- Split View
-
Views
-
Cite
Cite
Hai-Yan Li, Guo-Dong Yang, Huai-Rui Shu, Yu-Tao Yang, Bao-Xing Ye, Ikuo Nishida, Cheng-Chao Zheng, Colonization by the Arbuscular Mycorrhizal Fungus Glomus versiforme Induces a Defense Response Against the Root-knot Nematode Meloidogyne incognita in the Grapevine (Vitis amurensis Rupr.), Which Includes Transcriptional Activation of the Class III Chitinase Gene VCH3, Plant and Cell Physiology, Volume 47, Issue 1, January 2006, Pages 154–163, https://doi.org/10.1093/pcp/pci231
- Share Icon Share
Abstract
Inoculation of the grapevine (Vitis amurensis Rupr.) with the arbuscular mycorrhizal (AM) fungus Glomus versiforme significantly increased resistance against the root-knot nematode (RKN) Meloidogyne incognita. Studies using relative quantitative reverse transcription–PCR (RQRT–PCR) analysis of grapevine root inoculation with the AM fungus revealed an up-regulation of VCH3 transcripts. This increase was greater than that observed following infection with RKN. However, inoculation of the mycorrhizal grapevine roots with RKN was able to enhance VCH3 transcript expression further. Moreover, the increase in VCH3 transcripts appeared to result in a higher level of resistance against subsequent RKN infection. Constitutive expression of VCH3 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter also conferred resistance against RKN, but had no significant effect on the growth of the AM fungus. We analyzed β-glucuronidase (GUS) activity directed by a 1,216 bp VCH3 promoter in transgenic tobacco following inoculation with both the AM fungus and RKN. GUS activity was negligible in the root tissues before inoculation, and was more effectively induced after inoculation with the AM fungus than with RKN. Moreover, GUS staining in the mycorrhizal transgenic tobacco roots was enhanced by subsequent RKN infection, and was found ubiquitously throughout the whole root tissue. Together, these results suggest that AM fungus induced a defense response against RKN in the mycorrhizal grapevine roots, which appeared to involve transcriptional control of VCH3 expression throughout the whole root tissue.
Introduction
Arbuscular mycorrhizal (AM) fungi are ubiquitous soil inhabitants, forming mycorrhiza with the roots of most agricultural, horticultural and hardwood crop species. It is well known that the establishment of mycorrhiza eventually confers resistance against pathogens, including plant parasitic nematodes, in the host plant (Pozo et al. 2002). These parasitic nematodes have been acknowledged as one of the most serious pathogens, causing a significant loss in crop production worldwide. Because both AM fungi and nematodes commonly occur together in the roots and rhizosphere of the same plant, considerable attention has been paid to the potential role of mycorrhiza as biocontrol agents (Hussey and Roncadori 1982, Jothi et al. 1998, Li et al. 2002a, Diedhiou et al. 2003). To date, a variety of AM fungi have been shown to limit nematode development, hence reducing disease symptoms and improving the growth of nematode-infected plants (Smith et al. 1986, Jothi et al. 1998, Li et al. 2002b, Diedhiou et al. 2003). However, the molecular mechanisms of how the symbiotic association results in the bioprotection against nematodes remain poorly understood.
AM fungi induce a variety of physiological and molecular biological changes in the host plant. Of the latter, qualitative and quantitative changes in the expression of different isogenes for chitinases have been widely studied, as these enzymes may have a role in defense and stress responses of the host plant (Azcón-Aguilar and Barea 1996, Dumas-Gaudot et al. 1996, Pozo et al. 1996, Pozo et al. 1998, Pozo et al. 2002), in addition to their roles in the establishment and regulation of symbiosis (Lambais and Mehdy 1995, Lambais and Mehdy 1996, Dumas-Gaudot et al. 1996, Liu et al. 2003, Salzer et al. 2004). Plant chitinases [poly (1,4-(N-acetyl-β-d-glucosaminide)) glycanohydrolase; EC 3.2.1.14] catalyze the hydrolysis of chitin, a linear homopolymer of β-1,4-linked N-acetylglucosamine residues. They are grouped into the classes I–VI, each being characterized by a common primary structure (Collinge et al. 1993, Meins et al. 1994) and consensus sequences characteristic of the isoprotein (Levorson and Chlan 1997). In general, chitinase activity is maintained at low levels in unstressed plant tissues. However, it is induced to high levels in response to a variety of stimuli, including ethylene (Chen and Bleeker 1995), salicylic acid (Margis-Pinheiro et al. 1994), virus infection (Ohme-Takagi et al. 1998) and pathogenic microorganisms (Kästern et al. 1998, Mohr et al. 1998). In the mycorrhiza established between leeks and the AM fungus Glomus mosseae, and also between alfalfa and Glomus intraradices, chitinase activity increased during the early stages of the fungus colonization, but decreased during later stages (Spanu et al. 1989, Volpin et al. 1994), suggesting that the chitinase activity may be involved in an early phase of symbiosis. However, several reports indicate that different chitinase isogenes respond differentially to the same stimulus. In tobacco, for example, the expression of two acidic chitinases was up-regulated, while that of a basic chitinase was down-regulated after the colonization by the AM fungus G. intraradices (Dumas-Gaudot et al. 1992, David et al. 1998). Salzer et al. (2000) reported that the class III chitinase genes Mchitinase III-2, Mchitinase III-3 and Mchitinase III-4 from Medicago truncatula were all strongly induced by the AM fungus G. intraradices. They also found that Mchitinase III-2 and Mchitinase III-3 were not induced by pathogens or rhizobia, whereas Mchitinase III-4 was induced by the pathogenic fungus Ascochyta pisi (Salzer et al. 2000). Although the exact roles of chitinase isoforms in mycorrhizal plants are still unclear, it is hypothesized that certain chitinase isoforms may be bioprotective against plant–pathogen interactions.
