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Diplocarpon rosae is the leading cause of rose black spot, one of the most devastating diseases on rose fields. Black spot infects plants very often; repeated infection cycles severely weaken the host plants. It will reduce the growth and plants eventually die. The only possibility to control black spot in field is the application of fungicides or planting the resistant varieties. However, application of fungicide is dangerous to both environment and our health. Therefore for these reasons, improving the resistance of rose varieties is the best choice in rose fields.
Wild varieties are more adaptable to the natural conditions and diseases than the modern varieties. They are the major sources of resistance to important diseases. Many resistance genes in wild varieties have been found out and transferred to modern varieties. In roses, there are many resistance genes, which can resist most of dangerous diseases (such as black spot, powdery mildew, etc.) have been used in breeding. Most of them come form wild varieties. High resistance to black spot was found in genotypes of R. banksiae Ait., R. carolina L., R. laegivita Michx., R. multiflora Thunb. Ex Murray, R. rugosa Thunb., R. roxburghii Tratt., R. Virginia Herrm. and R. wichura Crep (Roberts et al, 2003).
Located on chromosome A1 as a single dominant gene in duplex configuration (RRrr), Rdr1 is the first resistance gene that had been found out in R. multiflora Thunb. and reported by Malek and Debener since 1997 in the genus Rosa (Von Malek and Debener, 1998). It induced resistance to most isolations of black spot. Tight linking markers with the resistance gene Rdr1 that were found recently have opened the new stage in breeding new rose varieties that can resist to black spot.
The total length of the Rdr1 region is about 240kb. This region is fully sequenced but is considered too big for gene transfer to the susceptible varieties. Therefore, it had been splitted into nine smaller parts comprising as RGAs (Resistance Gene Analogues). These nine RGAs are the candidate genes that most likely carry the black spot resistance. Therefore, this study will be conducted to determine which of these RGAs are really the genes for black spot resistance.
II LITERATURE REVIEW
2.1 History of Rose as Important Ornamental Crops
Originally, the distribution of modern roses is in the northern hemisphere, from 20 to 70 degree latitude, and they did not grow in the southern hemisphere. Nowadays, the genus is distributed all over the world.
Roses have been cultivated as ornamental crops for more than 2000 years, and they are becoming one of the most economically important crops in the world (Philips and Rix 1988). Nowadays, the value of rose products is exceeding those for vegetables and fruits. The value for flowers was about 24 billions Euro per year (based on values from 1995 -2007) in 2007, rose accounted for about 22% (723 million Euro) for all ornamentation exported from EU into other countries. Imports of roses into the EU in 2001 reached a value of 447 million Euros. About 31% of all cut flowers traded at European auctions were cut roses, with total value about 857 million Euros (Heinrichs, 2008).
The most developed genus Rosa belongs to family Rosaceae, including more than 150 species and thousands of cultivars. Most of modern roses do not belong to a single rose species, but they are belonging to complex hybrids, derived from multiple species. The basic chromosome is 7 and the ploidy levels are ranging form 2x to 8x. Wild species are often diploid (2x =14) while almost cultivated roses are tetraploid (4x =28) (Wissemann and Hellwing, 1997).
2.2 Diseases on The Rose
Crown gall is caused by bacterium Agrobacterium tumefaciens (Smith and Townsend, 1907). This bacterium can be found in all rose families (apples, rose, raspberry, peach, etc.) and is worldwide in distribution. The organism is a Gram-negative, rod-Shaped, non-spore forming, and motile aerobic bacterium. Crown gall usually attacks on the roots, lower stems, and owner branches of infected roses. In some cases, it may occur in the upper branches.
Powdery mildew, caused by Podosphaera pannosa (Wallr.:Fr.) de Bary, belongs to the family Erysiphaceae in the class Ascomyctes. It can occur almost anywhere on rose plants. Powdery mildew infections reduce the quality of cut flowers, and make nursery stock less saleable to consumers.
