Cotton (Gossypium hirsutum) is a universally important crop. Cotton plants often suffer from many severe diseases (Liu et al., 2000). One of the most serious diseases of this crop plant is cotton wilt, which attacks seedlings and is caused by Verticillium dahliae. Verticillium wilt, caused by Verticillium dahliae, is a widespread disease that occurs in most cotton-producing areas. V. dahliae is a soil-borne pathogen that infects plants through the roots. Symptoms of infected cotton plants include stunting and wilting by some strains of V. dahliae and defoliation by other strains.The estimated loss of produce due to V. dahliae is 15-70%. Several classes of genes have been used in a genetic engineering approach to develop resistance in cotton to fungal pathogens. One group of genes, referred to as defence response genes encode proteins such as: b-1,3-glucanases, thaumatin-like proteins (tlps), ribosome-inactivating protein (RIPs), and chitinases. The defence response genes function in a variety of ways to inhibit fungal infection. Expression of these genes in transgenic plants has been shown to enhance fungal resistance (Muehlbauer and Bushnell, 2003). Although there are some tolerant cotton lines in Iran (e.g. Sahel), but their level of tolerance is not sufficient. It is therefore recommended to use genetic engineering approach to further enhance the resistance using genes like Chitinase (chi). As Iranian cotton varieties have low regeneration potential, most of the desirable genes are initially introduced into cultivar Coker which responds well to tissue culture. The resulting transgenic plants are then back crossed with other varieties.
The role of chitinase in plant defense against fungal attack has been very well documented (Lawrence and Novak, 2006; Adams, 2004). This enzyme is a glycanohydrolase which limits fungal growth by degrading poly [b-1,4-N-acetyl-b-D-glucozamine], i.e., chitin, the major structural polysaccharide of the fungal cell wall of V. dahliae (Adams, 2004). Basic chitinases isolated from bean (Schlumbaum et al., 1986) and acidic ones isolated from cucumber (Zhang and Punja 1994) have shown fungicidal activity in vitro. Hence, chitinases hydrolyse the chitin in fungal hyphae and kill the fungus without causing damage to the plant cell. Several studies have demonstrated that enhanced chitinase levels in transgenic plants increase the resistance of plants to fungal pathogens (Nandakumar et al., 2005; Rajasekaran et al., 2005; Tohidfar et al., 2005; Lorito et al., 1998; Tabei et al., 1998; Asao et al., 1997; Broglie et al., 1991; Toyoda, 1991). Molecular characterization and evaluation of transgenic cotton lines for resistance to V. dahliae and expressing chitinase gene was the objective of this study.
MATERIALS AND METHODS
Plant materials: Seeds of first and second progenies (T1 and T2) of transgenic T0 plants (cultivar Coker) were transformed using plasmid pBI121-CHI (Tohidfar et al., 2005). Along with seeds from non-transgenic control plants were used in this study. Seeds were sterilized in HgCl2, were rinsed in sterile water and were placed on the germination medium (MS; Murushige and Skoog, 1962) medium with 3% sucrose and 0.8% agar (w/v)) as described by Tohidfar et al., (2005). Plantlets were potted in soil for growth in the greenhouse. After 4 weeks, all plants and nontransformed control plants were inoculated by dipping the roots of separate groups of plants in spore suspensions of V. dahliae (106 spores/ ml) for 20 min. After six weeks, symptoms were appeared and their changes in plant height chlorosis, leaf number and flowering date were noted every week (Mcffaden et al., 2001).
