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Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines.
Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression.
This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth, development and response to Xf infection.
Plant cytochrome P450 monooxygenases are a group of redox proteins that catalyze various oxidative reactions . It is proposed that cytochrome P450 monooxygenases mediate synthesis and metabolism of many physiologically important compounds. Steroids, fatty acids, lignins, terpenes, alkaloids, phenylpropanoids, and phytoalexins are examples of these primary and secondary compounds that act as plant defense agents against a range of diverse pathogenic microbes and insect pests [2–4]. Cytochrome P450 enzymes are also involved in several biosynthesis pathways in leaf tissues of Arabidopsis and function as part of the highly sophisticated network of plant defense reactions . These defense responses include the hypersensitive response  and inhibition of growth of particular plant pathogens . Gene expression analysis has revealed that most cytochrome P450 genes are strictly regulated in response to phytohormones (salicylic acid, jasmonic acid, ethylene and abscisic acid), pathogens (necrotrophic fungal pathogens Alternaria alternata and A. brassicicola), UV damage, heavy metal toxicity, mechanical injury, drought, high salinity and low temperatures . Therefore, cytochrome P450 genes may be involved in plant defense responses to abiotic and biotic stresses. How cytochrome P450 genes are regulated in response to these stresses, especially to bacterial infection, is not understood.
The most widely cultivated grape species is Vitis vinifera. These grapevines are highly susceptible to many pests and pathogens, including Xylella fastidiosa (Xf), the bacterium responsible for Pierce's disease (PD). Xf is transmitted by xylem-feeding sharpshooters. PD has been an important but localized disease in California for over 100 years. Recently, the introduction of the glassy-winged sharpshooter vector into California resulted in new and range expanding epidemics of PD . PD susceptible grapevines infected with Xf exhibit inhibited periderm development in stems (green islands), leaf blade separation from the petiole (matchsticks), irregular leaf scorching, fruit cluster dehydration, stunting and eventual plant death [10–12]. While cultivars of V. vinefera are susceptible to PD, several species of grapevine are resistant. A breeding program based on resistance from V. arizonica has been developed and led to genetic mapping of a single locus for resistance (PdR1) in the 9621 population [11, 13]. This population is a cross of two half-sib genotypes, D8909-15 (V. rupestris 'A. de Serres × V. arizonica b42-26) × F8909-17 (V. rupestris 'A. de Serres' × V. arizonica/candicans b43-17) . Two progeny from this population, one resistant (9621-67) and the other susceptible (9621-94), were characterized for transcriptomic profiles , which implicated involvement of cytochrome P450 genes in a defense response to Xf infection http://cropdisease.ars.usda.gov/vitis_at/main-page.htm.
To further characterize how cytochrome P450 monooxygenase genes are regulated in response to Xf infection, the genotypes 9621-67 and 9621-94 were used to study temporal and spatial expression and transcriptional regulation in inoculated greenhouse-grown plants. This report characterizes the structure, expression and transcript maturation of a cytochrome P450 monooxygenase encoded by the CYP736 gene.
Genomic DNA sequences of grape cytochrome P450 monooxygenase genes cloned from PD- resistant and susceptible grapevines using the PCR primer P450F1 and P450R1 (Table 1) were 2,927 bp (Accession FJ620897) and 2,892 bp (Accession FJ620898), respectively. The cDNA sequences of grape cytochrome P450 monooxygenase genes cloned from leaf tissues of control PD-resistant and -susceptible grapevines using the PCR primers P450F1 and P450R1 (Table 1) were 1488 bp (Accessions FJ828517 and FJ828518). BLAST analysis of grapevine genomic DNA sequences revealed that the grape P450 monooxygenase gene has three homologous copies (CYP736A, CYP736B and CYP736C) in tandem on grape chromosome 7 (Figure 1). These three copies share 89%, 98% and 78% nucleotide sequence identity, respectively, compared to the closest grape genomic DNA contig sequence (Accession AM475392.1). The grape P450 monooxygenase genes we cloned from PD-resistant and susceptible grapevines were CYP736B genes that shared 93.6%, 99%, 98.6%, 90.3% genomic DNA sequence identity with each other in the 5' untranslated, exon, intron, and 3' untranslated regions (UTR), respectively. The grape P450 monooxygenase CYP736B genes cloned from PD- resistant and -susceptible grapevines have the same structure. Main features of these two CYP736B genes are: two exons, one intron with GT as 5'splice site and AG as 3' splice site. Compared to the CYP736B gene sequence from the PD-resistant grapevine, there are three C to T substitutions and one insertion (CTAT) in the upstream UTR region (1000 bp), one A to G and one T to C substitution in the exon 1 region, one G to A, one T to G and one C to G substitution in the intron region, one C to A, one A to C, one C to T, and one T to C substitution and a large deletion (39 bp) in the downstream non-coding region (300 bp) of the CYP736B gene from the PD-susceptible grapevine. DNA motif analysis indicated that there are nine major transcriptional regulatory motifs in the upstream non-coding region of the CYP736B gene, including one TATA box, five CAAT box, one I box, one G box, and one W box (Figure 2).