The conidia of Podosphaera pannosa are mostly dispersed by wind. The optimal conditions for germination are 22Â°C, and the humidity is nearly 100%. When temperature is more than
30Â°C the germination of the conidia will be prevented. Conidia can bear long periods of 0Â°C without loss of viability, they can survival at 0 - 3Â°C for three months. Eventhough the optimal humidity for the germination of conidia is 100%, they still germination when the humidity down to 50%, but with lower frequency. The direct contact with large drop of free water in the first 6-8h after infection reduces the number of germinated conidia. Nearly 40% of the fungicide sprayed on roses is to control powdery mildew (Linde and Shishkoff, 2003).
Black spot is one of the most dangerous fungal diseases in the field of roses. It is caused by fungus Diplocarpon rosae (Wolf). Black spot was first reported in 19th century by Fries (1815) and Libert (1826). Aronescu (1934) and Frick (1943) are the first persons described the infection cycle of Diplocarpon rosae. They reported in details most of the infection structures on sensitive plant genotypes. Especially, they described the shape and size of conidia and micro conidia, penetration of hyphae, the typical parallel subcuticular hyphae, haustorial mother cells, and haustoria.
Diplocarpon rosae can be easily found on most rose cultivars and some wild species. It will make the plant become weaker and weaker, and eventually defoliation follows. Under natural conditions, Diplocarpon rosae, a hemibiotrophic fungus grows only on roses, where it is mostly found on the adaxial side of the leaves.
Black spot is spreaded by two cells asexually produced conidia, and sexual structures. Spores (conidia) can be spreaded by water splash, insects, or arachnids (Drews Alvaraze, 2003). After that, they germinate on the surface of the leaves and penetrate into the cuticle. After two days, the first haustoria are formed and the fungus spread out on the leaf (under favorable conditions, these conidia can germinate on susceptible leaf surfaces within a day (Blechert and Debener, 2005). Five days later, the new generation of conidia start, and three more days later, the first conidia will be released from acervuli through the breaking cuticle to start a new life
cycle. At this point, the damages to the leaves can be seen under macroscope. Black or brownish spots with flattened margins appear on the adaxial side of the leaves. After two weeks, most rose varieties drop their infected leaves.
Diplocarpon rosae can grow on artificial media. Isolates of the fungus can be differentiated by morphological traits, or according to their interaction with different rose.
Figure 1. Life cycle of Diplocarpon rosae Wolf. Adapted from Martinez (1962)
2.3 Control Methods of Black Spot
Base of this method, the black spot conidia need to be in contact with water before they germinate. Black spot could be controlled by keeping the required plant spacing to allow air movement, and watering early in the morning may have significance to limit the spread by reduce the humidity. But controlling insect, moisture, and humidity are difficult in the gardens.
With this method, the fungicides should be applied to kill newly spread conidia. The fungicides must be used repeatedly at 10 -14 day intervals when new leaves appear. This method is not effective if black spot established on rose plants and fungicides may take some problems with the environment and our health.
Breeding for Black spot resistance
With this method, developing new varieties contained resistance gene, that can resist to black spot is the best way to control this disease in the field. These plants could be inhibiting germination of conidia or to stop the growth of the fungus after it has invaded the plant tissue.
2.4 Rose Genetic Engineering
2.4.1 Plant resistance genes
Plant disease resistant genes (R genes) encode proteins that defense against infection of many pathogens. There are two type of resistance gene: vertical resistance and horizontal resistance. With vertical resistance, there are single genes for resistance in the host plant, and there are single genes for parasitic ability in the parasite. This is a very important phenomenon known as the gene for gene relationship, (The gene-for-gene relationship was firstly reported by an American scientist, Flor in 1940). Vertical resistance can be a strong type of resistance to the particular pathogen. However, vertical resistance can be easily broken down. Horizontal resistance on the other hand, does not involve a gene for gene relationship. It exhibits the resistance with many pathogens and the resistance degree is between a minimum and a maximum. It is hard to break down, and the resistance can remain in a long time.
