Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (chitinase and glucanase).

One way of enhancing and broadening resistance of plants to different biotic and abiotic stresses is to combine transgenes expressing several genes into a single line. This can be done using different strategies such as crossing, single vector with multiple genes, co-transformation, sequential transformation and IRES elements. In the present study conventional crossing method was used. Parental transgenic lines transformed via Agrobacterium tumefasciens-mediated gene transformation with pGreenII binary vector harbouring a bar gene as selectable marker in combination with the family 19 chitinase gene from Streptomyces olivaceoviridis for one line and 1,3-β-glucanase from barley (Hordeum vulgare) for the other line were used for crossing. Both chitinase and glucanase genes were cloned into pGreenII vector under the control of the constitutive double 35S-promoter from cauliflower mosaic virus. Progenies expressing the two genes were characterised at the molecular level using PCR, RT-PCR and Southern blot analysis, as well as segregation and stability studies of the respective expression levels. Leaf paint assay was used as functional test for herbicide resistant gene. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. The synergistic effect of crossed plants was tested using in vitro assay which shows higher inhibition of spore germination.


Introduction
Diseases are considered the most important cause of high yield losses with fungal diseases being the most important, yield losses can range between 50-75%. [1][2][3][4] Constitutive expression of individual PRs in transgenic plants can lead to reduced pathogen growth and symptom development, consistent with a role of PRs in the expression of acquired resistance. 5,6 Dana et al. 7 generated transgenic tobacco (Nicotiana tabacum) lines that overexpress the endochitinases CHIT33 and CHIT42 from the mycoparasitic fungus Trichoderma harzianum and evaluated their tolerance to biotic and abiotic stresses. Both CHIT33 and CHIT42, individually, conferred broad resistance against fungal and bacterial pathogens as well as tolerance to salinity and heavy metals. Transgenic plants overexpressing chitinases of several origins have been shown to exhibit enhanced levels of resistance to fungal infection and delayed disease symptoms when challenged with fungal pathogens. [8][9][10] Most frequently described antifungal proteins are probably chitinases and β-1,3-glucanases. The expression of glucanase and chitinase genes respectively in plants enhances their resistance One way of enhancing and broadening resistance of plants to different biotic and abiotic stresses is to combine transgenes expressing several genes into a single line. This can be done using different strategies such as crossing, single vector with multiple genes, co-transformation, sequential transformation and IRes elements. In the present study, conventional crossing method was used. parental transgenic lines transformed via Agrobacterium tumefasciens-mediated gene transformation with pGreenII binary vector harboring a bar gene as a selectable marker in combination with the family 19 chitinase gene from Streptomyces olivaceoviridis for one line and 1,3-β-glucanase from barley (Hordeum vulgare) for the other line were used for crossing. Both chitinase and glucanase genes were cloned into pGreenII vector under the control of the constitutive double 35s-promoter from cauliflower mosaic virus. progenies expressing the two genes were characterized at the molecular level using pCR, RT-pCR and southern blot analysis, as well as segregation and stability studies of the respective expression levels. Leaf paint assay was used as a functional test for herbicide resistance genes. stable inheritance of the antifungal genes in the transgenic plants was demonstrated. The synergistic effect of crossed plants was tested using in vitro assay which shows higher inhibition of spore germination.
Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (chitinase and glucanase) against fungal pathogens. [11][12][13] It has also been demonstrated that these hydrolases act synergistically in both in vitro and in vivo assays, resulting in a very strong antifungal activity. 14-16 A high level expression of these genes has been detected in transgenic rice and barley leading to a high resistance. 8,17 The co-expression of chitinase and glucanase genes in tobacco enhanced resistance against Cercospora nicotianae. 8,[17][18][19] In tomato, simultaneous expression of the basic tobacco chitinase PR-3d and glucanase PR-2e provided substantial protection against Fusarium oxysporum f.sp. lycopersici, whereas transgenic plants expressing either one of these genes were not protected. 20 Two or more transgenes can be sequentially introduced into a plant by conventional iterative procedures, e.g., a plant containing one transgene is crossed with individuals harboring other transgenes 21 or alternatively, is re-transformed with new genes, 22 co-transformation 23 and dicistronic vector with IRES elements. 24 The crossing techniques have been used at the research level to combine or reinforce existing transgenic traits. It has been demonstrated that crossing plants expressing different Bacillus thuringiensis (Bt) toxins provided an effective way of delaying the emergence of Bt-resistant pests. Cao et al. 25 illustrated this in genes in this study were chitinase and β-1,3-glucanase, two of pathogenesis-related proteins, produced in response to microbial infection. These enzymes hydrolyse chitin and glucan, respectively of fungal cell walls. 30 Successful introduction of the chitinase and glucanase genes into the pea genomic DNA was analysed using specific primers for the chitinase and glucanase in the F 0 and subsequent generations ( Fig. 1).