Meloidogyne incognita, the most frequently found root-knot nematode (RKN) species in vineyards, suppresses the yield of susceptible grape cultivars by up to 80% and that of resistant crops by approximately 40%. We reported previously that establishment of mycorrhiza with the AM fungus Glomus versiforme in the roots of the grapevine Vitis amurensis confers resistance against the RKN M. incognita in the host plant (Li et al. 2002b). Our preliminary study revealed that the inoculation with the AM fungus induced chitinase activity in the mycorrhizal grapevine roots, suggesting an effect on chitinase gene expression. However, grapevine has at least five different chitinase genes, classified as class I chitinase gene, VCHIT1b (GenBank accession number, Z54234), class III chitinase gene, VCH3 (GenBank accession number, Z68123) and three class IV chitinase genes, VvChi4A, VvChi4B and VvChi4D (GenBank accession numbers U97521, U97522 and AF532966). It is therefore necessary to identify which chitinase gene(s) is induced following inoculation with AM fungus, and to confirm whether this increase in gene acivity mediates subsequent resistance of the mycorrhizal grapevine to RKN.
In this study, we show that transcripts of the class III chitinase gene, VCH3, accumulate in mycorrhizal grapevine roots colonized by the AM fungus G. versiforme, and that this increase in gene activity coincides with the development of resistance against the RKN M. incognita. The constitutive expression of VCH3 cDNA in transgenic tobacco plants under the control of the cauliflower mosaic virus (CaMV) 35S promoter enhances the resistance against the RKN M. incognita, but has no significant effect on the level of mycorrhizal colonization by AM fungus. These results suggest the involvement of VCH3 in a bioprotective mechanism against the RKN. To understand better the expression of VCH3 in response to the AM fungus and the RKN, we have isolated a 1,216 bp VCH3 promoter and subsequently generated transgenic tobaccos expressing a VCH3 promoter–GUS (β-glucuronidase) chimera gene. GUS activity is strongly induced in the mycorrhizal transgenic tobacco roots colonized by the AM fungus G. versiforme, and the level is enhanced further following a challenge with the RKN M. incognita. On the basis of these results, we hypothesize that the AM fungus G. versiforme induces a defense response, which results in the growth restriction of the RKN M. incognita in the mycorrhizal grapevine roots, and which involves transcriptional activation of the class III chitinase gene, VCH3.
Results
Inoculation of the grapevine with the AM fungus G. versiforme significantly increased resistance against the RKN M. incognita.
We first investigated the mutual effects of AM fungus and RKN when colonizing the same grapevine roots. The results showed that the increase in colonization of RKN-infected mycorrhizal grapevine (AMR) plants with the AM fungus G. versiforme was not significantly different from that in mycorrhizal grapevine (AM) plants for at least 13 d following RKN inoculation (Fig. 1A). This suggested that there was no significant effect of the RKN on the growth of the AM fungus within the same mycorrhizal grapevine roots. In contrast, the level of RKN infection in the roots of AMR plants was considerably reduced 5 d after RKN inoculation, but began to increase 5 d after RKN infection in the RKN-infected non-mycorrhizal grapevine (R) plants (Fig. 1B). These results suggest that the presence of AM fungus inhibited the growth of the RKN in the mycorrhizal grapevine roots. Furthermore, in AMR plants, the percentage of RKN infection was negatively correlated with the percentage of AM fungus colonization within the same roots (Fig. 1C). Taken together, these results indicate that colonization by the AM fungus G. versiforme in the grapevine confers resistance against the RKN M. incognita.
