JXB Advance Access originally published online on April 18, 2005
Journal of Experimental Botany 2005 56(416):1525-1533; doi:10.1093/jxb/eri145
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RESEARCH PAPER |
Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance
Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
* To whom correspondence should be addressed. Fax: +1 519 837 0442. E-mail: pgoodwin{at}uoguelph.ca
Received 10 October 2004; Accepted 28 February 2005
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Abstract |
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Four glutathione S-transferase (GST) genes, NbGSTU1, NbGSTU2, NbGSTU3, and NbGSTF1, were amplified from cDNA of Nicotiana benthamiana leaves infected with Colletotrichum destructivum using primers based on conserved regions of N. tabacum GST sequences. Expression of NbGSTU1 and NbGSTU3 increased progressively during infection by either C. destructivum or Colletotrichum orbiculare, except for a slight decrease by NbGSTU1 late in the infection, whereas NbGSTU2 and NbGSTF1 expression remained relatively constant. Each of the four genes was cloned into a PVX vector for virus-induced gene silencing, and reduced expression of the four genes was detected by RT-PCR. A statistically significant increase in susceptibility of N. benthamiana to infection following gene silencing was found only for NbGSTU1-silenced plants, which had 130% more lesions and 67% more colonization by C. orbiculare compared with control plants. These results demonstrate that the different GST genes respond in different ways to fungal infection, and at least one plant GST gene has an important role in disease development.
Key words: Fungal infection, glutathione S-transferase, hemibiotrophy, virus-induced gene silencing
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Introduction |
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During infection by fungal pathogens, plant cells respond by expressing a battery of disease response genes, which can result in the production of various toxic plant products, including active oxygen species and phytoalexins (Marrs, 1996
; Lamb and Dixon, 1997). In addition, an invading fungus may produce stress-inducing chemicals, such as phytotoxins, resulting in significant stress and damage to the host cells. One response of plants is the increased expression of glutathione S-transferase (GST) genes following infection by pathogens (Mauch and Dudler, 1993; Hahn and Strittmatter, 1994; Wagner et al., 2002).
GSTs are dimeric enzymes that catalyse the conjugation of electrophilic molecules to glutathione (GSH). In plants, these conjugates are sequestered in the vacuole where they are further processed and detoxified (Gullner and Komives, 2001; Dixon et al., 2002). In addition to catalysing GSH conjugation reactions, GSTs can function as carriers of auxin and phenylpropanoids, transporters of anthocyanin into the vacuole, and enzymes in tyrosine catabolism (Droog et al., 1995; Mueller et al., 2000; Yu and Facchini, 2000; Smith et al., 2003; Kitamura et al., 2004). GSTs can serve as signalling molecules, activating phenylpropanoid metabolism following exposure to UV light (Loyall et al., 2000). Stress-inducible GSTs also have glutathione peroxidase activity, thereby protecting cells from oxidative injury by detoxifying organic hydroperoxides of fatty and nucleic acids (Dixon et al., 2002). Organic peroxides are created in plants during processes such as photosynthesis, pathogen attack (Mauch and Dudler, 1993), detoxification of microbial toxins (Edwards et al., 2000), and detoxification of phytoalexins produced during the hypersensitive response (Li et al., 1997). If not reduced, these peroxides will be converted to cytotoxic derivatives that can damage plant cells.
Based on sequence similarity, plant GSTs have been divided into classes phi and tau, which are found exclusively in plants, and classes theta and zeta, which are found in all five kingdoms (Edwards et al., 2000). Nicotiana species are good subjects for examining GSTs because nine GSTs have already been studied in Nicotiana tabacum (van der Zaal et al., 1987, 1991; Takahashi et al., 1989; Takahashi and Nagata, 1992a, b; Ezaki et al., 1995), and a GST gene has also been studied in N. plumbaginifolia (Dominov et al., 1992). Although N. tabacum GSTs are known to be involved in responses to cold, salt stress, and aluminum toxicity (Roxas et al., 1997; Ezaki et al., 2001), little is known about their role during pathogen infection.