Powdery mildew resistance
The description of a single dominant resistance gene (Rpp 1) to powdery mildew, which base on the repeated inoculations with single conidial isolates has been published before (Linde et al., 2004). In addition, major loci were described by analyses of natural infections on field sites, in greenhouses or by artificial infections with polysporous isolates (Zhang, 2003; Xu et al., 2005). The Rpp1 was mapped in a diploid population of R. multiflora hybrids and a SCAR marker was generated from a closely linked AFLP marker (Schulz, M. Linde, O. Bleachert and T. Debener, 2009).
Black Spot Resistance
Three major resistance genes have characterized in tetraploid and diploid populations, two of which are linked within 10 cM on the same linkage group (Malek and Debener, 1998; Kaufmann et al., 2003; Zhang, 2003; Yan et al., 2005; Gebreiyesus and Linde, personal communication).
Identified in Rosa multiflora, Rdr1, the black spot resistance gene, has been found on chromosome A1 as a single dominant gene in duplex configuration (RRrr) in a tetraploid breeding line 91/100-5 ( Malek and Debener, 1997). On the figure 2, the origin of the gene is a diploid genotype (2n =14) 8/124- 46. To make the breeding process easy, it was treated with colchicines, which resulted in a tetraploid line (4n=28) CT40. The breeding genotype 91/100-
5 was finally developed from a cross between C40 and a susceptible cultivar "Caramba". To speed up introduction the gene to cultivars, molecular markers linked to Rdr1 have been identified. The first closest marker that had been indentified is M10 (1,1 cM) AFLP (Amplified Frangment Length Polymorphism), which latter has been converted to a PCR based SCAR (Sequence Characterized Amplified Region) marker with 0.76 cM from Rdr1 (Malek et al, 2000). In addition, new CAPS (Characterized Amplified Polymorphic Sequence) markers to characterize genotypes base on variation on HaeIII restriction sites on the locus has been identified at a distance 0.093 cM to the telomeric side of Rdr1 (Von Malek, Debener,1998, Kaufmann et all, 2003).
Figure 2. Origin of the resistance breeding line 91/100-5 (Von Malek and Debener, 1998)
A contig around Rdr1 covering a physical distance of about 200kb was established containing a cluster of nine highly similar resistance gene analogues of the nucleotide binding site (NBS)-leucine rich repeat (LRR) type. All nine RGAs, Rdr1A, to Rdr1I, have a common gene structure with four exons and three short introns, the first and the second exon representing TIR and NBS domains, respectively, while the LRR region on exon three and four is divided by the third intron (Terefe et al., 2009). Apart from Rdr1I, the
RGAs encode open reading frames (ORFs) of approximately 1100 amino acids and resemble in size to other plant resistance genes of the TIR- NBS-LRR type as for example N of tobacco and L6 of flax (Whitham et al., 1994; Lawrence et al., 1995). Rdr1I contains a stop codon within the third exon that reduces the ORF to 794 amino acids. As at least parts of the LRR region are present in the putative protein. Rdr1I could encode a functional resistance protein and is considered an Rdr1 candidate gene. Another RGA (Rdr1D) probably represents a pseudo gene as it is disrupted by a transposon insertion of about 7kB. Although this transposon is located in the non coding region in the first intron of Rdr1D, inactivation of this element is highly possible due to the size of the
insertion (Kaufmann et al, 2009).
Fig.3 The BAC contig around the black spot resistance gene Rdr1
A: The contig around Rdr1
B: Intron-exon structure of RGAs
(Kaufmann et al., Act horticulture 2010, in press)
2.4.2 Regeneration system
220.127.116.11 Callus and embryogenesis induction
Callus culture of rose is the first step in genetic transformation system. Nearly all parts of the plant can be used as starting material for somatic embryogenesis, but the success with particular explant types strongly depends on the genotype. Each laboratories have other
protocols but all of them are based on some main medium: MS, N6 with the concentration of auxin, cytokine but they have been modified to be appropriate with each species or varieties. However, for some rose genotypes they might be of advantage to use other formulations. Particular rose cultivars respond better to glucose, fructose or maltose rather than sucrose. Furthermore, several rose genotypes exhibit enhanced frequencies of somatic embryogenesis.
