47
RESULTS
3.1 Construction of SSH libraries and identification of over-expressed genes
It was showed in previous works that in the standard experimental conditions (ten µl droplets of CP 1.5 x 10
-4M applied to the lower leaf surface) CP protein concentration diminished from droplets 48 hours after treatment and caused a rapid accumulation of phytoalexins together with a contemporaneous restriction of the mycelial growth following Cfp conidia addition. CP was still active at the concentration of a little more than 10
-5M, and the inhibition of the Cfp growth was effective at least until to 72 hours after treatment (Fig.1, 2 and 3) (Carresi et al., 2006).
In consequence of this results we have chosen to use tester and driver cDNA in SSH technique obtained from the mRNA from leaves treated with CP 1.5 x 10
-4M for 48 hours and with sterile water for the same time, as described by the manufacturing.
0 1 2 3
0.0 0.5 1.0 1.5
0 1 2 3 4
Time (d) after treatment
Residual CP concentration in 10µµµµl droplets added to the leaves, x 10-4 M(••••) Umbelliferone equivalents
nmol ml-1(o)
Fig. 1 Time course of relationships between disappearance of CP from 10 µl droplets added
to plane leaves and production of umbelliferone equivalents. Values are the means of 8 data
from two independent experiments ± SEM (Carresi et al., 2006)
48
Fig. 2 Growth of Ceratocystis fimbriata f. sp. platani on plane leaves 48 hours after the pre- treatment with ten µl droplets of CP at 10 µl droplets of CP 15 x 10
-5M (B) and control treated with sterile water (A) (Carresi et al., 2006).
Fig. 3 Growth of Ceratocystis fimbriata f. sp. platani on plane leaves 48 hours after the pre-treatment with ten µl droplets of CP at various concentration values. Data were collected two days after the addition of the fungal conidia (Carresi et al., 2006).
The ligation efficiency was tested by PCR analysis to verify that at least the 25% of the cDNAs have the adaptors on both ends, this condition guarantee a perfect subtraction efficiency. The experiment was performed as described in the PCR-Select cDNA Subtraction Kit User Manual from Clontech.
A B
49 The PCR product using one gene specific primer and the PCR Primer 1 should have the same intensity of the one obtained using two gene specific primers.
If the band intensity differ by more than 4 fold, the ligation was less than 25%
reducing the subtraction efficiency.
SSH was performed using as tester mRNA isolated from treated leaves and as driver mRNA isolate from control leaves, and the contrary. In this way they have been obtained: the forward library, containing products of activated or increase genes by CP treatment and the reverse library established from genes whose transcription is suppressed or diminished under CP treatment. The forward and reverse libraries showed several bands with a differential complexity (ranging from 300 bp to 1200 bp) (Fig.4).
The efficiency of subtraction was evaluated by PCR amplification using 5,8S ribosomal gene, if the subtraction is efficiency the transcripts detection should be reduced. The 5.8 S gene is detectable as faint band in the unsubtracted sample (T–C) only after 8 amplification cycles of PCR, while in the unsubtracted (C-T) sample after 15 cycles. No amplification was detected in subtracted libraries at the same number of PCR cycles.(Fig.5). After this evidence, the forward and reverse cDNAs were cloned and about 1600 independent positive clones were picked out from forward and reverse subtractive libraries. Subtracted cDNAs may contain some common sequences or to have the similar level in both samples; to exceed this difficulty we have used the PCR-select cDNA subtracting, a powerful technique that permits to identify differentially expressed genes.
We have performed a cDNA differential screening to eliminate the false positive clones and to have a primary information about the relative expression levels of the cDNA cloned.