Using heterologous primers based on N. tabacum GST sequences, four GSTs were amplified and cloned from N. benthamiana. The expression of these four GST genes was determined in response to infection by the causal agent of tobacco anthracnose, Colletotrichum destructivum (Shen et al., 2001a), as well as C. orbiculare, which can also infect Nicotiana spp. (Shen et al., 2001b). Both of these fungi produce intracellular hemibiotrophic infections in N. benthamiana (Shen et al., 2001a, b). To determine if the four GST genes are involved in the host response to fungal infection, they were silenced by virus-induced gene silencing (VIGS) using a potato virus X (PVX) gene-silencing vector (Ruiz et al., 1998). The susceptibility of the plants to infection by C. destructivum and C. orbiculare following silencing was then determined.
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Biological materials and inoculations
Nicotiana benthamiana plants were grown at 22 °C in Pro-mix (Premier Horticulture Inc., Red Hill, PA) until the 8-leaf stage, with a photoperiod of 8/16 h dark/light at 120 µmol m–2 s–1. Colletotrichum orbiculare isolate A20767P1 and C. destructivum isolate N150P3 (Chen et al., 2003a
) were cultured on potato dextrose agar (Difco Laboratories, Detroit, MI) or SYAS (Mandanhar et al., 1986) at 20 °C under continuous fluorescent lighting, and conidia were harvested after 7–12 d. After the plants had been placed in a plastic-lined container for 3 h, they were sprayed with a 2x106 conidia ml–1 suspension and incubated in the plastic-lined container. After 72–96 h, leaf samples were collected and immediately frozen in liquid nitrogen and stored at –80 °C for RNA extraction.
Primer design and cloning of GST and other genes from N. benthamiana
To design conserved primers, GST nucleotide and protein sequences from the classes phi, tau, and zeta were obtained from GenBank (Table 1). The protein sequences were aligned using Clustal X (Thompson et al., 1997). The mammalian theta GST from Rattus norvegensis (Gst1-rat) was used as an outgroup. Dendrograms were generated using both distance and parsimony methods. The aligned sequences were subjected to bootstrapping using the program SEQBOOT in the PHYLIP package (Felsenstein, 1989). The 100 bootstrap replicates were then examined using the PHYLIP programs PROTPARS and PROTDIST. The distance matrices from PROTDIST were then analysed with the PHYLIP program NEIGHBOR using the Neighbor–Joining algorithm. The data sets were analysed with PHYLIP CONSENSE to obtain bootstrap values that represent the consistency of tree branching patterns, and dendrograms were created using the DRAWGRAM program.
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Based on the groups of sequences observed in the protein alignment, nucleotide sequences were selected for further analysis and primer design. Primers Gst1S (5'-GATGGCAGAAGTGAAGTTG-3') and Gst1A (5'-CTCCTAGCCAAAATSCCA-3') were designed based on N. tabacum GSTs in cluster tau 1 (Fig. 1). Primers Gst2S (5'-YTRSARATGAAYCCWRTY-3') and Gst2A (5'-SAGSWARRGACTTWGMRAC-3') were designed based on N. tabacum GSTs in cluster tau 2 (Fig. 1). Primers Gst3S (5'-CTGGKGAWCACAAGAAGS-3') and Gst3A (5'-GCCAARATATCAGCACACC-3') were designed based on N. tabacum GSTs in cluster phi (Fig. 1). Degenerate bases are coded as follows: Y=C or T/U, M=A or C, K=G or T/U, W=A or T, S=C or G, R=A or G. As there were no known N. tabacum GSTs in the cluster zeta, no primers were designed for this group.
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PCR amplifications were performed in 15 µl reactions with 1 µl cDNA from N. benthamiana, 0.04 U µl–1 Tsg polymerase (Biobasic, Toronto, ON), 1x Tsg polymerase buffer, 2 mM dNTPs, 2.5 mM Mg2+, and 0.5 µM primers. RNA was extracted according to Chen et al. (2000)
and reverse transcribed into cDNA using Moloney murine leukaemia virus reverse transcriptase (Invitrogen, Burlington, ON) according to the manufacturer's instructions. Amplification conditions were 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, and one cycle for 10 min at 72 °C. All RNA samples used for reverse transcription were tested for the presence of genomic DNA by using them directly as the PCR template, prior to cDNA synthesis, under the same PCR conditions. PCR of the cDNA was performed in a GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, CT).
