Enhanced resistance to Rhizoctonia solani by combined expression of chitinase and Ribosome Inactivating Protein in transgenic potatoes ( Solanum tuberosum L . )

Potato (Solanum tuberosum L.) is susceptible to many fungal pathogens including Rhizoctonia solani. In the present study, the potato cultivar Desirée was transformed via Agrobacterium tumefaciens strain GV3101 containing the binary plasmid pGJ132 harboring both the chitinase (chiA) and rip30 genes. The potato leaf disc was used as an explant for transformation. PCR, Southern blot and Western blot were used for characterization of the transgenic plants. In this study it was shown that not all the plants developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis confirmed that transgenic plants integrated 2-3 copies of chiA and rip30 genes respectively into their genome. The expression of the CHIA and RIP30 proteins was confirmed in the leaf extracts of the transgenic clones by Western blot analysis. Transgenic potato plants expressing rip30 and chiA genes showed enhanced resistance to R. solani in a greenhouse assay. Additional key words: fungal resistance; genes; proteins; southern blot; western blot.


Introduction
Potato (Solanum tuberosum L.), one of the most important food crops in the world, is susceptible to diseases caused by several fungi, bacteria and other pathogens, leading to considerable losses in yield and quality of products (Walter et al., 2001;Khan et al., 2008).Rhizoctonia solani is the causal organism of Rhizoctonia disease complex in potato, resulting in two different symptomologies of the disease, namely stems canker and black scurf.These are recognized as necrotic lesions on underground plant parts, and Resistance to Rhizoctonia solani in transgenic potatoes Fusarium and Pythium (Osusky et al., 2005).Also, lysozyme accumulation in transgenic potato plants considerably inhibited growth of several bacterial species, including Erwinia carotovora and Pseudomonas syringae (Kato et al., 1998).Potato plants expressing the AP24, thaumatin-like pathogenesis-related protein, transgene, showed increased resistance to P. infestans (Liu et al., 1994) and tomato plants transformed with tobacco AP24 and bean chitinase transgenes showed improved resistance to F. oxysporum (Ouyang et al., 2005).
In the present study, simultaneous integration and expression of the barley rip30 and the Serratia marcescens chitinase (chiA) genes was used, and the transgenic potatoes produced were evaluated in greenhouse conditions for their resistance to R. solani.

Plant material
Virus free potato tubers of the tetraploid cultivar 'Desirée' was used for transformation.Etiolated sprouts from these tubers were surface disinfected and single-node segments were excised and placed on MS medium (Murashige & Skoog, 1962) supplemented with 100 g L -1 inositol, 30 g L -1 sucrose, 0.7% (w/v) agar, the pH was adjusted to 5.6.Stems developing from these nodes were propagated in vitro by subculturing the top shoots or stem segments including axillary buds every 3-4 weeks.The shoots were grown at 23 °C and a photoperiod of 16 h under 3000 lux light intensity.
Plants are known to have defence systems that involve the production of pathogenesis-related (PR) proteins, such as antimicrobial enzymes; chitinases, glucanases and ribosome-inactivating proteins (RIPs) (Ward et al., 1991).Chitinase catalyzes the hydrolysis of β-1,4 linkages of the N-acetyl-D-glucosamine polymer, called chitin.Chitin is a major component of the cell walls of many fungi.In fact, plant chitinases have exhibited antibiotic activity against various fungal pathogen in vitro tests by causing lysis of hyphal tips as well as the inhibition of spore germination and germ tube elongation (Chye et al., 2005).
RIPs are plant enzymes that have 28 S rRNA N-glycosidase activity which, depending on their specificity, can inactivate foreign ribosomes, thereby shutting down protein synthesis.The most common cytosolic type I RIP from the endosperm of cereal grains do not act on plant ribosomes but can affect foreign ribosomes, such as those of fungi (Stirpe et al., 1992;Hartley et al., 1996).
Several transgenic plants harbouring PR genes have already been produced, aiming at producing fungal disease-resistant varieties in a variety of crops (Jach et al., 1995;Tabei et al., 1997;Bieri et al., 2000;Datta et al., 2001;Jayaraj & Punja, 2007;Xiao et al., 2007;Almasia et al., 2008;Esfahani et al., 2010), including the transgenic tobacco that was engineered with the RIP encoding DNA sequence of barley and exhibits resistance to R. solani (Logeman et al., 1992).Many of the PR proteins may act synergistically in vivo and also enhanced inhibition of fungal growth when tested in combination in vitro (Jach et al., 1995;Chye et al., 2005).Transgenic plants expressing more than one PR protein genes were also developed in potato, rice and tobacco (Zhu et al., 1994;Zhu et al., 2007;Esfahani et al., 2010).Such transgenic plants showed better resistance than those having a single gene.
Transgenesis has proved its value as a method to enhance disease resistance in potato and related species.For example, potato plants transformed with the dermaseptin analog MsrA2 exhibit broad-range resistance to phytopathogenic fungi, including Cercospora, A. tumefaciens, GV3101 strain, was grown in Luria-Bertani (LB) medium at 28 °C.For leaf infection, bacteria cells were grown overnight at 28 °C with constant shaking (200 rpm) in YEB medium (Hooykaas et al., 1979) supplemented with 100 mg L -1 rifampycin, 50 mg L -1 of kanamycin, and 100 mg L -1 streptomycin.When OD 600nm reached 0.8-1, the culture was diluted 1:10 in MS medium.The bacterial cells were collected by centrifugation and resuspended in MS medium supplemented with 50 mg L -1 sucrose, pH 5.6.