Using forward and reverse subtracted cDNA probes, obtained by labelling with
the DIG-DNA Labelling Kit Nonradioactive (Roche), we can bypasses the
problem of losing low abundance sequences. Only the clones representing the
sequences truly differential expressed will hybridise only with forward cDNA
subtracted probe, the clones, that hybridise with the reverse cDNA subtracted
probe, will be consider background. Before to proceed with the hybridisation it
50 was necessary to remove the adaptors from the probes through a restriction enzyme to avoid a high background. The screening of the positive clones was carried out by colony PCR, using as primers the same nested primers used for the libraries amplification. Two identical blots were created by spotting PCR products onto positively charged nylon membrane and hybridised with cDNAs derived from forward and reverse subtracted libraries (Fig.6). Differentially expressed genes were determinate when signals were detected or were more intense in the forward-subtract pool than reverse subtracted one. We have also controlled the clones chosen with the previous method in the colony PCR gel to verify the presence of a single band avoiding the problem of the double or multiple clones. We analysed sequences from the forward library
Fig.4 SSH libraries: M= marker IX; R= reverse; RU= unsubtracted reverse;
F= foward; FU=unsubtracted foward
M R R U
F F
U
51 Fig.5 The efficiency of subtraction. It was evaluated by PCR amplification of the 5.8 S rRNA gene.
The 5.8 S gene is detectable as faint band in the unsubtracted sample (T–C) only after 8 amplification cycles of PCR, while in the unsubtracted (C-T) sample after 15 cycles.
M 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
5 cycles 8 cycles 11 cycles 15 cycles
Figure 7. PCR analysis of the amplification of the 5,8S RNA.
1- Library T-C; 2- unsustracted T-C;
3- Library C-T; 4- unsubtracted C-T
Fig.6 PCR products from the clones of the forward- subtracted library after hybridisation with cDNAs derived from reverse–subtracted library (A) and with cDNAs derived from forward–subtracted library (B).
A
B
52 sequencing 78 differential clones selected from among 576 clones screened as describe above. Seventy two putative, differentially expressed genes were identified based on through FASTA, BLASTN and BLASTX programs, plus six showing unknown function (Table 1). The Relative PCR analysis of 6 selected clones confirmed that they were over-expressed in plane leaves treated with CP for 48 hours (Fig. 8). The clones were the following: the Chlorophyll a/b-binding protein for the PSII (L2B clone), the selenium binding protein (L1B clone), tubby like proteins (Q7H clone), the RARI protein (L1A clone), EF-1 alpha (Q11H clone) and Ferredoxin (L2D clone).
In this study the primers for the studied gene were used together with the ones for the housekeeping gene. The internal control expression must be the same in all the analysed samples and to be used to normalise obtained data. Among the commonly used internal control genes (ß-actin, GAPDH and 18S rRNA) 18S rRNA was chosen because the other genes are up regulated in the same experimental conditions (not shown data).
Table 1.
Clone Accession no.
Accession no.
of mactching sequence
E valueb
Putative identificationc
DNA/RNA synthesis and metabolism
L1C AM260493 AAM19887.1 1e-79 Histone deacetylase R1B AM293618 BAA97391 1e-142 DEAD box helicase protein A10D AM260513 AAM61323 1e-81 Nucleotide sugar epimerase
C3D AM286242 AAF22455 1e-11 MADS box protein
C3F AM286254 NP_197031 1e-81 Putative nucleic acid binding protein L4A AM293609 NP_193524 2e-87 Zinc-binding family protein R5C AM260507 CAB75429 1e-117 Oligouridylate binding protein.
