MATERIALS AND METHODS
2.1 Production and purification of cerato-platanin
Cerato-platanin was purified according the procedure described by Carresi et al (2006).
2.2 Plane leaf treatments
Plane leaves were from a P. acerifolia tree living in Florence. Before treatment, leaves were abundantly washed, and the lower surface was carefully scraped and again washed with sterile distilled water. In a standard experiment numerous ten µl droplets of CP 1.5 x 10-4 M were applied to the right part of the lower surface (about five droplets per centimetre square), whereas the left part was treated with sterile distilled water (control). The leaves was maintained in a moisture chamber at 23°C in the dark.
At 48 hours after treatment, droplets were recovered, filtered through a 0.22 µm Millipore membrane and assayed for phytoalexin production as described by Carresi et al (2006). Fluorescence was recorded with a Perkin Elmer spectrofluorimeter 650-10S (Perkin Elmer, Wellesley, MA, USA), λex = 365
nm, λem = 460 nm and slit = 5. The standard calibration curve had a linear
correlation coefficient >0.99 using 0.1–10 µM pure umbelliferone (Sigma, St. Louis, MO, USA). The results were expressed as nmol/mL of umbelliferone equivalents.
The CP concentration was evaluated by ELISA according to the procedure described in Scala et al. (2004), with an anti-CP antiserum raised in rabbit against purified CP from culture filtrates of C. fimbriata f. sp. platani Cf AF 10. The standard calibration curves of A492 vs. the log of purified CP
concentration had a linear correlation coefficient >0.95 using purified CP over a concentration ranging from 3 ng to 1 mg per well.
The leaf portions pre-treated with CP or distilled water were separately recovered at -80°C before using for RNA isolation.
In some experiments different CP concentrations and treatment times were used as indicated in the captions to Figures.
2.3 Total RNA isolation
For the total RNA isolation approximately 2 g samples of the treated and control leaves were used.
The leaves were ground in liquid nitrogen then were homogenised in CTAB buffer extraction (NaCl 1.4 M, EDTA 20 mM, Tris-HCl 100 mM, pH 8.0, CTAB 3% (p/v), 2-Mercaptoetanolo 0.2% (v/v)) in ratio 6:1 (v:w). The mixture was incubate for 20 minutes at 60C and then twice isoamyl alcohol-chloroform extraction. After adding isopropyl alcohol to the surnatant the RNA was precipitated 1 hour at 4C The pellet obtained after centrifugation was washed by absolute ethanol and was dissolved in SSC 1x. The RNA was layer onto cushion of CsCl (CsCl 5,7 M in Sodium acetate 25mM, pH 6.4) and the gradient was centrifuged at 40K rpm with Kontron TY65 rotor at 20°C for 24 hrs. The pellet obtained after centrifugation was dissolved in DEPC water and precipitate by incubation for over night at -20C after addition of 3M sodium acetate (pH 5.2) and cold absolute ethanol. After washing the pellet with cold 70% DEPC ethanol the RNA was dried and then dissolved in DEPC water. The samples are stored at -80C.
2.4 SSH library construction.
Poly (A)+ RNAs were isolated from the pools of total RNAs of control and treated leaves using the PolyATract mRNA Isolation Systems (Promega,
Madison, Wi, USA) according to the protocol described by the manufacturer. Subtractive suppressive hybridisation (SSH) was performed according to Diatchenko et al. (1996) using the BD PCR-Select cDNA Subtraction Kit (BD Biosciences) after the of use the BD SMARTTM PCR cDNA Synthesis Kit (BD Biosciences Clontech).
enzyme RsaI and then ligated to different adaptators. The cDNAs were used as tester and driver respectively for the forward and reverse subtraction according to the BD PCR-Select cDNA Subtraction Kit.
The fragments that are expressed in the tester but not in the driver were then specifically amplified in two PCRs. Amplified cDNA sequences from the
forward and reverse subtraction were directly inserted into a T/A cloning vector and then transformed into Escherichia coli using the TOPO TA Cloning
Kit (Invitrogen). Positive transformants based on blue/white color selection were arrayed into 96-well microplates. All technical details followed the protocol described by the manufacturer.
2.5 Differential screening of the subtracted cDNA libraries
The recovered clones were cultured in LB medium with 10 mg/ml ampicillin in 96-well plates at 37°C. The cDNA inserts were amplified by PCR with nested PCR primers 1 and 2R, that are complementary to the adaptators, to check the presence and size of individual inserts.