Jacobs et al. (1968) reported that a modified medium, based on those of Knop and Berthelot (White, 1954) with different concentrations of IAA and kinetin, resulted in fairly rapid callus formation from pith explants of hybrid tea rose (cv. Super Star). In 1981, Tabaeezadeh and Khosh- Khui reported the induction of callus from haploid and diploid anthers of hybrid cultivars. In 2008, Zakizadeh et al. reported the induction callus from leaves in their experiments. Recently, Suk Weon Kim et al. (2009) reported the induction of callus and got the regeneration plants from root explants culture of R. hybrida L. Frequency of explants exhibiting somatic embryogenesis may vary from below 1% to nearly 100% (Schum and Dohn, 2003). All cultures will be kept in the dark condition at 23Â±2Â°C.
18.104.22.168 Plant regeneration
For plant regeneration, the embryos are transferred onto the new medium to make shoots and regenerating shoots. Different media including various concentrations of phytohormone are used by researchers for germination, and shoot regeneration. Roberts et al. (1990), Mathews et al. (1991), and Noriega and Sondahl reported lowering the concentration of 2,4-D from the culture medium helped embryo development and germinations as reported in many cultivars
of Rosas species. Rout et al. (1991) reported that the exposure of embryogenic calli to 80C for
four days enhanced the germination rates form 0 to 12% on half strength MS medium supplemented with 0.5mg/l BA, 0.1mg/l GA3 and 1.5mg/l adenine sulfate. However, on the experiments of Roberts et al. (1995) he reported that chilling at 40C for 2 weeks improved the germination rates from 12 to 24%.
There are two diffrent shoot types that may be observed in this step. Morphologically normal shoots develop into proper plantlets. While morphological abnormal shoots remain undeveloped. The ratio of the normal shoots could be 70% in the experiments of Zakizadeh et al. (2008). Secondary somatic embryos were also observed in this experiment.
These shoots are transferred to a rooting medium. And after that, they were planted on the green house.
2.4.3 Gene-transfer methods
Modern biotechnology had provided new tools of tissue culture and genetic engineering to either manipulate in vitro cell differentiation and regeneration and to develop gene transfer protocols for rose. The same as other plant species, rose transformation is achieved both via agrobacterium and particle bombardment. Reported on 1991 by Firoozabady et al., the first rose transformation was created by using Agrobacterium transformation of embryogenic tissue including from filament culture of R. Hybrida cv (Korban, 2007).
Nowadays, there are a lot of methods for introduction DNA in to plant cells. However, particle bombardment and Agrobacterium mediated transformation are the two methods used very common in the laboratory for large number of plant species.
22.214.171.124 Particle Bombardment
For this method, gold or tungsten micro are coated with plasmid DNA and delivered to plant tissue by mean of a particle gun. When particles enter the target cell, the DNA is released from the particles. There is a small percentage of particle t h a t becomes associated with the chromosomes. If proper conditions exist, the foreign DNA integrates into the chromosomes of the target cell.
The DNAs are physically delivered into the cells which by pass any potential biological incompatibilities. So they are also damages the host DNA by shearing. It would give the opportunities for foreign gene incorporation by the plant's DNA repair mechanisms (the introduction of particles should from 0.6 - 3 Âµ m, the bigger size can be damages the host cells ).
Integrated DNA resulting from particle bombardment mediated DNA transfer is often present in high copies and fragmented. High copy transgenes can show variation or loss of expression due to gene silencing. The major advantage of this method is wide applicability even some of the most "recalcitrant" species can be transformed. The main disadvantages of this method are low transformation efficiency, DNA fragmentation, and collateral genetic damage (James and Lee, 2001; Finer et al., 2006).
126.96.36.199 Agrobacterium tumefaciens
Among the various vectors, which used in plant transformation, Ti plasmid of Agrobacterium tumefaciens has been widely used. These bacteria can be naturally transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell, known as crown gall. This bacterium is also known as " natural genetic engineer" of plant.