R7A AM260511 U9SA34 1e-136 Inosine monophosphate dehydrogenase L9B AM293617 BAB83610 5e-09 Putative transcription factor Q1E AM397239 AAF64454 3e-45 DnaJ-like protein Q1A AM397240 ABE87461 9e-05 Putative transcription factor Protein synthesis/turnover
F1G AM260500 AAG51475 5e-74 Serine carboxypeptidase II F5G AM293611 CAB81384 3e-17 Putative ribosomal protein S10 L4F AM286233 AAM64318 6e-70 Plastid ribosomal protein PRPL5 L5A AM260506 XP_468840 2e-61 Hydrolase
Q11H AM293616 AAC39447 1e-100 Elongation factor 1 alpha A5H AM260517 CAI11456 1e-45 Glycosyltransferase
C3C AM286146 AAK15322 2e-106 FtsH protease, chloroplast precursor C6G AM286248 ABD66517 5e-122 Translation elongation factor 1 alpha R4B AM260495 NP_198638.2 3e-57 Catalytic protein with α/β hydrolase domain R1F AM260496 AAZ43369.1 1e-96 Alanine aminotransferase
F10F AM260499 BAD81372 7e-168 Histidinol dehydrogenase R2F AM260502 S30145 9e-46 Ketol-acid reductoisomerase
Q6G AM293614 NP_564815 7e-61 D-alanyl-D-alanine endopeptidase/ peptidase C6F AM397234 Q8L7H3 2e-05 Putative xyloglucanendotransglucosylase/hydrolase Energy
L2B AM286231 AATO8647 1e-117 Chloroplast chlorophyll a/b-binding protein, PSII F10C AM286234 CAA45523 2e-67 Chloroplast chlorophyll a/b binding protein, PS I L2D AM286232 P09911 4e-25 Ferredoxin A
Q1B AM293623 X60008 2e-42 Photosystem I reaction center subunit II Q2D AM293608 BAB71853 8e-30 Phosphoenolpiruvate carboxylase kinase C5G AM286245 AAW21667 4e-33 Ribulose-biphosphate carboxylase A4H AM260516 AAM6833 5e-54 Phospho-glycerate dehydrogenase
53
Cellular metabolism
A11H AM293622 AY162465 1e-122 Glutamine synthetase F4C AM286255 CAA63981 7e-108 Cytosolic Glutamine synthetase A11A AM260515 AAL69511 3e-85 AMP-binding protein
Q2B AM260498 T52308 7e-61 Fatty acid condensing enzyme CUT1 F9E AM286237 AAL49750 2e-28 Aquaporin
L5B AM293612 CAA04451 1e-55 Potassium channel beta subunit F9A AM293607 T07808 2e-91 Inorganic phosphate transporter C6H AM286250 CAC84545 4e-93 Dicarboxylate/tricarboxylate carrier F9B AM260510 BAA88226.1 1e-66 Thiamine biosynthetic enzyme F4F AM293615 XM_479463 2e-140 Arginine N-methyltransferase protein L1B AM260504 CAC65501 2e-92 Selenium binding protein L5G AM260508 AAS17751 5e-105 Beta-xylosidase Q4G and Q7H AM2604505 AAM20254 1e-102 TUBBY-like proteins (TULPs) R12D AM260497 XT479475.1 3e-97 TGF-beta inducible protein F4D AM293613 AAK11299 5e-88 ADPglucose pyrophosphorylase Signalling
C7H AM286251 CAA54803 5e-47 Shaggy/glycogen synthase kinase-3 like prote Defence and/or stress related proteins
L3A AM286235 AAM62409 1e-54 Rar1 protein L8B AM260509 AAS44667 1e-80 β-1,3 glucanase R1C AM293619 AAM44961* 9e-15 Thaumatin protein
A11B AM286253 NP_849614 5e-33 Aldehyde lyase (threonine aldolase activity) A12D AM293606 AAK72616 2e-59 Actin-depolymerizing factor
C4G AM286244 CAB51533 8e-32 Galactinol synthase C5H AM286249 AAL27855 3e-40 Lipid transfer protein A6H AM260514 AAB81996 6e-04 translation initiation factor eIF-1A C3E AM286243 AAS80139 3e-17 Arachidonic acid-induced DEA1-like A9B AM286239 XP_470175 1e-19 Putative ubiquitin protein
L1E AM260494 AAV31238 4e-110 26S proteasome non-ATPase regulatory subunit 1 A12B AM286240 AAC24708 1e-40 Ribulose-phosphate 3-epimerase
C8H AM286252 AAOO33154 9e-121 Transketolase
C7G AM293605 CAB61246 1e-77 Putative serine/threonine kinase L8A AM260492 O22432 1.7e-25 Putative cell wall protein (glycine-rich protein) Other proteins
C6E AM397241 putative Ty1-copia reverse transcriptase and partial DfRedu pseudogene A10C AM286247 CAB79665 2e-56 Hypothetical protein
A12F AM286241 BAB02703 1e-103 Hypothetical protein F3G AM286238 AAU44392 2e-58 Hypothetical protein FC9 CAL25343 AAC79135 1e-06 Hypothetical protein Q8E CAL25354 AY057515 8e-29 Hypothetical protein L1A AM286236 AAL76137 1e-36 Hypothetical protein L4G AM260503 XP_464048 7e-35 Hypothetical protein
F3F AM397237 EST
F3H AM397238 EST
L4B AM397236 EST
F9F AM293624 EST
C7F AM397235 EST
F9H AM397242 EST
* BLASTP
a) Accession number and source of the best match sequence.
b) E value of best match sequence, calculated by BLAST analysis.
c) Functional assignment based on sequence similarity.