PCR reactions (25µl) contained : 18.5µl sterile water, 0.6µl each of primers (10µM each), 2.5µl 10X reaction buffer (Euroclone), 1µl MgCl2 (Euroclone),
0.6µl of dNTP mix (2.5mM each), 0.25µl Euro taq polymerase (Euroclone) and 1µl bacterial culture.
The samples were first denatured at 94°C for 10 min, followed by 30 cycles of 94°C for 30s, 68°C for 30s and 72°C for 1.30 min, with a final extension at 72°C for 10 min. All PCR products were analysed by agarose-gel electrophoresis.
Two identical blots were created by spotting heated-denatured PCR products (1µl) from each of the clones of the subtractive libraries onto nylon membranes positively charged (Roche) and cross-linked by UV.
The membranes were pre-hybridized for 3 hours in pre-hybridization buffer (DIG easy Hyb, Roche) and then incubated overnight at 42°C in hybridisation buffer (DIG easy Hyb containing the labelled cDNAs obtained by DIG-DNA
The fragments that hybridised only with the labelled forward cDNA or showed at least higher signals compared to the signals obtained with the reverse labelled cDNA were subjected to sequencing analysis.
2.6 Clones sequencing and analysis.
Differentially expressed clones were sequenced by automated sequencing (MWG Biotech Ebersberg, Germany).
Homology searches of all sequences were compared to the GeneBank
EMBL-EBI database using the BLAST algorithms.
2.7 Semi quantitative determination of transcript levels by RT-PCR.
Reverse transcriptions were carried out with total RNA (2µg) isolated from CP treated and control leaves of Platanus acerifolia with iScript cDNA Synthesis
kit (Bio-Rad) according to the manufacturer’s instructions. 1 µl of first-strand cDNAs were used for each PCR amplifications and PCRs were performed using gene-specific primers (chlorophyll a/b-binding protein for the PSII-L2B
(right: 5’-ACGAAGTTGGTGGCATAGGC-3’; left
5’-GAGATCAAGAACGGGAGACTG-3’), the selenium binding protein-L1B
(right: 5’-TCTGAGGGTTCCTTCTTTGC-3’, left:
ACTATGGCCTGGTTTCTCCC-3’), tubby like protein-Q7H ( right: 5’-CTCATGCTGGAGCCACAGT-3’, left: 5’-GCATCCATGACACACTGCAT-3’), the RARI protein-L3A (right: 5’-GCCGGTCAATAGGAATCAGA-3’, left: 5’-ATCCTGGCCCTGCTATTTTC-3’), Ferredoxin A-L2D (right: CAGGTTCTTGCTCTTCCTGC-3’, left:
5’-AGGTCCTCTTCCTTGTGGGT-3’) and Elongation factor 1α-Q11H (right:
5’-TCTCCACGCTCTTGATGACTCC-3’, left:
5’-CGGTGATGCTGGGTTTGTGAAG-3’) and the 18S ribosomal gene as the housekeeping gene. We have used the Ambion’s Competimer™ technology (QuantumRNA™ Universal 18S Internal Standard) to modulate the efficiency of amplification without affecting the performance of other targets in the
amounts of 18S Competimers, the overall PCR amplification efficiency of 18S cDNA can be reduced without the primers becoming limiting and without loss of relative quantification. For each analysed gene we have identified the cycles of the exponential phase, we have done the same thing with the 18S rRNA using different mixes of primers-competitors and testing which mix was in the exponential phase in the same range of cycles of the analysed gene. The relative amounts of each PCR product were readily quantified by direct scanning with a densitometer of ethidium bromide-stained 2% TBE-agarose gels with Quantity One® Software (Bio-Rad). To normalize for equal amounts of total RNA and efficiency of cDNA synthesis from various tissue samples, the band intensities were normalized with the average intensity of 18S product across the samples investigated. The ratio between the value of the analysed gene product level and the18S product level of each sample was calculated from three independent experiments performed for each gene. The GraphPad
Prism 4.00 software has been used to analyse the data. The analyse of the data is carried out using Student’s t-test (p< 0.05).
2.8 In situ hybridisation
RNA probes were obtained using DIG RNA labelling Kit (SP6/T7) (Roche) from PCR product inserted into a T/A cloning vector and then transformed into
Escherichia coli using the TOPO TA Cloning Kit (Invitrogen), all technical details followed the protocol described by the manufacturer. The PCR product was obtained using primers that permit to amplify a specific platanus tubby region (right primer: AGGTCCCGGTTGGAGATG, left primer: GATGCCATCCCTGCCTCT).