The amplification products were purified as described in Sambrook et al. (1989) and cloned into the TA vector, pGEMT-easy (Promega, Madison, WI) to obtain the plasmids, pGST1, pGST2-16, pGST2-17, and pGST3. Based on the sequence of the inserts in the plasmids, the genes were designated NbGSTU1, NbGSTU2, NbGSTU3, and NbGSTF1, respectively. The GenBank accession numbers of the sequences are listed in Table 1. The predicted protein sequences of all four genes were aligned with the 26 GST sequences mentioned earlier using Clustal X, and dendrograms were constructed using the PHYLIP programs.
Primers TobefiS (5'-CTCCAAGGCTAGGTATGATG-3') and TobefA (5'-CTTCGTGGTTGCATCTCAAC-3') were designed from conserved regions of the translation elongation factor 1
(EF-1) genes of N. tabacum and N. paniculata. Primers Pr1aS (5'-TGGSATTTRTTCTCTTTTCAC-3') and Pr1aA (5'-CCTGGAGGATCATAGTTGC-3') were designed from conserved regions of the N. tabacum and N. glutinosa acidic PR (pathogenesis-related) 1a genes. Primers Pr2S (5'-CATCACAGGGTTCGTTTAGGA-3') and Pr2A (5'-GGGTTCTTGTTGTTCTCATCA-3') were designed from conserved regions of the N. tabacum, Lycopersicon esculentum, and Solanum tuberosum basic PR2 genes. These genes were amplified and cloned as previously described. The identity of these genes was confirmed by sequencing, and they were designated NbEF-1, NbPR1a, and NbPR2, respectively.
Relative RT-PCR analysis
For relative RT-PCR (reverse transcriptase PCR), the gene of interest was co-amplified with primers TobefiS and TobefA as described by Dean et al. (2002) in order to include a constitutively expressed NbEF-1 gene as an internal control in each reaction. Primers Gst1S (5'-GATGGCAGAAGTGAAGTTG-3') and Gst1A (5'-CTCCTAGCCAAAATSCCA-3') were used to amplify NbGSTU1, and primer Gst216iA2 (5'-TAGGCATAAAACCAGCTGTAGT-3') or primer Gst217iA2 (5'-CATAAGAATAAAACCAACTAGTAA-3') was paired with primer GST216/7iS (5'-TGAGTACATTGAWGAAGTTTGG-3') to amplify NbGSTU2 and NbGSTU3, respectively. Primer Gst3iS (5'-GGCTTCAAGATTAACCTGGGA-3') was paired with primer Gst3A (5'-GCCAARATATCAGCACACC-3') for relative RT-PCR of NbGSTF1. The identity of the RT-PCR products was confirmed by direct sequencing.
Quantification of the relative RT-PCR products was performed as described by Dean et al. (2002). PCR reactions were performed in 15 µl reactions with 1 µl cDNA from N. benthamiana, 0.04 U µl–1 Tth polymerase (Interscience, Markham, ON), 1x Tth polymerase buffer, 2 mM dNTPs, 5.0 mM MgCl2, 0.5 µM EF-1 primers and 1.0 µM GST gene-specific primers. Amplification conditions were as previously described. RNA samples were tested for the presence of genomic DNA as described above. PCR of the cDNA was performed in a GeneAmp PCR System 2400. The relative RT-PCR was repeated three times for each GST gene using different RNA samples from different fungal inoculations for each replication. The data were subjected to analysis of variance, and Fisher's Protected LSD at P=0.05 was used to separate the means.