Agrobacterium utilizes a highly adapted gene-transfer and integration technique to integrate single stranded DNA into plant cells. This method is widely used since it gives predictable results. Agrobacterium transformation produces genetically transformed plant cells or tissue that is highly stable, because the foreign gene is inserted into the main plant genome. The typical procedure for Agrobacterium mediated transformation includes co-culture of plant cells with the bacteria, antibiotic treatment to disinfect the culture and kill the contaminating bacteria, selection of transformed plant cells using the conferred resistance trait, and identification of high producing clones through callus culture under continued selection pressure (James and Lee, 2001).
For Agrobacterium-mediated transformation, plant tissues are cultured in the presence of Agrobacterium, which is a bacterium that has the unique ability to introduce part of its DNA into plants. Because Agrobacterium is a natural plant pathogen, some biological incompatibilities exist when using certain plant species or stages of plant growth. However, most of these biological incompatibilities have been removed or at least lessened as more has been learned about the mechanism of DNA transfer. With the addition of signal compounds to the medium where Agrobacterium and the plant tissues are co-cultivated, and enhancing exposure of cells to the invading bacteria, the process of DNA transfer has become quite efficient for most plants.
Although antibiotics must be applied to eliminate the bacterium after DNA transfer, this method of delivery has two distinct advantages over particle bombardment. First, no instrumentation is required and the cost of performing DNA introductions is minimal. Second, the DNA transfer process, which is mediated by the bacterium, generally results in more consistent integration events. The transferred DNA (T-DNA) is usually defined by specific borders and genes of interest can simply be engineered between those borders. The resultant
integrated DNA can be single copy or show somewhat more complex integration patters
(Finer et al., 2006).
188.8.131.52 Other methods
Particle bombardment and Agrobacterium mediated transformation are methods that can be used nearly every plant species can be transformed effectively. However, these methods may result in limited transformation efficiency for some cell types. For this reason, the use of alternative gene-transfer methods is an active area of research for all cell types. Alternative gene-transfer techniques include electroporation, microinjection, liposome fusion, direct transfer into protoplasts, and laser treatment.
3.1 Generation of Roses Transgenic for Individual Candidate Genes
(At least three (RGA1, RGA3, and RGA8), if possible five (RGA 1, 3, 7, 8 and 9) different genes will be introduced into susceptible Pariser Charme roses).
ïƒ˜ Induction of somatic embryos on in vitro leaves of Pariser Charme.
ïƒ˜ Transformation of the embryos with different constructs.
ïƒ˜ Regeneration of transgenic plants.
3.2 Phytopathological Analyses for Resistance to Different Black spot Isolates
ïƒ˜ Degree of resistance to race 5 (Dort E4).
ïƒ˜ Degree of resistance to races 6 and others.
3.3 Molecular Analysis of the Transgenic Plants
ïƒ˜ Presence of the transgene in the putative transgenic via PCR.
ïƒ˜ Expression of the transgene via RT-PRC.
ïƒ˜ Copy number of the transgenes via either PCR or Southern blot.
ïƒ˜ One or two of the TNL-Genes will confer resistance to the race five (Dort E4).
ïƒ˜ None of the TNL-genes will confer resitance to race 6.
ïƒ˜ The type of resistance will be a gene for gene resistance and will be based on a HR.
V MATERIALS AND METHODS
5.1 Plant Materials
The rose genotype to be used in this study is susceptible Pariser Charme roses. It is a part of the genotype collection of the institute of Plant Genetics of Hannover University.
5.2.1 Callus and somatic embryos induction
Leaves taken from in vitro plant will be injured by several cuts across their midribs and placed onto MS basal medium in 90Ã-15 mm petri dishes with abaxial side down. These media in this experiment are base on MS medium, and use other kind of agars and phytohormones ( Table 1).