They have been used paraffin sections. After the treatments with CP and fungus conidia the leaves were cut and fixed with formaldeyde and then paraffin embedded in according to standard procedure. They were cut 7µm from paraffin embedded material and place the sections onto coated glass slides. The sliced were dried over night at 40°C and then dewaxed with fresh xylene. The slides were rehydrate in ethanol series and DEPC water.
The paraffin was removed from the sections with Histoclear (National Diagnostics) or Citra-Solv (Fisher) and the sections are rehydrated through an
ethanol series: 100 absolute ethanol (1-2 minutes), 90% ethanol(1-2 minutes), 70%ethanol(1-2 minutes), 40% ethanol(1-2 minutes), sterile water (1-2 minutes) and SSC2X DEPC.
Tissue sections were proteased to increase the accessibility of the target RNA. Sections were treated with proteinase K (1 microgram/ml in 100 mM Tris pH 8 and 50mM EDTA) at 37°C for 30 minutes. The tissues were refixed to prevent disintegration of the tissue following protease treatment: 2mg/ml glycine in 1 X PBS (2 minutes), 1 X PBS (2 minutes), 4% paraformaldehyde, pH 7 (made fresh) (10 minutes), 1 X PBS (5 minutes).
The sections were later treated with acetic anhydride to reduce non-specific binding of the probe (0.1 M triethanolamine (pH 8, made fresh) and acetic anhydride for 10 minutes with stirring) and dehydrated (1X PBS 5 minutes, twice; 40% ethanol 30 seconds, 70% ethanol 30 seconds, 90 ethanol 30 seconds and 100% ethanol 30 seconds, twice.). Before the hybridisation the sections were dried at room temperature.
Hybridization: Slides were sandwiched together as pairs. Probe was applied to each individual pair (Hybridization solution: 100µl 10X in situ salts, 400µl deionized formamide, 200µl 50% dextran sulfate, 20µl 50 X Denhardt’s, 10µl 100 mg/ml tRNA, 70µl DEPC-treated water, total volume: 800µl). For each pair of slides, the desired amount of probe (about 200µl) should was added to 50% formamide so that the volume was 40 µl. Heated this solution to 80°C for 2 minutes. Ice, spinned down and keeped on ice. Added 160µl of hybridization solution to the 40µl of probe solution. Mixed without generating air bubbles. Applied probe/hybridization solution mixture to slides (200 µl) Elevated the slide sandwiches above wet paper towels in an airtight plastic container. Plastic serological pipettes was used to raise the slides above the paper towels. The container was placed at 55°C overnight. The next day slides washed with: 0.2X SS(1hour at 55°C, twice), NTE (10 minutes. at 37°C, twice), RNAse (20 micrograms/ml in NTE) at 37°C 30 minutes, NTE (10 minutes. at 37°C, twice), 0.2X SSC (30 minutes at 37°C, twice), 1 X PBS 5 minutes at room temperature, 45 minutes with 1% Boehringer block (cat. #1096176) (made fresh following manufacturers directions) in 100 mM Tris, pH 7.5, 150 mM
(Boehringer, cat#1093274) was diluted 1 to 1250 in the BSA/Tris/Triton solution. Maked slide “sandwiches”. Dip the long edge of the sandwich in the antibody solution and allow capillary action to pull the solution up into the gap between the slides and allowed to sit at room temperature for 2 hours. The slides were washed at room temperature, 15 minutes each time, with the BSA/Tris/NaCl/Triton solution, ten minutes with 100mM Tris (pH 9.5) 100 mM NaCl and 50 mM MgCl2. Then dipped each slide in fresh Tris 9.5/NaCl/MgCl2 solution to ensure that all of the detergent is washed off. Pour 10 ml of the NBT/BCIP premix (Western Blue from Promega, cat #S3841) into a plastic weigh boat. Sandwich slides together, placed edge of sandwich in solution and allowed capillary action to draw the solution up between the slides. Drain on a Kimwipe and repeated. The slides were placed in an airtight plastic container and stored in total darkness for three days. When sufficient signal had developed the slideswere put into a bath of TE to separate them and to stop the reaction. The sections were then dehydrate in a ethanol series.