Silencing of GST genes and effect on susceptibility
To amplify internal fragments from pGST1, pGST2-16, pGST2-17, and pGST3, primer Gst1iA (5'-CTCTTGCTCCTCTCCTTT-3') was paired with Gst1S, primer Gst216iA (5'-CCAATCAGAGCAATATCCAC-3') was paired with Gst216/7iS, primer Gst217iA (5'-CCCTTTACTAGTTGGTTTTATT-3') was paired with Gst216/7iS, and primer Gst3iA (5'-AAGCACTCACACGACGGC-3') was paired with Gst3S, respectively. These fragments were purified and cloned into pGEMT-easy vectors. They were then digested with NotI (MBI Fermentas, Burlington, ON), and cloned into the PVX-based VIGS vector, pGR106, which is a derivative of pGR107 (Jones et al., 1999). The pGR106-GST clones were transformed into E. coli strain DH5 and then into Agrobacterium tumefaciens strain GV3101 via electroporation. Transformed A. tumefaciens was grown at 28 °C on LB agar containing 50 mg l–1 kanamycin and 5 mg l–1 tetracycline.
At the 6-leaf stage (5–6 cm tall), N. benthamiana plants were inoculated using a toothpick at four sites per leaf along the main veins of the two largest leaves. The toothpick inoculum contained A. tumefaciens with either a pGR106-GST construct, a pGR106 vector without an insert (PVX-vector control) or water instead of A. tumefaciens (water control). After 2 weeks, plants were tested for gene silencing by relative RT-PCR and inoculated with conidial suspensions of C. orbiculare (5x105 conidia ml–1 or C. destructivum (1x105 conidia ml–1) as described previously. At 96 h post-inoculation (HPI) for C. orbiculare-inoculated and 72 HPI for C. destructivum-inoculated plants, three or four leaves were collected for lesion counts and to determine fungal biomass, or the tissue was immediately frozen in liquid nitrogen, stored at –80 °C and later used for RT-PCR analysis of silencing. The leaf area was measured using the Leaf Area Meter, Model 3100 (Li-Cor, Lincoln, NE). The biomass of GFP-marked strains of C. orbiculare and C. destructivum was quantified in the leaves according to Chen et al. (2003a). These procedures were done two or more times for C. destructivum and C. orbiculare, and two sets of at least five plants were assessed in each procedure. The data were subjected to analysis of variance, and Fisher's Protected LSD at P=0.05 was used to separate the means.
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Cloning of four GST genes from N. benthamiana
Based on alignments of N. tabacum GST genes obtained from GenBank, three pairs of degenerate primers were designed in conserved regions to amplify GST fragments from N. benthamiana cDNA taken at 72 HPI with C. destructivum. Single PCR products of the predicted sizes were cloned and sequenced, and both distance and parsimony analyses of the predicted protein sequences showed that they all belonged to the predicted groups of GST genes (Fig. 1). One cloned gene, designated NbGSTU1, was most similar to other members of the tau 1 group of GST sequences, and two other clones, designated NbGSTU2 and NbGSTU3, were most similar to the tau 2 group of GST sequences. A fourth clone, designated NbGSTF1, was most similar to the phi group of GST sequences. In both distance and parsimony analysis, bootstrap support for groups zeta and tau was over 99% (Fig. 1). Group phi showed greater variation among its members with bootstrap support at 68% and 72% in distance and parsimony analyses, respectively (Fig. 1).
Role of GST in the response to infection by C. destructivum
Relative RT-PCR was done for each of the four GST genes, and sequencing of the RT-PCR products confirmed the specificity of the amplifications for each GST gene using leaf samples infected with C. destructivum as well as C. orbiculare that is described later. Following infection by C. destructivum, the relative expression of NbGSTU1 increased 5-fold to a maximum at 96 HPI followed by a significant decrease of 30% (Fig. 2). NbGSTU3 expression increased at a similar rate to NbGSTU1 up to 96 HPI. However, unlike NbGSTU1, NbGSTU3 expression did not show a significant decrease at 120 HPI. By contrast, expression levels of NbGSTU2 and NbGSTF1 changed relatively little following infection (Fig. 2). NbPR1a expression increased from 24 to 96 HPI and then declined from 96 to 120 HPI (Fig. 3). NbPR2 expression increased progressively from 0 to 120 HPI when the expression level was five times higher than that prior to inoculation by C. destructivum (Fig. 3). The NbPR2 gene expression was induced faster and showed a greater increase by 120 HPI than any of the GST genes tested.
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At 0 HPI, prior to fungal inoculation, all the GST genes except NbGSTU1 in GST-silence plants showed significant reductions in expression compared with either the water or PVX-vector controls, with average reductions of 57% and 58%, respectively (Tables 2, 3). Although NbGSTU1expression was significantly less than the water control, it was not significantly less than the PVX vector control.