Accumulation of the VCH3 transcripts in the mycorrhizal grapevine roots is correlated with resistance against RKN infection
Our previous work demonstrated that chitinase activity was induced in mycorrhizal grapevine roots colonized by the AM fungus G. versiforme (Hai-Yan Li and Cheng-Chao Zheng, unpublished data). It is known that grapevine expresses at least five chitinase genes. We attempted to determine which isogene(s) is induced following inoculation with AM fungus by RNA gel blot analysis, using gene-specific probes. However, we failed to detect any expression. We therefore performed relative quantitative reverse transcription–PCR (RQRT–PCR) analyses using gene-specific primers for each of the five chitinase genes. The result demonstrated that only a 1.0 kb band, representing the VCH3 transcripts, was amplified from samples derived from the roots of AM plants (data not shown), whereas no expression of VCH3 could be detected in uninfected grapevine roots. Similarly, no 1.0 kb band was seen when using genomic DNA from G. versiforme or M. incognita as template with the same sets of VCH3-specific primers, thus confirming the specificity.
The amplification profiles of the VCH3-specific transcript were subsequently examined by RQRT–PCR, using root RNA samples from UC (uninoculated control), AM, R and AMR plants (Fig. 2A). Amplification of the 1.0 kb band was observed 1 d after RKN infection in the AM or the R plants. This band was amplified more strongly from AM than R grapevine root RNA samples, but maximally from both samples around 5 d after RKN infection. Expression of this transcript was highest in the root RNA samples of the AMR plants. Noticeably, the maximal amplification of the VCH3-specific transcript occurred in the AMR plants 5 d after RKN infection. This peak in transcript expression preceded the rise in infection level seen in non-mycorrhizal grapevine roots inoculated with RKN (Fig. 1B), and correlated with no significant increase in the percentage of RKN infection in the AMR plants. These results suggest a possible link between the AM fungus-induced accumulation of VCH3 transcripts and the induction of resistance against RKN in grapevine roots.
Constitutive expression of VCH3 in transgenic tobacco enhances resistance against RKN
To evaluate the potential role of the class III chitinase gene VCH3 in the defense response against the RKN M. incognita, the tobacco Nicotiana tabacum cv. NC89, a good host for the AM fungus G. versiforme and a cultivar sensitive to the RKN M. incognita, was used for the Agrobacterium tumefaciens-mediated leaf disc transformation. A number of transgenic tobacco plants constitutively expressing the VCH3 cDNA under the control of the 35S CaMV promoter were generated (CE plants). The T1 transgenic tobacco plants harboring the chimeric gene 35S:VCH3 were confirmed by Southern blot (data not shown). Nine of the T2 transgenic tobacco lines constitutively expressing VCH3 were subsequently chosen for inoculation with the RKN and AM fungus (Fig. 3A). The result showed that there was a considerable reduction in the percentage of infection of RKN in roots from the CE plants when compared with control plants (CK) transformed with the vector pBI121 (Fig. 3B). However, constitutive expression of VCH3 in the transgenic tobacco plants had no significant effect on the mycorrhizal colonization level of AM fungus when compared with the CK plants (Fig. 3C), in accordance with the previous findings of Gianinazzi-Pearson et al. (1994) and Vierheilig et al. (1995). These results demonstrate that constitutive expression of VCH3 in the transgenic tobaccos results in enhanced resistance against RKN but does not affect growth of the AM fungus, suggesting that this gene may play a critical role in AM fungus-induced defense responses against RKN in the mycorrhizal grapevine.
VCH3 promoter responds more to the AM fungus than to the RKN
To examine the mechanism of induction of the VCH3 gene in grapevine by G. versiforme or the RKN M. incognita, a 1,216 bp VCH3 promoter fragment was isolated by adaptor-PCR, with reference to the transcriptional start site determined by primer extension analysis (Fig. 4). Sequence analysis revealed that the VCH3 promoter sequence contains CAAT and TATA motifs located at nucleotides –122 and –29 relative to the transcriptional start site, respectively (Fig. 5). Each of these motifs is characteristic of eukaryotic gene promoters. The 1,216 bp VCH3 promoter fragment was then inserted into the plant expression plasmid pBI121 to construct the VCH3 promoter–GUS chimera. Using an A. tumefaciens-mediated leaf disc transformation method, this construct was transferred to the tobacco N. tabacum cv. NC89 mentioned above.