L2D clone
Q11H clone
18S
18S L2D clone
Q11H clone
18S
18S
L1B clone 18S
18S L2D clone
L1B clone 18S
18S L2D clone
18S
18S L3A clone
Q7H clone
18S
18S L3A clone
Q7H clone
Fig.7 The Relative PCR analysis of 6 selected clones.
54 We have also analysed some clones by relative PCR using total RNA extracted from treated leaves with fungus conidia and cerato platanin for 6, 24 and 48 hours to investigate possible differences in the genes expression between these systems (cerato platanin-plant and fungus-plant).
In the cerato platanin treatments we have observed a significant increment for all clones at 48 hours confirming the SSH results; from the time course we have observed a significant increment only for the Q7H clone at 24 hours.
In the fungus treatments we have observed a significant increment at 48 hours for L1B and L2B clones and at 6 hours for Q7H and L3A clones (Fig.8 and Tab.2).
M 6T 6C 24T 24C 48T 48C M 6T 6C 24T 24C 48T 48C
18S Q7H
18S Q7H 18S
L1B
18S L1B 18S
L2B
18S L2B 18S
L3A
18S L3A
Fig.8 Time-course expression in the CP and fungus treated leaves after 6, 24 and 48
hours using relative PCR. The relative expression levels of each analysed transcripts
were evidenced with respect to the 18S products. C, controls; T, treated leaves.
55 Many of the over-expressed clones could be classified into more than one category defined on the basis of the metabolic processes they are associated with. Thus, the up-regulated clones listed in Table 1 have been classified in 6 macro putative groups taking in account the functional categories established for Arabidopsis (T.A.G. Initiative, 2000). The macro-groups concerned defence and/or stress related proteins (19.2%), proteins involved in protein synthesis/turnover (18.0%), DNA/RNA synthesis and metabolism(14.1%), cell primary metabolism (19.2%), energy (9.0%) and signalling pathways (1.3%).
Clones machting with genes encoding hypothetical proteins and EST were included in the group “Other proteins”.
The percentage of the over-transcribed genes involved in defense responses and signalling was significantly high and contained genes known to be involved in resistance reaction in many plant-microbe interactions. This group concerned genes encoding, for example, the Rar1 protein, a Beta 1-3 glucanase, the thaumatin, a lipid transfer protein translation initiation factor eIF-1A and a serine/threonine kinase.
* n.s.
n.s.
* n.s.
n.s.
Chlorophyll a/b binding proteins L2B
n.s.
n.s.
*
* n.s.
n.s.
RAR1 L3A
* n.s.
n.s.
* n.s.
n.s.
Selenium binding protein L1B
n.s.
n.s.
*
* n.s.
n.s.
Tubby Q7H
48h 24h 6h
48h 24h 6h
Fungus CP
Putative identification Clone
Table 2. Schematic representation of the statistical data. The analyse of the data is carried out using Student’s t-test (p< 0.05).
* significant data; n.s. not significant data
56 A significant presence of genes is involved in the DNA synthesis (histone deacetylase, DEAD box helicase and various transcription factors), in RNA processing (oligouridylate binding protein) and in protein synthesis/turnover (Putative ribosomal protein S10, Elongation factor 1 alpha, FtsH protease, chloroplast precursor, Catalytic protein with α/β hydrolase domain).
Importantly, the primary metabolism pathways are involved in the response of plane leaves to CP, like photosynthesis (ribulose-biphosphate carboxylase, chloroplast chlorophyll a/b-binding protein, PSII and PSI and photosystem I reaction center subunit II), nitrogen metabolism (glutamine synthetase and cytosolic glutamine synthetase), pentose phosphate pathway (ribulose- phosphate 3-epimerase and transketolase), lipid metabolism (AMP-binding protein and fatty acid condensing enzyme CUT1) and amid synthesis (ADPglucose pyrophosphorylase, a key enzyme in its synthesis).