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For plants inoculated with C. destructivum, all the genes showed silencing at 72 HPI except NbGSTU1, which appeared not to have any silencing (Table 2). Among the three which showed silencing, NbGSTF1 had the highest level of silencing with an average 21% expression compared with water and PVX vector controls (Table 2). NbGSTU2 and NbGSTU3 had expression levels around 50% compared with the controls (Table 2). Cross silencing among the three tau GSTs was observed only for expression of NbGSTU3, which was at 0.38±0.04 in NbGSTU2-silenced plants following inoculation with C. destructivum. No cross silencing was observed among the tau GSTs when NbGSTF1was silenced.
When inoculated with C. destructivum, none of the GST-silenced plants had significantly different lesion numbers compared with the PVX-vector control, but the NbGSTU2, NbGSTU3, and NbGSTF1-silenced plants did have significantly more lesions than the water control (Table 4). GFP measurements revealed that all of the infected GST-silenced plants contained a significantly greater biomass of C. destructivum than the water control, but none had a significantly greater biomass of C. destructivum than the PVX-vector control.
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Role of GST in the response to infection by C. orbiculare
Following infection by C. orbiculare, the relative expression of NbGSTU1 progressively increased 10-fold from 0 to 96 HPI before declining slightly at 120 HPI (Fig. 4). The relative expression of NbGSTU3 also increased at a similar rate to that of NbGSTU1 throughout the infection, except that it continued to increase at 120 HPI (Fig. 4). Expression of NbGSTU2 remained relatively unchanged during the infection, except for a small increase at 96 HPI followed by a decrease at 120 HPI. Expression of NbGSTF1 increased slightly, but continually, throughout the infection cycle, becoming approximately 1.5-fold higher at 120 HPI than at 0 HPI (Fig. 4). By comparison, the expression levels of NbPR1a did not increase until between 24–48 HPI, after which the expression remained relatively unchanged (Fig. 5). The increase in NbPR1a expression due to C. orbiculare infection was less than that of NbGSTU1 and NbGSTU3 (Figs 4, 5). The relative expression of NbPR2 doubled from 0 to 24 HPI but then remained relatively unchanged until it increased again at 96 to 120 HPI, (Fig. 5).
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The level of expression of all of the four GST genes in the silenced plants was significantly reduced compared with the water and PVX vector controls at 96 HPI following infection by C. orbiculare (Tables 2, 3). The levels of silencing in the C. orbiculare-inoculated plants were quite similar to those prior to inoculation with the greatest level of silencing at approximately 24% of the control levels for NbGSTF1, 36% for NbGSTU2, 43% for NbGSTU1, and 58% for NbGSTU3 (Tables 2, 3). Some cross silencing among NbGSTU1, NbGSTU2, and NbGSTU3 was observed. Expression of NbGSTU3 was at 0.49±0.08 in NbGSTU1-silenced plants, expression of NbGSTU3 was at 0.59±0.04 in NbGSTU2-silenced plants, and expression of NbGSTU2 was at 0.87±0.16 in NbGSTU3-silenced plants following inoculation with C. orbiculare. However, no cross silencing was observed for any of the three tau GSTs when NbGSTF1was silenced.
When inoculated with C. orbiculare, NbGSTU1-silenced plants had a significantly greater number of lesions compared with both the water and PVX-vector controls (Table 4). However, plants silenced for NbGSTU2, NbGSTU3, and NbGSTF1 did not show any altered susceptibility to C. orbiculare. As an indicator of the amount of fungal biomass in the C. orbiculare-inoculated plants, the amount of GFP expressed by C. orbiculare was measured and was also found to be significantly higher in NbGSTU1-silenced plants compared with the controls and the other GST-silenced plants (Table 4). None of the other inoculated GST-silenced plants had a greater biomass of C. orbiculare than the controls.