Plant transformation
The leaf disc transformation method was used.Five mm leaf discs were cut from in vitro plants.A total of 40 explants were used.The explants were infected with 30 mL of the A. tumefaciens and placed at 25 °C in dark conditions for 1 hour.Infected explants were cocultivated for 2 days, in the dark, on a MS basal medium without antibiotics to facilitate transformation and supplemented with 100 g L -1 of inositol, 30 g L -1 sucrose, 0.7% (w/v) agar.The pH was adjusted to 5.6.Subsequently, the leaf discs were washed with MS basal medium containing 500 mg L -1 cefotaxime to eliminate bacteria.After drying on sterile filter paper, they were transferred to Petri dishes containing 25 mL of the MS basal medium supplemented with 2 mg L -1 zeatin, 20 µg L -1 gibberellic acid, 20 µg L -1 α-naphthalene acetic acid (α-NAA), 500 mg L -1 cefotaxime and 100 mg L -1 kanamycin.They were maintained on this medium for 5 weeks at 23°C and 16 hour photoperiod for shoot regeneration and elongation.The resulting shoots, about 5 mm long, were regenerated during 4-5 weeks after transformation and were then transferred individually to MS medium containing kanamycin (100 mg L -1 ) and cefotaxime (500 mg L -1 ) for rooting.Transgenic plants were further transferred to greenhouse conditions.
In this experiment several parameters were recorded: the percentage of explants with induced callus proliferation, the percentage of regenerated calli, the number of shoots per callus and the percentage of transgenic plants.

Molecular characterization of transgenic plants
To detect rip30 and chiA genes by PCR, genomic DNA was extracted from leaf material according to Edwards et al. (1991).DNA fragment, containing the rip30 or chiA genes, of 500 and 700 bp respectively was amplified by PCR using specific primers.The complete sequence of these primers is as follows: rip30, 5'CAACCCGGCGCACTTCTC3'; reverse rip30, 5'GGCCTTCATCTCATTGCCG3'; forward chiA, 5'CTGAAAGAGATTGAAGGCAGCTTC3'; and reverse chiA, 5'CATGGCGGTGCCGACGAC3'.These primers were designed by the software Primer3 (Rozen & Skalestsy, 1999).PCR amplification reactions consisted of an initial denaturation at 94 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 1 min, hybridization at 55°C for 1 min, extension at 72 °C for 2 min and a post-extension at 72 °C for 10 min.Taq DNA polymerase was used in all PCR reactions.The resulting PCR products were separated by electrophoresis agarose gel 1% (w/v).
Southern blot analyses were employed to detect the integration of rip30 and chiA in transgenic plants.A total of 10 µg of DNA genomic was extracted from young leaves and digested with Eco RI and Hind III.The probe of 1936 bp corresponded to the insert from pGJ132, digested with Stu I and Sgf I, separated on a 0.9% agarose gel, transferred to a nylon membrane (Hybond N + , Amersham Pharmacia Biotech) and hy-  spRIP Resistance to Rhizoctonia solani in transgenic potatoes bridized with the DIG-labeled probe of the rip30 and chiA genes.Hybridization, washing and detection were performed using DIG Easy Hyb (hybridization solution) and DIG Wash and Block Buffer set following the supplier's instructions (Boehringer Mannheim).
Western blot analysis was used to evaluate the expression of the integrated rip30 and chiA genes in genomic DNA of transgenic plants.Total proteins were extracted from 300 mg of young leaves of potato plant according to Laemmli (1970).The homogenized samples were boiled for 3 min, extracts were centrifuged at 13000 rpm for 2 min, and then 40 µL of soluble proteins were loaded onto 12% SDS-polyacrylamide gel (SDS-PAGE) according to Laemmli (1970) and electroblotted onto a polyvinylidine difluoride (PVDF) membrane (Amersham).Immunodetection was performed using polyclonal anti-serum raised in rabbit against the RIP30 and CHIA proteins and goat-antirabbit IgG (Amersham) conjugated to horseradish peroxidise (HRP) as secondary antibody, at 1: 1000 dilution.