Table 1. The media for callus induction
2mg/l NAA 0.5 mg/l 2,4,5 T 1 mg/l 2,4,5 T
Plant agar 8.4g/l X X X
Phytogel 4g/l X X X
Gelrite 4g/l X X X
All treatments will be repeated three times, 15 petri dishes, and a single petri dish containing 10 leaf explants. All cultures will be kept in the dark condition at 25Â±2Â°C for about f o u r weeks, and then data on callus induction will be recorded.
After four weeks the callus will be cut into smaller parts and transferred onto the new medium, bases on MS medium, and supplement with 30g/l sucrose, 4mg/l zeatin, and 4g/l Gelrite, to make embryos. ( in this step, the leaf that callus does not grow and the roots on the callus will be remove before place on the new medium). A f t e r o n e m o n t h , e m b r y o s w i l l b e t r a n s f e r r e d t o t h e m u l t i p l i c a t i o n o f e m b r i o g e n i c c a l l u s m e d i u m w h i c h b a s e s o n M S a n d s u p p l e m e n t w i t h 0 . 2 5 m g / l N A A , 1 . 5 m g / l Ze a t i n , 1 m g / l G A 3 .
5.2.2 Regeneration of plants
For plant regeneration, the somatic embryos will be isolated from the callus and transferred onto MS medium with 0.1 mg/l IBA, 2.0 mg/l BAP, and 0.1 mg/l GA3. The regenerating shoots will be propagated on MS basal medium supplemented with 0.004mg/l NAA,
1.0mg/l BAP and 0.1mg/l GA3. Rooting of these shoots will be induced by subculture on half strength MS medium with 0.1 mg/l NAA and 0.05 mg/l IBA. The regenerating somatic embryos as well as the shoots w i l l b e incubated at 25 Â°C in a 16h photoperiod. For greenhouse adaptation, the rooted plants will be planted into a 1: 1 mixture of standard pricking soil and perlite after removal of the agar basal medium. For the following three weeks, the plants are kept in plastic boxes, which are progressively opened in order to adapt the plants to a lower humidity. The plants w i l l b e gradually transferred into 14 cm diameter containing with potting soil and raised to flowering.
5.2.3 Gene transfer
184.108.40.206 Agrobacterium mediated gene transfer
A single bacterial colony will be incubated in liquid YEP medium (Sambrook and Russell,
2001) supplemented with 50 mg/l kanamycine sulphate and cultivated at 29Â°C and
175rpm for 24h. The resulting bacterial suspension will be diluted 1: 20 in YEP medium or minimal A medium according to Miller (1972) and incubated at 29Â°C and 100 rpm. In preliminary experiments to determine the best infection time, the bacterial suspensions are cultivated for 2- 20h prior to use for transformation. For transformation, fully developed somatic embryos will be solated from the callus at the end of a 4 week subculture. The selected embryos will be wonded by shaking with sand and immersed into the Agrobacterium suspension for 1h. Before incubation of the explants on MS basal medium supplement with 0.01mg/l IBA, 2mg/l BAP, and 0.1mg/l GA3 f o r shoot induction excess bacterial suspension are removed with sterile filter paper. After a cocultivation period of 2 days for EHA 105 or of 6 days for GV 2260, the somatic embryos w i l l b e transferred onto fresh medium containing 500mg/l cefotaxime sodium and 50mg/l carbenicilline to inhibit further bacterial growth. The cultures w i l l b e grown at 25Â°C in a 16h photoperiod.