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Discussion |
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Based on the response of plant GSTs to stress and chemical treatments, it is believed that GST expression is primarily regulated at the transcriptional level (Dixon et al., 2002
). Expression of both NbGSTU1 and NbGSTU3 showed a major increase following infection by C. destructivum and C. orbiculare. The changes in NbGSTU1 and NbGSTU3 expression due to infection were similar to each other, except that NbGSTU1 expression declined late in both interactions whereas NbGSTU3 expression did not. The inducibility of NbGSTU1 and NbGSTU3 after infection contrasted with the lack of induction of NbGSTU2 and NbGSTF1 implying that the first two genes are more likely than the latter two to be involved in the plant response to infection by C. orbiculare and C. destructivum.
By comparison, expression of both acidic NbPR1a and basic NbPR2 from N. benthamiana increased following infection, with NbPR2 showing a greater increase. The expression of acidic PR1a genes is dependent on the accumulation of salicylic acid, which probably acts as a signalling molecule (Malamy et al., 1990), whereas basic PR2 genes are regulated by ethylene, a gaseous signalling molecule that is produced during interactions of various types of pathogens and their plant hosts (Kombrink and Somssich, 1997). Ethylene production in N. tabacum was induced by C. destructivum infection beginning at 24 HPI, peaking at 48 HPI, and then followed by a second peak at 120 HPI (Chen et al., 2003b). The rapid production of ethylene may explain the rapid induction of NbPR2, NbGSTU1, and NbGSTU3 expression. Ethylene treatment induced the expression of a GST gene, AtGSTF2, in Arabidopsis thaliana (Smith et al., 2003). Although none of the four GSTs followed precisely the pattern of expression of acidic NbPR1a or basic NbPR2, the patterns of NbGSTU1 and NbGSTU3 expression were more similar to that of NbPR2, indicating a possible role for ethylene in the induction of those two GST genes.
Following silencing treatment of the four N. benthamiana GST genes by VIGS, an examination of gene expression in the silenced plants showed that the four GST genes were always silenced compared with the water and PVX vector controls in the C. orbiculare-infected plants, but NbGSTU1 was not silenced in the C. destructivum-infected plants. This may reflect the higher level of infection by C. destructivum, which may have disrupted the plant cells more than C. orbiculare, thus affecting the metabolic machinery needed for silencing. A higher level of infection could also explain why higher levels of NbPR1, NbPR2, NbGSTU1, and NbGSTU3 expression were generally observed in the C. destructivum versus the C. orbiculare interaction.
NbGSTU1-silenced plants inoculated with C. orbiculare exhibited an increase in both fungal biomass and the number of lesions compared with the water and PVX vector control plants. Therefore, NbGSTU1 appeared to play a significant role in the plant response to the pathogen. However, genes with approximately 80% or higher similarity can also be silenced by the same VIGS construct (Baulcombe, 1999). Expression of NbGSTU3 was also reduced in the NbGSTU1-silenced plants following inoculation with C. orbiculare, and it is possible that other genes closely related to NbGSTU1 may also have been silenced, contributing to the altered disease reaction due to the effects of the NbGSTU1–VIGS construct. However, silencing of the other three GST genes, which mostly achieved similar or greater degrees of reduced target gene expression without causing cross silencing of NbGSTU1, did not show a significant effect on disease susceptibility compared with both the water and PVX vector controls. This demonstrates that not all of the N. benthamiana GST genes play a role in disease susceptibility. The most surprising was the lack of an effect of silencing of NbGSTU3 by VIGS on the susceptibility of the plants to C. destructivum or C. orbiculare since this gene showed a similar amount of induction as NbGSTU1 following infection by either fungus. This demonstrates that although induced gene expression may indicate involvement, it still is necessary that gene expression be altered, such as by VIGS during an infection process, to demonstrate the significance of a gene in the plant response to fungal infection.
The lack of a change in disease severity compared with the PVX vector control when NbGSTU1-silenced plants were inoculated with C. destructivum was surprising considering that C. destructivum and C. orbiculare both follow an intracellular hemibiotrophic mode of infection. One possible reason for this difference is that there was no significant reduction in NbGSTU1 transcript levels in NbGSTU1-silenced plants inoculated with C. destructivum, whereas silencing was observed in the C. orbiculare-inoculated plants. These two fungi appear to differ mainly in their manner of biotrophic growth. C. orbiculare infects and forms large primary hyphae growing through multiple epidermal cells, while C. destructivum forms a multi-lobed infection vesicle, which remains in one epidermal cell (Shen et al., 2001a, b). The infection strategy of C. orbiculare may result in a greater amount of host cell stress. Another difference is the greater virulence of C. destructivum to N. benthamiana, which may overwhelm the plant's defence and stress responses, and therefore NbGSTU1 may not be able to play an important role in the host response.