Resistance evaluation of the transgenic plants in greenhouse
Five individual transformants were selected.These clones with confirmed insertion of rip30 and chiA genes and untransformed plants were micropropagated in vitro.After 4-5 weeks, five plants for each clone and for each control were transplanted into individual pots and placed in the greenhouse for further screening for resistance to the fungus.The R. solani strain Ci96, provided by Neiker Institute (Vitoria, Spain) was grown on potato dextrose agar (PDA) medium [20% (w/v) potato, 1.5% (w/v) glucose and 1.5% (w/v) agar] at 25°C with light for 5 days, and subcultured as needed.The inoculation was done as described by Chand & Logan (1983).After 4 months, the crop was harvested and the tuber symptoms were assessed in five tubers per plant.The symptoms evaluated were the appearance of black or brown sclerotia on the tuber surface, using the ADAS (Adams disease assessment scale) (Anonymous, 1976) ranging from 0 to 25% according to the infected surfaces covered by sclerotia.The inoculations were repeated three times for a more effective infection.
For the statistical analysis, an ANOVA was performed to evaluate the significance of the differences in resistance among the clones.Means were separated using the LSD test (p < 0.05).

Transformation and characterization of transgenic plants
Transformation with the pGJ132 showed that a total of 70% (28 out of 40) of explants induced callus proliferation.The number of explants with callus regeneration was 17.The total number of shoots was 218.Not all the plants growing on kanamycin-containing medium were positive in the identification of the corresponding gene present on the T-DNA using the PCR technique.Only 65% (141 out of 218) of the plants rooted in kanamycin medium displayed a 500 bp amplification fragment for rip30 gene and a 700 bp for chiA gene.The specific primers did not amplify the corresponding fragments in the untransformed samples (Fig. 2).
Southern blot analysis was performed in 141 positive PCR clones and showed that the rip30 and chiA genes were integrated into the genome of the tested transformed plants.In these transformants, two and three hybridizing bands were detected for chiA and rip30 genes respectively.Fig. 3 presents a Southern blot for one selected positive PCR clone (C1) hybridized with the probes for chiA and rip30.Two bands are obtained for the chiA gene (lane C1a), indicating that there could be two copies inserted for this transgenic clone.A digestion with Hind III, on the other hand, gives three bands of the rip30 gene (Fig. 3, lane C1b).The size of the bands differed, indicating that this transformant have two and three copies of the chiA and rip30 genes respectively integrated at random sites in the genome.The pattern of integration was variable.Therefore, 84% (118 out of 141) of the transgenic clones showed two and three copies of the chiA and rip30 genes integrated, respectively, whereas 23 transgenic clones showed two copies of both genes.No transgene insertion was detected in non-transformed controls.
The Western blot analysis demonstrated that the rip30 and chiA genes are transcribed and translated into proteins in the tested transgenic clones.The 30 kDa peptide of the RIP30 protein was detected as a single band in the total protein extracts from the transgenic plants with varying levels of expression in different clones (Fig. 4a).Also, the 52 kDa peptide of the CHIA protein was expressed at different detectable levels in the tested transgenic plants (Fig. 4b).The control nontransformed plant did not express the RIP30 and CHIA proteins.

Resistance evaluation to virulent isolate of R. solani
The results of greenhouse evaluation of the transgenic clones expressing the rip30+chiA genes are presented in Table 1.The clones expressing chiA and rip30 genes showed an enhanced resistance to the pathogen and no symptoms were found in their tubers.The non-transgenic control presented damages of 10% of the surface tuber covered by sclerotia, compared with the high level of protection observed in the transgenic clones.(Anonymous, 1976), assigning values between 0 and 25% according to the infected surface covered by sclerotia.The mean of a total of 25 tubers for each transgenic clone and for the control is shown.Values followed by different letters are significantly different according to the Tukey's b test (α ≤ 0.05).