GUS activity was assayed in the roots of the transgenic tobacco plants inoculated with either AM fungus or RKN alone, and also in those of the mycorrhizal transgenic tobacco plants after infection with RKN. At each time point, GUS activity was approximately 2-fold higher in the roots colonized by the AM fungus G. versiforme than in those infected by the RKN M. incognita (Fig. 6A), suggesting that AM fungus may have a stronger inductive effect on VCH3 than RKN. In the roots of mycorrhizal tobacco plants after RKN infection, GUS activity increased by approximately 2-fold at 1 d, and 3-fold at 5 d when compared with activity at 0 d. Interestingly, the enhancement of GUS activity appeared to be positively correlated with the degree of colonization by the AM fungus, but negatively correlated with the percentage of infection of RKN (Fig. 6B, C). These results show that the colonization of transgenic tobacco roots by AM fungus induces a high level of GUS expression, and that this is enhanced further by subsequent RKN infection. This demonstrates that the 1,216 bp VHC3 promoter is able to respond to a signal(s) from the AM fungus G. versiforme or the RKN M. incognita, and further suggests a more significant effect of the AM fungus on the induction of VCH3.
Histochemical analysis of GUS activity in transgenic tobacco roots expressing a VCH3 promoter–GUS chimera gene construct
The induction of GUS activity by AM fungus and RKN infection was examined further by performing histochemical analysis on transgenic tobacco plants 5 d after infection by the RKN. GUS staining in the transgenic plants inoculated with AM fungus was seen more strongly in epidermal tissues and both outer and inner cortexes than in vascular tissues (Fig. 7A). In contrast, GUS staining was stronger in vascular tissues than in epidermal tissues and outer and inner cortexes in the transgenic tobacco roots infected by RKN (Fig. 7B). This is probably due to the different pathways used by these two microorganisms to invade plant roots. The AM fungus extensively colonizes the outer and inner cortexes of the plant roots. However, the sedentary endoparasitic nematode, RKN, is only able to traverse the root cortexes to reach their feeding sites, the plant vascular tissues. Therefore, considering a possible correlation between the degree of colonization by the AM fungus and VCH3 transcript expression (Fig. 1, 2), it is perhaps not surprising that there is higher GUS activity in AM fungus-colonized transgenic tobacco tissues than in those infected by RKN. In addition, it was noted that GUS staining was enhanced in the mycorrhizal transgenic tobacco roots following RKN infection, and that this expression was detected ubiquitously in the root tissues, including the vascular tissues, epidermal tissues and outer and inner cortexes (Fig. 7C). These results suggest that such ubiquitous expression of VCH3 may confer protection against attack by RKN in the mycorrhizal grapevine roots.
Discussion
The application of protective microorganisms provides a durable and environmentally friendly alternative to the use of agrochemicals in the control of plant disease. AM fungi are an interesting group of microorganisms that form a symbiotic association with a wide range of plant species, and effectively reduce root diseases caused by a number of soilborne pathogens, particularly nematodes and fungi (Linderman 1994, Bodker et al. 2002). However, the cellular, molecular and physiological mechanisms of root pathogen control in mycorrhizal plants are still not well understood. Thus, to optimize the effectiveness of AM fungi as potential bioprotective agents, it is essential to characterize their mechanisms of action further.
Plant chitinases are considered to be one of the important components of plant defense systems. Their transient activation, induction and differential expression have been reported in different mycorrhizal plants (Spanu et al. 1989, Lambais and Mehdy 1993, Vierheilig et al. 1994, Volpin et al. 1994, Vierheilig et al. 1995, Pozo et al. 1996, Pozo et al. 1998, Salzer et al. 2000, Pozo et al. 2002, Liu et al. 2003, Salzer et al. 2004). As one of the early defense responses of plants to invading fungi, expression of chitinase was considered to be important in the regulation of symbiosis (Salzer et al. 2000, Liu et al. 2003, Salzer et al. 2004). However, the mycorrhizal fungi appear to be insensitive to plant chitinase (Arlorio et al. 1992). Furthermore, chitinases appear neither to have direct contact with the intracellular structures of AM fungi nor to bind to their external hyphae (Spanu et al. 1989). In addition, constitutive expression of chitinase genes in transgenic tobacco plants does not affect the establishment and functioning of mycorrhiza, while such plants show an increased resistance to root fungal pathogens (Gianinazzi-Pearson et al. 1994, Vierheilig et al. 1995). It therefore seems likely that some chitinases induced by AM fungi have a bioprotective role against soilborne pathogens. However, to date, there is no experimental evidence demonstrating that the chitinase genes induced by AM fungi are involved in the resistance to any pathogen including the nematode.
In this study, we have shown that transcripts of the class III chitinase gene VCH3 accumulate in mycorrhizal grapevine roots colonized with the AM fungus G. versiforme, and that this coincides with the development of resistance against the RKN M. incognita. We have also demonstrated that constitutive expression of VCH3 cDNA in transgenic tobacco plants under the control of the CaMV 35S promoter enhances resistance against the RKN M. incognita, but has no significant effect on mycorrhizal colonization with the AM fungus, strongly suggesting that VCH3 is involved in a bioprotective mechanism against the RKN.