220.127.116.11 Selection and regeneration of transgenic plants
About 3 weeks after transformation, the selection of transgenic tissue will be started. The transformed embryos will be incubated on MS basal medium contained 0.01mg/l IBA,
2mg/l BAP, and 0.1mg/l GA3 for shoot induction and supplemented with 150mg/l TIMENTIN (Duchefa) to inhibit the bacterial growth as well as 60mg/l kanamycine sulphate for selection. Regenerating shoots will be separated from the original explants, transferred onto MS basal medium with 0.004mg/l NAA, 1.0mg/l BAP, and 0.1mg/l GA3, and
supplemented with the same antibiotics. Rooting of these shoots will be induced by subculture on half strength MS medium with 0.1mg/l NAA and 0.05mg/l IBA. The regenerating somatic embryos as well as the shoots will be incubated at 25Â°C in a 16h photoperiod. For greenhouse adaptation, the rooted plants will be planted into a 1: 1 mixture of standard pricking soil and perlite after removal of the agar based medium. For the following 3 weeks, the plants will be kept in plastic boxes, which are progressively opened in order to adapt the plants to a lower humidity. The plants will be gradually transferred into
14 cm diameter containers with potting soil and raised to flowering.
5.2.4 Molecular Techniques
18.104.22.168 DNA extraction
CTAB extraction procedure using NucleoSpinÂ® Plant II Kit (MACHEREY-NAGEL GmbH
& Co.KG) will be used to extract genomic DNA. In this procedure, 45-70 mg of young leaf samples will be dried overnight in oven (370C), homogenized in 2ml eppendrof tubes using steel beads and rotor-stator homogenizer. According to the manufacturer's instruction, the resulting powder will be treated with 600 Î¼l lysis buffer (PL1) and 10Î¼l RNase A, thoroughly vortexed, and incubated in 650C for 10 min. The lysate then centrifuged for 2 min. at 11,000 x g on the filter column. The supernatant will be transferred to new tube and equal volume of DNA binding buffer added before separating with filter column. After successive washings, DNA was recovered from the membrane by adding 50Î¼l buffer heated to 700C, incubating for
5 min. in 700C and centrifuge for 1 min. at 11,000 x g.
22.214.171.124 RNA Extraction
RNA will be extracted with the InvisorbÂ®Spin Plant RNA Mini Kit (Invitek, Germany) according to the manufacturer's instructions summarized as follows. About 30mg leaf material will be collected in Eppendorf tubes and frozen in liquid nitrogen and then ground immediately in a bead mill to a fine powder for 3min. Then 900Î¼l Lysis Solution RP will be added into the tube and mixed thoroughly by vortexing and incubated for 20 min at 25Â°C, in continuous shaking. The solution will be then centrifuged at 13,000 g for 1 min and the clean supernatant was transferred to a filter column. The filter will be discarded after centrifuge for
1min at 10,000 rpm. 500Î¼l absolute ethanol will be then added to the filtrate, and mixed by pipetting up and down. The lysate will be transferred to an RTA-Binding Spin Filter Set, incubated for 1 min and centrifuged twice at 10,000 rpm for 1 min. Next, 500Î¼l Wash Buffer
R1 will be added once and 700Î¼l Wash Buffer R2 twice, onto the Spin Filter. Each time it will be centrifuged for 30 sec at 10,000 rpm and finally centrifuged for 3 min at 12,000 rpm to eliminate any traces of ethanol in the Spin Filter. 30 - 60 Î¼l of Elution Buffer R will be added directly into the membrane of the Spin Filter, incubated for 2 min and centrifuged for
1 min at 10,000 rpm. The Spin Filter will be discarded and the eluted total RNA will be immediately placed on ice.
To digest any contaminating DNA in the isolated RNA, DNase Kit (InvitrogenÂ®) will be used as follows. For 50Î¼l RNA, 0.1 volumes of DNase buffer and 1Î¼l rDNase (2U/Î¼l) were added and incubated at 37Â°C for 25 min. Then, 0.1 volume of DNase inactivation reagent will be added and incubated at room temperature with occasional mixing. Finally, the reaction tube will be centrifuged at 10,000 g for 1.5 min at 4Â°C. The supernatant is then used as a purified RNA.