Genomic organization and structure of cytochrome P450 CYP736 genes in grapevines. There are three cytochrome P450 genes, CYP736A, CYP736B and CYP736C, flanked on the left with a RE-LTR gene on grape chromosome 7. Numbers on the top indicate order of exons, and the arrow indicates direction of transcription. Exon size and distance between genes and exons were drawn to approximate scale. The DNA coding sequence identity of each CYP736 gene derived from a large genomic contig CU459237.1 (4,713,370 bp) was compared with the target CYP736B gene cloned from PD- resistant 9621-67 grape selection and is shown below each gene member. The directions of PCR primers are shown in arrows at specific locations.
Molecular structure and functional regulatory domains of grape CYP736B. A representative genomic DNA sequence from the PD- resistant selection 9621-67 was used as an example. The 5' upstream region, intron and 3' down-stream region are in lower case, exons are in upper case. Several regulatory elements were boxed with highlight in yellow in the 5'UTR-promoter region. The DNA fragment in red within the 5'UTR-promoter region was not present in PD- susceptible selection 9621-94. The transcription initiation regions are highlighted with red and the specific transcription start sites (TSS1, TSS2, and TSS3) in green are indicated with stem-arrows to indicate the direction of transcription. Three Poly(A) signal motifs were boxed with highlight in yellow and specific poly(A) sites, poly(A)1, poly(A)2 and poly(A)3, were highlighted in green and indicated by vertical arrows at 3'UTR. The DNA fragment in red at 3'UTR was not present in PD- resistant plants. The translation start codon was marked in bold and stop codon was marked with star (*). The donor site (gt) and receptor site (ag) for pre-mRNA splicing are marked in blue.
Expression of CYP736B in stem and leaf tissues of both PD-resistant and -susceptible grapevines was detected at different stages of growth and disease development in response to Xf infection using a pair of specific primers P450F1 and P450R1 (Table1) for the CYP736B coding region. Quantitative Real-time PCR analysis results showed that expression of CYP736B was much higher in leaf tissues than in stem tissues of both PD-resistant and -susceptible grapevines, ranging from 30 - 138.6 fold greater depending on growth stage (Table 2). In stem tissues of control PD-resistant plants, CYP736B expression at weeks 1 and 10 was similar but slightly lower at week 6 when expression of CYP736B was elevated by a factor of 1.3 (p < 0.05). In contrast, expression of CYP736B was increased by factors of 5.3 (p < 0.01) and 2.9 (p < 0.01) in stem tissues of the control PD-susceptible grapevine at week 6 and week 10. However, Xf infection did not result in a significant change in CYP736B expression in stem tissues of the PD-susceptible grapevine at week 1. Infection did cause a significant decrease of CYP736B expression in stem tissues of PD-susceptible grapevine to 4.08-fold less (p < 0.01) and 1.71-fold less (p < 0.05) at weeks 6 and 10, respectively. Interestingly, CYP736B expression was elevated up to 112.9-fold and 74.9-fold in leaf tissues relative to stem tissues of both control PD-resistant and -susceptible grapevines at week 1 post-inoculation, respectively, but down-regulated in leaf tissues with progression of plant growth under normal conditions without Xf infection. For example, CYP736B expression was decreased by 1.31-fold (p < 0.01) and 2.29-fold (p < 0.01) at weeks 6 and 10 in leaf tissues of the PD-resistant grapevine, whereas CYP736B expression decreased by 1.79-fold (p < 0.01) and 2.53-fold (p < 0.01) at weeks 6 and 10 in leaf tissues of the PD-susceptible grapevine. However, Xf infection resulted in increase of CYP736B expression up to 1.6-fold (P < 0.01) in leaf tissues of the PD-resistant grapevine and a decrease of CYP736B expression by 2.11-fold (P < 0.01) in leaf tissues of the PD-susceptible grapevine.
The cDNA sequences of CYP736B transcripts from leaf samples of both PD-resistant and -susceptible genotypes with or without Xf infection were determined. The results indicated that amino acid sequences encoded by CYP736B transcripts were highly similar (99% identical) within each PD-resistant and -susceptible sampling group and between the two groups, suggesting that involvement of CYP736B in the defense response against Xf infection could be regulated at transcriptional and posttranscriptional levels.