Plant chitinases induced by AM fungi may be assigned to one of two groups: the first is mycorhiza specific but not induced by any pathogen, and the second is induced by both AM fungi and pathogens, such as the class III chitinase gene Mchitinase III-4 from M. truncatula (Salzer et al. 2000). The former may be involved in the regulation of symbiosis in mycorrhizal plants, and hence considered as a hallmark for the establishment of arbuscular mycorrhiza (Salzer et al. 2000), whereas the latter may be involved in defense mechanisms induced by AM fungi (Pozo et al. 1996, Pozo et al. 1998, Pozo et al. 2002). Here, we have demonstrated that both the AM fungus G. versiforme and the RKN M. incognita induce expression of the grapevine class III chitinase gene VCH3, which is 62.3% homologous to Mchitinase III-4. In addition, we have shown that the AM fungus is more effective than the RKN in this role. At each time point, the level of the VCH3 transcripts appears to be most abundant in mycorrhizal grapevine roots subjected to RKN infection, which supports the theory that symbioses with AM fungi could result in a more rapid response to subsequent pathogen attack (Gianinazzi 1991, Benhamou et al. 1994, Gianinazzi-Pearson et al. 1996). Furthermore, GUS activity directed by the VCH3 promoter in transgenic plants inoculated with the AM fungus is stronger than in those infected with the RKN. Interestingly, our present work has also shown that GUS activity is strongly induced in the roots of mycorrhizal transgenic tobacco expressing a 1,216 bp VCH3 promoter–GUS chimera gene, and that this is enhanced further after challenge with the RKN M. incognita. Moreover, the increase in GUS activity seems to be positively correlated with the degree of colonization by the AM fungus, and occurs widely throughout the whole root tissues. Taken together, this would suggest that the AM fungus G. versiforme induces defense responses in the mycorrhizal grapevine roots resulting in resistance to attack by the RKN M. incognita, and that the defense response probably includes transcriptional regulation of the class III chitinase gene VCH3 throughout all root tissues.
Pozo et al. (2002) previously demonstrated that the AM fungus G. mosseae is effective in reducing disease symptoms produced by Phytophthoraparasitica, through local or systemic induction of chitinase activity. Our present results support these findings and also suggest a specific role for local and/or systemic chitinase gene VCH3 expression in G. versiforme-induced protection against RKN infection. Further experiments to study the differential expression of VCH3 in response to the AM fungus and/or RKN using a split root system (Pozo et al. 2002) are required to confirm this hypothesis.
Materials and Methods
Plant materials, AM fungi and nematode inoculations
Seeds of grapevine (V. amurensis Rupr.) cv. ‘Shuangyou’, a variety susceptible to the RKN M. incognita race 3, were surface sterilized with 0.1% HgCl2 for 40 min and germinated on sterile wet filter paper at 27°C for 5 d. Each seedling was aseptically planted in a pot (500 ml) filled with a mixture of sands that had been steam sterilized at 121°C for 2 h and inoculated with 8,000 inoculum potential units (IPU) of the AM fungus G. versiforme (Liu and Luo 1994). The control grapevine seedlings were grown in pots containing a mixture of steam-sterilized sands and the root segments of tobacco plants which were free of AM fungi. Seedlings in pots were managed in a greenhouse at day/night temperature of 25/15°C with a 16 h photoperiod at a photon flux density of 300 µmol m–2 s–1 under a relative humidity of 60%. Pots were watered daily and fertilized with Hoagland’s solution every 3 d. Twenty-five days after germination, seedlings were inoculated with 3,000 second-stage juveniles (J2) of the RKN M. incognita, which was kindly provided by the Bioprotection Institute of the Chinese Academy of Agricultural Science. Root samples were harvested at 0, 1, 3, 5, 7, 9, 11 and 13 d after inoculation with the RKN, and immediately frozen in liquid nitrogen and stored at –80°C prior to analysis. Experimental treatments were arranged randomly with 10 replicates.
Quantification of the mycorrhizal colonization and RKN infection
Mycorrhizal colonization of the AM fungus was estimated as the percentage of colonized cortex in the root system according to the procedure described by Biermann and Linderman (1981). Briefly, mycorrhizal plant roots were made transparent by incubation with 5% KOH at 90°C for 45 min and stained with trypan blue. The number of typical appressoria, intracellular hyphae, arbuscules and root vesicles was counted under a microscope (BX 50, Olympus, Tokyo, Japan) equipped with the PM-30 Automatic Photomicrographic System (Olympus, Tokyo, Japan). The level of RKN infection was evaluated as the percentage of root penetration by RKN, as previously described (Liu 1995). The whole root system was stained with acid fuchsin in lactophenol for 3 min and the number of nematodes in the root systems of each plant was counted under a microscope (BX 50, Olympus) after the roots were excised with a sharp scalpel.