3.2 Clones with complete CDS sequence.
During the screening some clones (L4A, A12D, C5H, L8A) contained the complete CDS of the following genes: putative zinc-binding protein, actin- depolymerizing factor, lipid transfer protein and cell-wall protein, respectively.
L4A clone (accession number:) shows two domain: DUF59 and zf-B_box (Fig.9).
DUF597 is a conserved region in several uncharacterised plant proteins and it is unknown its function. zf-B_box is found essentially in transcription factors, ribonucleoproteins but no function is clearly assigned to this domain. Among the seven possible ligands for the zinc atom contained in a B-box, only four are used and bind one zinc atom in a Cys2-His2 tetrahedral arrangement (showed in blue characters in Fig.9).
A12D clone (accession number:) shows ADF/cofilins domain (Fig.9).
ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic
cells so far examined. Members of this family remodel the actin cytoskeleton,
for example during cytokinesis, when the actin-rich contractile ring shrinks as
it contracts through the interaction of ADF/cofilins with both monomeric and
57 filamentous actin. ADF/cofilins sever actin filaments (F-actin) and/or bind to actin monomers, or G-actin, thus preventing actin-polymerization by sequestering the monomers.
C5H clone (accession number:) shows the plant lipid transfer protein/seed storage/trypsin-alpha amylase inhibitor domain (Fig.9).
This domain has been found in several proteins, including plant lipid transfer proteins, seed storage proteins and trypsin-alpha amylase inhibitors The domain forms a four-helical bundle in a right-handed superhelix with a folded leaf topology, which is stabilised by disulphide bonds, and which has an internal cavity. Plant cells contain proteins, called lipid transfer proteins (LTP) transfer phospholipids, glycolipids, fatty acids and sterols from liposomes or microsomes to mitochondria. These proteins, whose subcellular location is not yet known, could play a major role in membrane biogenesis by conveying phospholipids such as waxes or cutin from their site of biosynthesis to membranes unable to form these lipids. The eight cysteines are a plant characteristic (showed in blue characters in Fig.9).
L8A clone (accession number:) shows glycine and serine rich-regions.
Fig. 9. They are showed the complete protein sequence with the domains.
L4A (Accession N° …………):
MAIDNQEPTVKEIKPKSRRIMGAGGPEDEDNKWPPWLSPLLQTRFFVQCKFHADSHKCECNMYCLDCMNGALCSLCLSYHKDHRAIQIRRSSYHDVIRVS EIQKVLDISGVQTYVINSARVVFLNERPQPRPGKGVTNTCEVCERSLLDSFRFCSLGCKIVGTSKNYQKKKRSQATTSDSEESYSGRSLASEKIKVQSFTPSTPPPTVVNY
PHRAPMGGLIIEF
Two domains are present: DUF597 (red characters) and zf-B_box (yellow-highlighted).
A12D (Accession N° …………):
MANAASGIAVHDDCKLKFLELKVKRTYRFIVFKIEDKQKQVVVEKVGEPTQSYEDFSASLPADECRYAVYDFDFVTAENVQKSRIFFIAWSPDTSRVRSK MIYASSKDRFKRELDGIQVELQATDPTEMGLDVIRSRAS
The ADF/cofilins domain is present (blue-highlighted).
C5H (Accession N° …………):
MAFSRVAKLACLLLACMVATAPHAEAAITCGTVVTRLTPCLTFLRSGGAVAPACCNGVKALNNDAKTTPDRQAACGCLKTASTSISGIQLGNAASLAGKC GVNLPYKISPTIDCSKVK
Plant lipid transfer protein/seed storage/trypsin-alpha amylase inhibitor domain (violet characters).
L8A (Accession N° …………):
MARLKSVTLLALLVAVFAIVAESRVARKDLSLDLGGGLGVGVGAGIGLGLGGGSGSASGSGSGSGSGSGSGSGSGSGAGSGAGSYAGSGAGSGSGHGQGQ GAGSGSGRGQGEGSGYGSGSGHGEGYGEGSGTGRGSGSGYGEGSGYGSGYGSGHGK
This protein shows glycine and serine rich regions.