The cDNA sequences of CYP736B transcripts from leaf and stem tissue of both PD-resistant and -susceptible grapevines at different stages of growth and development with or without Xf infection were cloned using CYP736B coding region specific primers P450F1 and P450R1. These cDNA sequences were further examined to determine if gene structure and/or posttranscriptional processing patterns changed over time and tissues. The results showed that CYP736B transcripts were spliced with GT as a donor site and AG as an acceptor site within the intron sequence of CYP736B pre-mRNAs in both PD-resistant and -susceptible samples (Figure 2). To study the dynamic pattern of pre-mRNA splicing of CYP736B, we designed a pair of Real-time PCR primers, SYBR-P450F2 and SYBR-P450R2 (Table 1), for quantitative analysis of the relative frequency of unspliced CYP736B transcripts in leaf tissues during growth and development in response to Xf infection. As shown in Table 3, the relative frequency of unspliced CYP736B transcripts was 3 to 4-fold less in leaf tissues of control PD-resistant and -susceptible grapevines at week 1 when genomic DNA was used as a control reference for Real-time Q-PCR analysis. The results indicate that most CYP736B pre-mRNA transcripts were correctly spliced in leaf tissues of both control PD-resistant and -susceptible grapevines at week 1 in plants not inoculated with Xf. In comparison, relative frequency of unspliced CYP736B transcripts was significantly increased up to 16.22- and 4.51-fold greater in leaf tissues of control PD- resistant plants at week 6 and 10, respectively. In contrast, relative frequency of unspliced CYP736B transcripts was as low as 1.18- and 2.21-fold less in leaf tissues of control PD-susceptible plants at week 6 and 10, respectively. However, when the leaf tissues of the PD- resistant grapevines was inoculated the relative frequency of unspliced CYP736B transcripts decreased to as low as 1.79- and 1.95-fold at week 1 and 6 and then increased to 3.59-fold greater at week 10. However, when leaf tissues of the PD- susceptible grapevines were inoculated, the relative frequency of unspliced CYP736B transcripts increased to 2.73- and 358.95-fold greater at week 1 and 6, respectively, and then decreased to 1.23-fold less at week 10. Representative cDNA sequencing confirmed that both spliced and unspliced CYP736B transcripts were generated in both PD-resistant and -susceptible grapevines irrespective of Xf infection (data not shown).
Genomic DNA was used as a reference for unspliced/spliced transcript analysis.
Due to lower levels of CYP736B expression in stem tissues at all growth stages analyzed and the most significant change of CYP736B gene expression in leaf tissues at week 6, the 5'RACE method was used to determine transcription initiation sites in leaf tissues of both PD-resistant and -susceptible grapevines at week 1 and 6 under inoculated and uninoculated conditions. As shown in Figure 3a, there were 1 - 3 major cDNA bands generated from leaf RNA templates from both PD-resistant and -susceptible grapevines at week 1 and week 6, with or without Xf inoculation. To elucidate the nature of these major multiple 5'RACE cDNA bands, we combined promoter and transcription start site prediction and experimental verification methods. Bioinformatics-aided promoter prediction revealed that there were potentially three major transcription start sites (Figure 2): TSS1 (-290 to -240), TSS2 (-136 to -86), and TSS3 (-69 to -19). 5'RACE cDNA cloning and sequencing analysis revealed that all of predicted transcription start sites were found in CYP736B transcripts as we predicted from the agarose gel analysis of the 3'RACE-amplified PCR products (Figure 3). We further quantified the relative abundance of major transcription start site usage using Real-time PCR analysis with a pair of primers SYBR-P450F1 and SYBR-P450R1. As summarized in Table 4, usage frequency of both TSS2 and TSS3 varied, from 0.66- to 9.20-fold for TSS2 and from 0.35- to 8.67-fold for TSS3, respectively, in leaf tissues of both PD -resistant and susceptible grapevines at different stages of growth without Xf infection. Compared to control plants, however, usage of TSS2 increased significantly in leaf tissues of resistant plants at week 1 (17.65-fold greater than susceptible control plants, 26.74-fold greater than resistant control plants, n = 3, p = 0.001854) and at the growth stage week 6 (37.12-fold greater than susceptible control plants, 4.03-fold greater than resistant control plants, n = 3, p = 0.017017) after Xf inoculation. In contrast, usage frequency of TSS2 increased significantly after Xf inoculation at week 1 (2.22-fold greater than control plants, n = 3, p = 0.003532) in leaf tissues of susceptible plants, but remained unchanged (1.42- and 1.56-fold for control and Xf infected plants, respectively, compared to the susceptible control plant for Xf infection at week 1, n = 3, p = 0.354589) at week 6 after Xf inoculation. Similarly, usage frequency of TSS3 remained unchanged at growth stage week 1 after Xf inoculation (0.62- and 0.50-fold, respectively compared to the susceptible control plants, n = 3, p = 0.204438), but greatly increased at the growth stage week 6 after Xf inoculation (8.67-fold higher than susceptible control plants, 24.77 fold higher than resistant control plants, n = 3, p = 0.000483) in leaf tissues of resistant plants. Usage frequencies of TSS3 were low in leaf tissues of susceptible plants at week 1 (0.4-fold compared to control plants, n = 3, p = 0.000203) and week 6 (0.23-fold compared to control plants, n = 3, p = 0.017818) after Xf inoculation.