RQRT–PCR analysis
RQRT–PCR analyses were performed according to the method of Burleigh (2001). A 1 µg aliquot of total RNA was reverse-transcribed using murine reverse transcriptase (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The primer oligo(dT)12–18 and the primer NS21 for 18S rDNA, 5′-AATATACGCTATTGGAGCTGG-3′, were used to prime the reverse transcription reactions. Then, the reverse transcription products were amplified by PCR using the primers NS21 and NS1, 5′-GTAGTCATATGCTTGTCT-3′, under the following thermal cycle conditions: 94°C for 5 min followed by 4–22 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 30 s. The PCR products were subjected to Southern blot analysis using a 32P-labeled probe specific to 18S rDNA, and the radioactive bands were quantified with a phosphoimage analyzer (BAS-2500, FujiXerox, Tokyo, Japan). A cycle number that gave rise to a linear phase of increases in the intensity of the rDNA-derived band was determined for each set of RNAs. An appropriate volume to standardize each treatment was also determined empirically and used in all the subsequent PCRs. For quantifying the level of the VCH3 transcripts, PCR was conducted using the primers A, 5′-ATTCGGCACGAGTGCAAGAAT-3′, and B, 5′-TCCATACATTCATCGAGGATG-3′. The RQRT–PCR products from all root samples obtained by this method were blotted onto nitrocellulose membranes. VCH3 cDNA was labeled with [α-32P]dCTP using a Random Prime labeling kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Pre-hybridization was performed at 65°C overnight in a solution containing 6× SSC (1× SSC is 0.15 M NaCl, 0.015M sodium citrate), 5× Denhart’s solution, 0.5% SDS and 0.1 mg ml–1 single-stranded cDNA, and hybridizations were performed at 65°C for 18 h in a solution containing 6× SSC, 0.5% SDS and 0.1 mg ml–1 single-stranded DNA, according to standard protocols.
Isolation of the chitinase gene VCH3 promoter
The VCH3 promoter fragment was isolated by adaptor-PCR using a TaKaRa LA PCR in vitro cloning kit (TAKARA SHUZO CO., LTD, Japan), according to the manufacturer’s protocol. Genomic DNA was completely digested with PstI to produce a cohesive end. About 5 µg of digested DNA was ligated to the dephosphorylated PstI adaptor. Half of the ligation product was amplified by the first round of PCR using the 5′ adaptor primer C1, 5′-GTACATATTGTCGTTAGAACGCGTAATACGACTCA-3′, and the 3′-specific primer S1, 5′-GGTTAGGGTCCCTTCATTGCCGTT-3′, which is derived from the VCH3 DNA sequence. The second round of PCR was performed using the first PCR product as template, the second 5′ adaptor primer C2, 5′-CGTTAGAACGCGTAATACGACTCACTATAGGGAGA-3′, and the second 3′-specific primer S2, 5′-ATTGCCGAACTTGTTGAGGAAGGC-3′, which is based on the upstream sequence of the S1 primer in the VCH3 gene. Of the PCR products, the largest fragment was purified, cloned into the pGEM-T easy vector and sequenced.
Primer extension analysis
Primer extension analysis was performed using the primer P, 5′-GTAGATTGCGATGCCACCAGCATAAGAGGTCTA-3′, that is complementary to a region downstream of the ATG start codon in the VCH3 gene. The primer was labeled with 10 µCi of [γ-32P]ATP (3,000 µCi/mmol) using T4 polynucleotide kinase, and the labeled primer was annealed with 50 µg of total RNA at 42°C for 18 h. The primer extension reaction was carried out at 42°C for 1 h, using 200 U of reverse transcriptase (Promega, Madison, WI, USA) according to the published procedure (Sambrook et al. 1989). The extension product was separated on a 6% polyacrylamide gel containing 7 M urea. For the 5′ upstream sequence ladders, the VCH3 gene was sequenced using the primer P, the VCH3 promoter fragment as a template, and a SequiTherm EXCEL™ II DNA sequencing kit (Epicentre, Madison, WI, USA).
Plasmid construction for the VCH3 cDNA constitutive expression and the VCH3 promoter–GUS gene expression
A 1,022 bp fragment of VCH3 cDNA was amplified by PCR. The resultant VCH3 cDNA fragment was digested with XbaI and SmaI and then inserted between the CaMV 35S promoter and the GUS coding sequence in the binary vector pBI121.
To generate the VCH3 promoter–GUS construct, the 1,187 bp VCH3 promoter fragment relative to the transcriptional start site was amplified by PCR. The resultant fragment was blunted using the Klenow fragment and then digested with BamHI. The plasmid pBI121 was cleaved with PstI, blunted using the Klenow fragment and then digested with BamHI. The blunt/BamHI VCH3 promoter fragment then replaced the 35S promoter and was inserted in blunt/BamHI pBI121.
