbells V8 juice or minimal media (Tinline et al., 1960) containing 1% glucose for 5 d or on B. napus leaf-extract media (prepared by macerating mature leaves, boiling them for 30 min and filter sterilizing the extract) for 2 d. Isolate A22 was cultured on 10% V8 juice and harvested after 5 d. Cotyledons of 14-d-old seedlings of B. napus were punctured with a 25 gauge needle (two wounds per cotyledon) and pycnidiospores (10 µl of 106 mL–1) of isolate IBCN18 were placed over the wounded area (Purwantara et al., 1998). Pycnidiospore suspensions in 1% glucose (four drops of 10 µL per drop) of isolate IBCN18 were applied onto intact leaves of A. thaliana lms1, chosen as it supports a large amount of fungal biomass, which maximizes detection of fungal transcripts. Infected cotyledons of B. napus and leaves of A. thaliana lms1 were harvested at 14 and 10 dpi, respectively, frozen in liquid nitrogen and freeze-dried. Samples were then extracted and RNA isolated using the TRIzol® reagent (Invitrogen) and total RNA was DNase treated and reverse transcribed as described by Elliott and Howlett (2006).
The expression level of ipa in L. maculans isolate IBCN18 in 10% V8 juice was estimated using a five-fold dilution series (1/1, 1/5, 1/25, 1/125, and 1/625) of cDNA to compare amplification of a fragment of ipa (with primers P9 and P10) to actin as a representative of the fungal biomass. Primers LmActinF and LmActinR (Table 1) used to amplify actin spanned an intron such that amplification of genomic DNA would result in a 193-bp band whilst amplification of cDNA would result in a 141-bp band. RT–PCR experiments were also performed to confirm the extent of the ipa transcript using primer sets indicated in Figure 5.
Accession Numbers
Sequence data from this article can be found in the GenBank data library under accession number EU266496.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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We thank AUSAID, Australia, for a scholarship to Harjono, and the Australian Grains Research and Development Corporation for funding this research.
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We thank Professor Christina Dixelius and Dr Jens Staal, Dr Shauna Somerville, and Professor Paul Schulze-Lefert for helpful discussions and for providing Arabidopsis thaliana mutants. We thank Anton Cozijnsen for assistance with the transcriptional analyses and Dr Adrienne Sexton for critically reading the manuscript.
No conflict of interest declared.
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2 These authors contributed equally to the work.
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