Copy numbers and integration patterns were investigated in the crossed transgenic F 1 to F 4 generations respectively using Southern blot analysis with different probes (chit and gluc). One or two copies were detected in the progenies which reflected the same copy numbers of the parental transgenic lines (Fig. 2).
RNA denaturing gel using formaldehyde was used to check the RNA integrity. Transcription of the glucanase and chitinase genes was confirmed using RT-PCR. Most of the crossed transgenic F 1 to F 4 generations clearly exhibited the transcription of the genes as shown in Figure 3.
Leaf paint assay was done to verify the expression of the bar gene. One week after application, clear effects of herbicide were observed (Fig. 4). The 600 mg/l of BASTA ® used in the present study is high in comparison to that used by other groups, for example 200 mg/l on pea 31 or 400 mg/l on faba bean (Vicia faba). 32 Recovery of herbicide resistant plants from sensitive parental plants through recombination in meiosis was observed in some lines. Table 1 shows a summary table of leaf paint results from F 1 to F 4 generations.
During the establishment of homozygous lines most of the transgenic lines which inherited the bar gene became sensitive to the herbicide BASTA ® in subsequent generations (Fig. 5). This result is similar to those observed by Richter et al. 33 In this study, broccoli where pyramided cry1Ac and cry1C Bt genes controlled diamondback moths resistant to either single protein and significantly delayed the evolution of resistant insects. 26 Similarly, Datta et al. 27 developed disease-and pest-resistant rice by crossing plants expressing the Xa21 gene (resistance to bacterial blight) with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and tolerance to sheath blight, respectively).
The objective of the present study was to enhance the resistance level of pea to fungal diseases by crossing two transgenic pea lines one expressing chitinase gene and the second expressing glucanase gene. The consequence was transgenic pea plants expressing both chitinase and glucanase genes.

Results and Discussion
One of the major problems impeding the advance of plant genetic engineering and biotechnology is the fact that the expression or manipulation of multiple genes in plants is still difficult to achieve. Gene pyramiding is emphasized to obtain many complex biochemical pathways in plants for crop improvement and durable resistance. Approaches can involve conventional sexual crossing, re-transformation, co-transformation and the use of linked transgenes. 27 The level of expression of a transgene is variable and is influenced by various factors, such as the site of integration or position effect. 28 Crossing of transgenic parental lines allows a direct evaluation of protective interaction between the transgenes in the crossed plants with that provided by each transgene alone at the same respective loci in the parental lines. 29 Gene 'pyramiding' or 'stacking' present advantages and offer the potential for providing higher resistance to diseases. Interesting PCR was carried out in a volume of 25 μl reactions consisting of 1 μl template DNA (30-50 ng), 5 μl 5x Green GoTaq ® Flexi PCR buffer, 2.5 μl MgCl 2 (25 mM), 1 μl of dNTPs (10 mM), 1 μl (10 pmol) each primer, 0.2 μl (1 U) GoTaq ® polymerase (Promega) and 13.3 μl sterile nuclease-free water. The amplification was carried out in a T3 thermal cycler (Biometra). Positive, negative and water controls were also run along with DNA samples of interest to avoid the detection of false positive or false negative results.
PCR conditions were: initial denaturing at 94°C for 1 min, 30 cycles at [denaturing at 94°C, 45 s; annealing at 60°C, polygalacturonase inhibiting proteins (PGIP) from raspberry and stilbene synthase (Vst) from grape were expressed in pea. The PGIP, as well as the bar gene, exhibited independent expression dynamics in different generations. They observed the silencing of the bar gene in the T1 of some lines.