Tobacco transformation
Leaf discs from N. tabacum cv. NC89 were transformed with A. tumefaciens LBA4404 harboring the binary vectors, and the transgenic plants were regenerated by standard methods (Horsch et al. 1985). For each construct, the presence of the target gene in transformed plants was verified by Southern blot analysis, about 80 and 180 independent transformants containing one copy of the chimeric genes 35S::VCH3 and VCH3 promoter::GUS were allowed to self-fertilize, and seeds were collected and germinated on MS agar medium containing 300 µg ml–1 kanamycin sulfate. Kanamycin-resistant T2 seedlings were used for the subsequent inoculation.
RNA gel blot analysis
RNA gel blot analysis was conducted according to the method described by Zheng et al. (1998). About 10 µg of total RNA was separated on a 0.8% formaldehyde–agarose gel and blotted onto a Nytran membrane (Schleicher and Schuell, Dassel, Germany). Blots were hybridized with the VCH3 cDNA probe labeled by the random priming method. Blots were washed three times for 20 min at 55, 60 and 65°C with 0.2× SSC and 0.1% SDS solution, and then autoradiographed at –80°C, using Kodak XAR-5 film and an intensifying screen (Cronex Lightning Plus, DuPont, Wilmington, DE, USA).
Transgenic tobacco inoculated with the AM fungus and the RKN
In the following experiments, each tobacco plantlet with 4–5 leaves was transplanted into a single plot for inoculation with AM fungus and RKN.
Transgenic tobacco seedlings constitutively expressing VCH3 cDNA (CE plants) or those transformed with the vector pBI121 (CK plants) were transplanted in pots (200 ml) filled with steam-sterilized (121°C, 2 h) sand. Each pot contained a single plantlet. Half of the CE and CK plants were inoculated with 8,000 IPU of the AM fungus G. versiforme. All of the plants were raised in a greenhouse at day/night temperature of 25/15°C with a 16 h photoperiod and at a relative humidity of 60%. At 25 d after transplanting, the remaining halves were inoculated with 1,500 J2 of the RKN. The root samples were harvested at 0, 1, 3, 5, 7, 9, 11 and 13 d after the RKN infection. All experiments were repeated three times, and nine plants were used for each treatment at each time point.
Transgenic tobacco plantlets carrying the VCH3 promoter–GUS construct were transplanted in pots (200 ml) filled with the steam-sterilized (121°C, 2 h) sand and inoculated with 8,000 IPU of the AM fungus G. versiforme. In uninoculated control (UC), the transgenic tobacco plantlets were transplanted into pots containing a mixture of steam-sterilized sands and non-mycorrhizal tobacco root segments. Plants were then raised in the greenhouse under the aforementioned conditions. At 25 d after transplanting, half of either the UC or AM plants were inoculated with 1,500 J2 of the RKN. Root samples were harvested at 0, 1, 3, 5, 7 and 9 d after RKN inoculation. All experiments were repeated three times, and 12 plants were used for each treatment at each time point.
GUS activity assays and histochemical analyses
Root samples were homogenized in 0.6 ml of chilled lysis buffer containing 0.1 M sodium phosphate (pH 7) and 1 mM EDTA to obtain the crude homogenates, and 10 µl aliquots of the homogenates were used for measuring GUS activity, using a flurorometric assay as described by Jefferson (1987). Results were expressed as the specific activity in the crude homogenates, i.e. pmol 4-methyl umbelliferone min–1 mg–1 soluble protein. Protein content was measured by the Bradford (1976) method using bovine serum albumin (BSA) as a standard.
Histochemical analysis of GUS activity was performed as follows (Stomp 1992): 1–2 cm long tobacco roots were fixed by immersing for 30 min in a fixing solution containing 0.1 M sodium phosphate (pH 7.0), 0.1% formaldehyde, 0.1% Triton X-100 and 0.1% 2-mercaptoethanol. Fixed samples were stained for GUS activity by immersing in a staining solution containing 0.1 M sodium phosphate (pH 7), 10 mM EDTA, 0.5 mM K ferrocyanide, 0.5 mM K ferricyanide, 1 mM X-glucuronide and 0.1% Triton X-100. Tissues were vacuum infiltrated in the staining solution in order to assure homogeneous infection of the substrate. After dehydration in 100% ethanol, the tissues were incubated in toluene for two 2 h passages at room temperature and then embedded in paraffin for sectioning (8–10 µm in thickness).
Acknowledgments
We would like to thank Dr. Runjin Liu for his generous gift of the AM fungus. This research was supported by grants from the National Basic Research Program (Grant No. 2006CB1001006), the ‘863’ project in China (Grant No. 2002AA224101) and the National Natural Science Foundation of China (Grant No. 30270145) to Z.C.C; and the National Natural Science Foundation of China (Grant No. 30400300), the Special Program for Research of Transgenic Plants (Grant No. 20040209–2) and the Science and Technology Development Project of Jilin province PRChina (Grant No.20030553-1, 20050117) to L.H.Y.