Other fungal–plant interactions have shown altered GST expression due to pathogen attack, and a variety of roles have been proposed for the GST genes in the host response. In potato, Prp1-1, a tau GST, was induced 2 HPI with Phytophthora infestans with maximum expression between 48 and 56 HPI (Hahn and Strittmatter, 1994). They speculated that Prp1-1 was induced during the disease as a result of auxin produced by P. infestans, which competitively binds PRP1-1, thereby inhibiting GST function and causing an increased need for GST. In wheat, a phi GST, Gsta1, was induced dramatically by 2 HPI with Erysiphe graminis f. sp. tritici, and the expression level remained high for at least 2 d in both compatible and incompatible interactions (Mauch and Dudler, 1993). The proposed function of GSTA1 involved the detoxification of organic peroxides to prevent continuing cell death caused by free radicals produced during the hypersensitive response in the incompatible interaction. In the compatible interaction, GSTA1 was proposed to detoxify active oxygen species produced as the plant was damaged by the pathogen. After inoculation of A. thaliana with a compatible or incompatible strain of Peronospora parasitica, higher expression of phi, tau, and zeta GST genes was observed, and these may have roles in restricting cellular damage by functioning in antioxidative reactions (Wagner et al., 2002). Treatment of poppy cell suspension cultures with a fungal elicitor extracted from Botrytis spp resulted in the induction of a class phi GST 1 h after exposure to the elicitor, and the GST was believed to be involved in the translocation or metabolism of phenylpropanoids both as part of the normal developmental physiology and the defence response (Yu and Facchini, 2000).
This report is the first to show that a plant GST gene plays a role in susceptibility to fungal infection. NbGSTU1, possibly in conjunction with closely related GST genes, could affect disease development in N. benthamiana following infection by C. orbiculare in several possible ways. For example, toxins have been reported from several Colletotrichum species and have been isolated from mulberry leaves infected by C. dematium (Bailey et al., 1992; Yoshida et al., 2000). GSTs are active in the process of binding of xenobiotics to produce less toxic metabolites, and chickpea GSTs may be involved in detoxifying two toxins produced by the chickpea blight fungus, Ascochyta rabei (Hamid and Strange, 2000). Therefore, NbGSTU1 might act by conjugating and detoxifying toxins produced by C. orbiculare. NbGSTU1 could also possibly reduce infection by C. orbiculare by maintaining auxin homeostasis. Auxin production by plant pathogenic fungi may be involved in pathogenesis, and it has been shown that higher auxin production by C. gloeosporioides f. sp. aeschynomene increases virulence (Cohen et al., 2002). A potato GST was competitively bound by auxin produced by P. infestans (Hahn and Strittmatter, 1994), and maintenance of auxin homeostasis has been proposed to be the role for the GST genes, ParB and Nt107, in N. tabacum (Droog et al., 1995; Takahashi and Nagata, 1992a). Many plant GSTs also have glutathione peroxidase activity that detoxifies cytotoxic alkenals and lipid hydroperoxides (Mauch and Dudler, 1993). Gullner and Komives (2001) concluded that although GSTs appear to have a variety of functions in plant metabolism, the most likely role for GSTs in pathogen-infected plants was to suppress necrosis by detoxifying lipid hydroperoxides produced by peroxidation of membranes. This may also apply to the role of GST in limiting C. orbiculare infection in N. benthamiana.
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Acknowledgements |
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This work was supported by the Natural Sciences and Engineering Research Council of Canada.
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Footnotes |
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Abbreviations: GST, glutathione S-transferase; HPI, hours post-inoculation; PVX, potato virus X; PR, pathogenesis-related; RT-PCR, reverse transcriptase PCR; EF-1
, translation elongation factor 1; VIGS, virus-induced gene silencing. |
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