Polymerase chain reaction (PCR): Genomic DNA of cotton plant was extracted from young leaves following the protocol of Haiwen et al. (2001), using the PCR Kit (Cinagen, Tehran, Iran) with following pairs of primers:
nptII F 5´-GAACAAGATGGATTGCACGC-3´
nptII R 5´- GAAGAACTCGTCAAGAACGC-3´
chi F 5´-GAGTGGTGTGGATGCTGTTG-3´
chi R 5´- GCCATAACCGACTCCAAGCA-3´
The PCR profile included a 95°C for 5 min followed by 30 cycles for 30 s at 94°C, 1min at 60°C (chi) and 55°C (nptII) for annealing, 1min at 72°C for elongation and final extension at 72°C for 5 min. Expected PCR products size were about 785 bp and 870 bp for nptII and chi genes respectively.
Southern blot hybridization analysis: Southern blot analysis of plant genomic DNA was carried out using standard protocols (Sambrook and Russell 2001) to confirm the transgenic status of plants showing a positive reaction in PCR analysis. Briefly, 20 mg of cotton genomic DNA was digested with XbaI and EcoRI, electrophorezed in 0.8% agarose gel and transferred onto Hybond- N+ nylon membrane (Amersham, Buckinghamshire, UK). A 860 bp PCR product of the coding sequence of the chi gene was labeled with DIG DNA labeleing kit (Boehring Mannheim, Germany) and used as probe.
Western blot analysis: Extraction of proteins from fresh leaves of the PCR-positive plants and western blot analysis were performed according to Sambrook and Russell (2001). Ten mg of proteins extracted from leaves of PCR positive and control plants were subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). After electrophoresis, the proteins were electrophoretically transferred onto a nitrocellulose filter. The rabbit anti-chi-I anti-serum (1:2,000 v/v) (a gift from Dr. Els van Deventer, Zeneca Mogen International, Leiden, The Netherlands) and alkaline phosphatase-conjugated goat antirabbit IgG (Promega, Madison, WI, USA; 1:5,000, v/v) was used as the primary and secondary antibody, respectively. Total soluble protein was measured using Bradford method (Bradford, 1976). BCIP/NBT (5-Bromo-4-chloro-3-indolyl Phosphate (BCIP)/Nitroblue Tetrazolium (NBT)) was used for visualization.
In vitro effect of transgenic plants on the growth of V. dahliae: SS4 isolate of V. dahliae was obtained from infected cotton fields in Gorgan, Iran. This isolate was used to produce a mycelial plug. The inhibitory activity of extracts from transgenic cotton was assessed in vitro following the method of Tohidfar et al., (2005).
In vivo effect of bean chitinase on the growth of V. dahliae: All T1 and T2 transgenic plants survived acclimatization and grew to maturity. Fifty-five percent of the examined transgenic plants could restrict V. dahliae growth and symptoms. There were no discernible differences in the phenotypic appearance, height, leaf number and flowering date of transgenic plants as compared to those of non-transgenic plants. However chlorosis (symptom of infection) were significantly less in transgenic plants compared to that of nontransgenic ones. Some of the transgenic plants developed into dwarves and could not restrict V. dahliae growth and symptoms (Fig. 1).
PCR and Southern blot analysis: PCR analysis was carried out initially to confirm the transgenic nature of the progeny plants. Bands of 870 bp (chi) and 785 bp (nptII) in length were amplified from nuclear DNA for 77 T1 and T2 transgenic plants (Table 1). Seventy five plants showed both chi and nptII bands (Figs. 2 and 3). The c2 (chisqure test) test of T1 plants (line 11) indicated that the inheritance of both chi and nptII genes follows the Mendelian ratio for a single copy gene (3:1). No amplification was obtained from nontransgenic plants (Table 2).
Southern blot analysis showed that the hybridization of probe to the undigested was exclusively at high molecular weight, indicating the integration of the gene into the cotton genome. When the DNA was digested with EcoRI, most of the hybridization was corresponded to an expected fragment of ~900 bp including the whole coding sequence of the chi gene. This result showed that there is at least one copy of the integrated chi gene in the genome (Fig. 4). When the DNA digested with XbaI was hybridized to the probe, only one band of different size was detected in each of the different transgenic lines examined. Since there is only one XbaI site in the T-DNA, this result indicated the presence of only one single copy of the transgene in each of the tested events. No hybridization signal could be detected for the DNA extracted from untransformed plant.