The upstream nucleotide sequence of the grapevine chitinase III gene which is reported in this paper has been submitted to the NCBI under the accession number AF441123.
Abbreviations
- AM
arbuscular mycorrhizal
- CaMV
cauliflower mosaic virus
- GUS
β-glucuronidase
- IPU
inoculum potential unit
- J2
second-stage juvenile
- RKN
root-knot nematode
- RQRT–PCR
relative quantitative reverse transcription–PCR
References
Arlorio, M., Ludwig, A., Boller, T., Mischiati, P. and Bonfante, P. (
Azcón-Aguilar, C. and Barea, J.M. (
Benhamou, N., Fortin, J.A., Hamel, C., St-Arnaud, M. and Shatilla, A. (
Biermann, B. and Linderman, R.J. (
Bodker, L., Kjoller, R., Kristensen, K. and Rosendahl, S. (
Bradford, M.H. (
Burleigh, S.H. (
Chen, Q.G. and Bleeker, A.B. (
Collinge, D.B., Kragh, K.M., Mikkelsen, J.D., Nielsen, K.K., Rasmussen, U. and Vad, K. (
David, R., Itzhaki, H., Ginzberg, I., Gafni, Y., Galili, G. and Kapulnik, Y. (
Diedhiou, P.M., Hallmann, J., Oerke, E.-C., and Dehne, H.-W. (
Dumas-Gaudot, E., Furlan, V., Grenier, J. and Asselin, A. (
Dumas-Gaudot, E., Slezack, S., Dassi, B. and Pozo M.J., Gianinazzi-Pearson, V. and Gianinazzi, S. (
Gianinazzi, S. (
Gianinazzi-Pearson, V., Dumas-Gaudot, E., Gollotte, A., Tahiri-Alaoui, A. and Gianinazzi, S. (
Gianinazzi-Pearson, V., Gollotte, A., Dumas-Gaudot, E., Franken, P. and Gianinazzi, S. (
Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. (
Hussey, R.S. and Roncadori, R.W. (
Jefferson, R.A. (
Jothi, G., Rajeshwari, S. and Sundarababu, R. (
Kästern, B., Tenhaken, R. and Kauss, H. (
Lambais, M.R. and Mehdy, M.C. (
Lambais, M.R. and Mehdy, M.C. (
Lambais, M.R. and Mehdy, M.C. (
Levorson, J. and Chlan, C.A. (
Li, H.Y., Liu, R.J. and Shu H.R. (
Li, H.Y., Liu, R.J. and Shu, H.R. (
Linderman, R.G. (
Liu, J.Y., Blaylock, L.A., Endre, G., Cho, J., Town, C.D., Vandenbosch, K.A. and Harrison, M.J. (
Liu, R.J. and Luo, X.S. (
Liu, W.Z. (
Margis-Pinheiro, M., Marivet, J. and Burkard, G. (
Meins, F., Jr, Fritig, B., Linthorst, H.J.M., Mikkelsen, J.D., Neuhaus, J.M. and Ryals, J. (
Mohr, U., Lange, J., Boller, T., Wiemken, A. and Vögeli-Lange, R. (
Ohme-Takagi, M. Meins, F., Jr and Shinshi, H. (
Pozo, M.J., Azcón-Aguilar, C., Dumas-Gaudot, E. and Barea, J.M. (
Pozo, M.J., Cordier, C., Dumas-Gaudot, E., Gianinazai, S., Barea, J.M. and Azcón-Aguilar, C. (
Pozo, M.J., Dumas-Gaudot, E., Slezack, S., Cordier, C., Asselin, A., Gianinazzi, S., Gianinazzi-Pearson, V., Azcón-Aguilar, C. and Barea, J.M. (
Salzer, P., Bonanomi, A., Beyer, K., Vogeli-Lange, R., Aeschbacher, R.A., Lange, J., Wiemken, A., Kim, D., Cook, D.R. and Boller, T. (
Salzer, P., Feddermann, N., Wiemken, A., Boller, T. and Staehelin, C. (
Sambrook, J., Fritsch, E.F. and Maniatis, T. (
Smith, G.S., Roncadori, R.W. and Hussey, R.S. (
Spanu, P., Boller, T., Ludwig, A., Wiemken, A., Faccio, A. and Bonfante-Fasolo, P. (
Stomp, A.M. (
Vierheilig, H., Alt, M., Lange, J., Gut-Rella, M., Wiemken, A. and Boller, T. (
Vierheilig, H., Alt, M., Mohr, U., Boller, T. and Wiemken, A. (
Volpin, H., Elkind, Y., Okon, Y. and Kapulnik, Y. (