126.96.36.199 cDNA synthesis
High Capacity cDNA Reveres Transcription Kit (Applied Biosystems) will be used to prepare total cDNA. As recommended by the company; 10 Âµ l of 2x RT master mix for each reaction will be prepared by 2 Âµ l of 10x RT buffer, 0.8 Âµ l of 25x dNTP mix (100 mM), 2.0 Âµ l of 10x RT random primers, 1 Âµ l of reverse transcriptase (MulliScribeTM), 4.2 Âµ l of Nuclease-free H2O. In the marster mix, 10 Âµ l of RNA (1 Âµ g) sample will be added to make a total volume of 20 Âµ l solution. It will be then incubated in a thermal cycler programmed to
25Â°c for 10 min, 37Â°c for 120 min and 85Â°c for 5 sec. The RT-PCR product will be checked by actin primers.
188.8.131.52 Southern blot analysis
Genomic DNA will be isolated, using the Nucleon Phytopure Plant DNA isolation kit (Scotlab, Coat- bridge, UK), from 1g fresh weight of leaf tissue fully expanded leaflets of terminal shoots from all of the putatively transformed plants and from nontransformed controls. The procedure used for Southern blot border analysis will be based on that described by McCabe et al. Genomic DNA (10g) will be digested with s u i t a b l e restriction enzyme, precipitated in ethanol, redissolved in TE buffer and electrophoresed on an 0.8% agarose (Molecular Biology Grade Agarose; NBS Biologicals, Hunting- don, UK) gel (25V for 16h). DNA will be transferred onto Hybond N positively charged nylon membrane (Amersham International, Amersham, UK) by alkaline transfer. A polymerase chain reaction digoxigenin-
labelled fragment of the RCH10 coding sequence (labelled according to Lion and Haas) will be used as a hybridization probe. Hybridization (37 0 C for 16 h) and washing are according to standard procedures. Chemiluminescent detection will be undertaken using CDP-Star substrate (Tropix, Bedford, USA). The membrane will be subsequently exposed to Hyperfilm MP X-ray film (Amersham International) for 1-4 h at room temperature.
5.2.5 Inoculation Test
This study will use the inoculation procedures as described by Blechert and Debener (2005). This will be done by collecting young and disease-free leaves grown in the greenhouse. New and fully developed healthy leaves will be selected for inoculation to reduce the effects of mechanical resistance prior to the infection by similar or different pathogens other than the inoculums used in the study.
Cleaning the leaf surface with 50% of Ethanol and subsequent washing with distilled water will be done after collection. This method removes any contamination from the field and improve spore suspension droplets binding to the leaf surface. Leaves ready for inoculation will then be prepared in inoculation boxes with moistened tissue paper.
Inoculum will be collected either from freeze stored propagating materials or newly multiplied spores on infected leaves. Spores propagated on Pariser Charme will be washed and collected with sterile tap water. In most of the inoculation experiments, newly propagated conidia rather than freeze stored spores are more preferable to be used. A standard concentration spore suspension of 10 5/ml will be used to infect the disease to each genotype. The concentration of the inoculums will be determined by counting under a microscope the estimated total concentration of viable conidia. This will be done by treating an aliquot (15Âµl) of collected spore solution with 15Âµl of 0.05% phenosa phranin (3.7- diamino 5-phenylphenazinium chloride, Sigma-Aldrich), which selectively stains non viable spores. The concentration of the spores collected will be then adjusted to 105 / ml for inoculation. Ten micro liters of spore suspensions will be applied with four to ten droplets to the upper side of each leaf. Three to five sample leaves will be used per treatment. Inoculation boxes will be placed in an incubator which is maintained 16 light hours and
20Â°C temperature. After three days, excess spore suspensions will be dried with towel paper, and boxes will be placed back to the incubator.
Disease evaluated scores
The first data of genotypes' reactions against specific isolates will be recorded after seven
days of inoculation while the second data will be taken after 14 days. It will be evaluated using the following system.
0 - Absence of any disease symptom at the site of inoculation
1 - Some mycelia growth limited within the site of infection and formation of some dark or brown spots, no acervuli formed
2 - Well-established pathogen growth but limited production of acervuli
3 - High infection and formation of masses of reproductive structures comparable to growth on the susceptible control
CALENDAR OF RESEARCH ACTIVITIES
Inoculation test and molecular analysis