Western blot analysis: Forty four transgenic plants were analyzed for chi expression using Western blot analysis. Extracts of thirty nine plants had one protein band of expected size (32 kDa) that reacted with a bean chitinase antibody (Fig. 5). The other 5 plants had no detectable chitinase. The level of chitinase expression varied among the transgenic plants. Untransformed control plants did not show any positive signal for the chitinase protein.
In vitro effect of bean chitinase on the growth of V dahliae: Leaf tissue extracts from T1 and T2 plants inhibited mycelial growth of V. dahliae, whilst the extracts from negative control did not have any inhibitory effect (Fig. 6). There was no significant difference between mycelial growth when different amounts of leaf extracts from transgenic plant were used at 50-100 µg range. Leaf tissue extracts from transgenic plants lysised the hyphal tip; hyphal morphology was distored, and hyphae became gradyally thinner and shorter, eventually turning dark and not sporulating (Fig .7).
In the last two decades efforts have been made on the transgenic expression of plant fungal chitinases in crop plants, and significant improvements in the resistance to fungal diseases have been recorded (Nandakumar et al., 2005; Rajasekaran et al., 2005; Pappinen et al., 2002; Lorito et al., 1998; Tabei et al., 1998; Asao et al., 1997; Broglie et al., 1991). Plant chitinases have been shown to inhibit fungal growth in vitro by degrading chitin polymers in fungal cell walls (Noel et al., 2005; Nielsen et al., 1994; Toyoda et al., 1991; Hüttermann and Cwielong 1982). However, the genes which have been used in these studies encode basic chitinases. Acidic chitinases have also been shown to have antifungal activity in vitro (Nishizawa et al., 1999; Raharjo et al., 1996; Zhang and Punja, 1994; Schlumbaum et al., 1986).
Our study also shows the antifungal effect of chitinase in our transgenic progenies. There are some reports on the production of transgenic plants with improved resistance to fungal diseases using a chitinase gene (Grisson et al., 1996; Lin et al., 1995; Jach et al., 1992; Broglie et al., 1991). Several reports have also stressed the advantages of using chitinases for plant protection because these fungicidal enzymes are part of the plant defense system and not harmful to plants as the substrate chitin is not found in plants (Lorito et al., 1998; Collinge et al., 1993). Copy number of the transgene (chi) is one of the most important factors implicated in the stability of gene expression in transgenic plants (Katleen, 2005). Our results showed the integration of a single copy of chi for line#11 and several other independent events (data not shown).
Significant differences in the level of expression of the chitinase gene among the transgenic T0 cotton plants have been reported earlier (Tohidfar et al., 2005). Our western blot analysis showed the differential expression of chitinase among the progenies. When the transgenic T1 and T2 cotton plants were challenged with V. dahliae, they developed less necrotic areas than nontransgenic plants, resulting in an overall improved resistance. A positive correlation was established between the level of chitinase expression and restriction of fungal growth and damage. It has also been suggested that glycosidic fragments released by degradation of chitin can serve as elicitors of additional plant defense responses (Kurosaki et al., 1987). Five of the progeny plants had no detectable chitinase. This could be resulted from a gradual differential gene silencing process (Jerzy et al., 2001).
We also tested the chitinase transgenic cotton against Verticillium dahliae, we observed a low level of resistance against this pathogen. This may be due to the fact that the chitinase gene alone may not provide effective protection agains the pathogen where chitin is either not a major constituent of the fungal cell wall or is not easily accessible to enzyme action. It may be possible to confer resistance to this pathogen by combining chitinase genes with other antifungal genes such as beta 1,3 glucanase. Regardless, our results with Verticillium dahliae provide the first convincing demonstration of the usefulness of a bean-derived endochitinase in conferring an effective resistance against a soil-born in cotton.