In some cases, this may be due to gene inactivation or silencing, methylation, co-suppression or due to the physical loss of the gene due to incomplete T-DNA transfer to the plant genome, since the bar gene is located next to the chitinase and glucanase genes near the left border of T-DNA. 34,35 Crude extracts of different transgenic F 2 and F 3 progenies showed inhibitory effects on spore germination of Trichoderma harzianum in contrast to extracts from isogenic transgenic lines, untransformed pea line (negative control) or Na-acetate buffer as shown in Figure 5. 36
Cross pollination. Transgenic seeds were grown at 17-22°C in a 16/8 h day night interval. At flowering stage, after 6-8 weeks of planting, mature pollen was transferred manually from the donor plants to the stigma of emasculated recipient flower for pollination, the flowers were closed, labelled and let to continue growing until setting the seeds. Mature crossed seeds were harvested and dried before germination in greenhouse for multiplication and further analyses. Detection of integrated genes into transgenic pea plants was done by polymerase chain reaction (PCR).   DNA (30-50 ng), 10 μl 5x Green GoTaq ® Flexi PCR buffer, 5 μl MgCl 2 (25 mM), 0.25 μl of dNTPs (10 mM), 1 μl (10 pmol) each primer, 2 μl PCR Dig labeling mix (Roche) 1 μl (1 U) GoTaq ® polymerase. The quality of the probe was checked on 1% agarose gel and compared with control probe without adding PCR Dig labeling mix.
Expression analysis via RT PCR. Total RNA was isolated from very young pea leaves using Trizol reagent (RNAtidy G, AppliChem) according to the manufacturer's protocol and quantified by spectrophotometer, RNA integrity was checked by denatured agarose gel electrophoresis in MOPs buffer. Five micrograms of total RNA was used for cDNA synthesis and RT-PCR according to the manufacturer's manual (MBI Fermentas). cDNA was used as template for the normal PCR as mentioned earlier.
Leaf paint assays. Leaf paint assay was done to verify the expression of the bar gene. The bar gene activity in crossed transgenic plants was assayed according to Schroeder et al. 38 The upper surface of a leaflet was thoroughly wetted by painting with an 45 s; extension at 72°C; 45 s]; final extension at 72°C, 5 min. The PCR products (20 μl of sample per lane) along with 100 bp DNA molecular weight marker were electrophoresed on 1.0% agarose gel stained with EtBr and visualized by UV transillumination.
Southern blot analysis. To analyse the integration pattern and copy number of introduced genes, Southern blot was performed. About 30 μg of genomic DNA was digested with a restriction enzyme (BamHI), separated by electrophoresis (0.8% agarose), depurinated, denaturated and blotted onto a positively charged nylon membrane (Roche Diagnostics GmbH). Non-radioactive detection method using digoxigenin (DIG) system according to the application manual for filter hybridization (Roche Diagnostics GmbH, 2000) was applied. Filters were prehybridized with DIG Easy Hyb for 3 h at 42°C then hybridized with PCR-DIG-labelled probes over night at 42°C, the probe was prepared according to the manufacturer's manual (Roche Diagnostics GmbH, Mannheim, Germany) briefly to 50 μl final volume the following compounds were added 1 μl template aqueous solution of herbicide Basta ® (Aventis GmbH, Frankfurt, Germany) with a final Phosphinothricin (PPT) concentration of 600 mgl -1 and 0.1% Tween20. The opposite leaflet of each pair was left untreated as a control, the herbicide effect was evaluated 1 week later.
In vitro bioassay. In vitro bioassays were performed for testing the synergistic effect of the F 2 and F 3 crossed transgenic plants to inhibit fungal spore germination using Trichoderma harzianum (T12 strain), 39 40 μl spore suspension of T. harzianum was mixed with 40 μl protein crude extract and incubated overnight at ambient RT. The effect of crude extracts on spore germination was examined under a light microscope.

Conclusion and Outlook
A successful combination of chitinase and glucanase transgenes in one pea line via conventional crossing was achieved in the present study. However, variation in expression and activity was observed with some lines having a higher activity than the parental transgenic lines. This may possibly be due to the hemizygous state of some of the crossed transgenic lines or due to the  subsequent studies for stacking of more genes and to understand the inheritance of transgenes since it is different from traditional Mendelian genetics.
constitutive expression of the 35S promoter. It would be interesting to test the antifungal effects in vivo under field conditions with different fungi. Furthermore, these lines may be used in