Discussion
In this study, the potato cv.'Desirée' was transformed with an association of chiA and rip30 genes isolated from Serratia marcescens and Hordeum vulgare L., respectively.This was attempted because application of multiple resistance genes to plant genetic manipulations could widen the spectrum and increase the level of resistance in many crops (McDowell & Woffenden, 2003;Zhu et al., 2007).
Potato leaf discs were used as explants for transformation with A. tumefaciens, which has been commonly used for this purpose in potato (Beaujean et al., 1998;Ducreux et al., 2005;Banerjee et al., 2006).In previous studies the potato micro-tuber discs, were found to be effective explants for transformation (Esfahani et al., 2010).In the laboratory, however, when micro-tuber discs were used there was a lack of response of some varieties and even a decrease in the regeneration rate (M'hamdi et al., 2003).In this study, using leaf disc explants, the regeneration rate was high and the transgenic shoots obtained were morphologically normal.
Differences between the number of plants grown on selective medium with kanamycin and those positive in the PCR assay were noted.Something similar was reported by Kumar et al. (1995), who observed many escapees despite the selective medium.This could be explained by the fact that calli are formed by a set of juxtaposed cells and, thus, some of these cells although not transformed, can escape the selective antibiotic and regenerate shoots (M'hamdi et al., 2003).Moreover, some plants may integrate only the gene of resistance to the antibiotic but not the transgene, as the transfer of T-DNA occurs from the right to the left border and the actual transgene is not integrated (Wang et al., 1984).Indeed, in the binary vectors that we used, the nptII gene conferring resistance to kanamycin is nearest to the right border than the chiA and rip30 genes.The transgenic clones (positive in PCR) were confirmed by Southern blot analysis.This showed that the PCR product was the result of T-DNA integration into the genome of transgenic plants.We observed integration of two and three copies of chiA and rip30 genes, respectively, into the genome of 118 clones.
The greenhouse assay was carried out to evaluate resistance to the pathogen in transgenic plants and showed enhanced resistance in transgenic clones expressing the rip30 and chiA genes.The combined expression of these two genes was effective in preventing disease development.The deployment of genetic engineering approaches that involve the expression of two or more antifungal gene products in a specific crop should provide more effective and broad-spectrum disease control (Jach et al., 1995;Melchers & Stuiver, 2000;Anand et al., 2003;Almasia et al., 2008;Esfahani et al., 2010).
A previous study by Esfahani et al. (2010) showed that a combination of chitinase (chit42) and glucanase (bgn13.1)genes isolated from different Trichoderma species increased the antifungal activity against R. solani AG-3 in transgenic potatoes.Chye et al. (2005) reported that transgenic potato plants co-expressing Brassica juncea chitinase (B j CHI 1 ) and Hevea brasiliensis β-1,3-glucanase (H b GLU) inhibited fungal growth better than extracts from transgenic potato expressing either B j CHI 1 or H b GLU, suggesting a synergistic effect.Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.
Our results revealed that the rip30 and chiA genes were successfully integrated into the genome of transgenic potato plants, and expressed as revealed by the production of the RIP30 and CHIA proteins.The expression of these two proteins together demonstrated enhanced resistance to R. solani in the transgenic potato.

Figure 2 .
Figure 2. PCR-amplified regions used to confirm the integration of the rip30 and chiA genes in transgenic plants of cv.Desirée.Amplification of rip30 (500 bp) and chiA (700 bp) genes in the transformed potato with the pGJ132.Lane M, size marker.Lane C -, non-transformed control.Lanes C1-C5, independent transgenic potato clones.Arrows indicate the amplified fragments of rip30 (500 bp) and chiA (700 bp) genes in transgenic clones.

Figure 3 .
Figure 3.Southern blot analysis of the cv.Desirée.Genomic DNA (10 µg) from transgenic and non-transgenic control potato plants was digested with EcoRI and HindIII enzymes, and hybridized with a DIG-labeled probe for chiA and rip30 genes.Lane M, DIG-labeled molecular weight marker III.Lane C -, non-transformed control.Lane C1a and C1b, independent transgenic clone transformed with the pGJ132 and genomic DNA digested with Eco RI and Hind III, respectively.Lane C + , the plasmid pGJ132 DNA template as positive control.The arrows indicate the transgenes copy number in the transgenic clone (bands with different locations).

Figure 4 .
Figure 4.Western blot analysis of RIP30 and CHIA in cv.'Desirée'.Protein extracts (10 µg) of young leaves from five independent transgenic clones (C1-C5) and non-transformed control (C -), resolved in a 15% polyacrylamide gel and subjected to immunoblot analysis using antibodies for rabbit polyclonal antiserum.Arrows indicate the 30 kDa band of the RIP30 protein (a) and the 52 kDa band of the CHIA protein (b).

C5
Resistance to Rhizoctonia solani in transgenic potatoes

Table 1 .
Symptoms caused by R. solani in the transgenic clones of potato 'Desirée' expressing rip30 and chiA genes, tested in greenhouse 1 Symptoms were assessed by ADAS scale