A. 5'RACE display of CYP736B transcripts in both PD- resistant and susceptible grapevines in response to Xf infection. B. 3'RACE display of CYP736B transcripts in both PD- resistant and susceptible grapevines in response to Xf infection. Names of grapevine genotype, sampling time are shown on the top, molecular sizes are shown on the left.
** Significant level at P < 0.01; * Significant level at P < 0.05.
Since transcription termination involves polyadenylation, the 3'termini of CYP736B transcripts were cloned using 3'RACE method to determine termination and poly(A) signals of CYP736B. Several major cDNA bands with different intensities were obtained from leaf tissues of both PD- resistant and susceptible grapevines at week 1 and week 6 (Figure 4). To elucidate the nature of these major 3'RACE cDNA bands, we isolated and cloned each major 3'RACE cDNA. Sequence analysis revealed that there were three major polyadenylation sites, as predicted from the bioinformatics-aided polyadnylation analysis (Figure 2): poly(A)1 (++53 to ++58, AATAAT), poly(A)2 (++153 to ++159, AATTAAA), and poly(A)3 (++238 to ++245, AATAAA).
Relationship between positions of 5'TSS/3'Poly(A) sites and CYP736B pre-mRNA splicing and the effect of transcription initiation and termination/polyadenylation on pre-mRNA splicing of CYP736B genes in leaf tissues of grapevines. Three 5'TSS sites (left three) and three 3'Poly(A) sites (right three) are shown at the bottom, splicing frequency (column in blue) or unsplicing frequency (column in red) of CYP736B transcripts corresponding to each 5'TSS site and 3'Poly(A) site are shown on the left.
Based on results of 3'RACE cDNA display (Figure 4), it appears that Poly(A)1 was the major polyadenylation site utilized for CYP736B transcripts under all conditions tested, while Poly(A)2 and Poly(A)3 were less frequently used as alternative sites and subject to change upon plant growth and Xf inoculation. To quantify relative levels of representative 3'RACE cDNAs, usage frequency of CYP736B transcripts with Poly(A)2 was determined using Real-time PCR. In leaf tissues of PD- resistant plants after Xf inoculation, total usage frequency of Poly(A)2 site was increased 12.38-fold at week 1 (n = 3, p = 0.000057) but decreased 2.75-fold at week 6 (n = 3, p = 0.005304) when compared to respective control plants. However, usage frequency of Poly(A)2 in leaf tissues of susceptible plants was not significantly increased (1.28-fold greater than control, n = 3, p = 0.075134) upon Xf infection at week 1, and at week 6 (1.12-fold greater than control, n = 3, p = 0.414483) (Table 5).
The objective of this study was to understand coordination of transcription initiation and termination of CYP736B and its impact on pre-mRNA splicing after Xf inoculation. First, we used a 5'UTR-defined long 3'RACE strategy to amplify full-length cDNAs of CYP736B transcripts with various 5' and 3' ends. Only two representative RNA samples were used in this test: PD-resistant and -susceptible leaf tissue harvested 6 weeks after Xf inoculation. The results revealed several distinguishable cDNA bands from each sample, as expected from 5' RACE and 3' RACE data (Figures 3a and 3b). Based on the 5' RACE and 3' RACE results and potential 5'TSS and 3'poly(A) site mapping results (Figures 2, Figure 3a and 3b), approximate size distributions of CYP736B transcripts with specific 5' and 3' termini were estimated, and corresponding cDNA bands excised from the gel for cloning. Then, we designed three upstream primers located at the three different 5'TSS regions: -300UF, -140UF, and -60UF (Table 1), and three downstream primers located at the three different 3'poly(A) regions: ++70UR, ++200UR, and ++260UR (Table 1). Different combinations of these primers were used to amplify full-length cDNA sequences of CYP736B transcripts with specific 5'TSS and 3'poly(A) termini. Finally, relative abundance of each CYP736B transcript with specific initiation and termination/polyadenylation sites were quantified by a Real-time PCR with primers SYBR-P450F1 and SYBR-P450R1. As shown in Table 6, relative abundance of CYP736B transcript in leaf tissues of resistant plants with a specific initiation site at the -300 position in the TSS1-containing region was increased as the distance of each transcription termination/polyadenylation site increased from poly(A)1 to poly(A)3 (P = 0.0000 ~ 0.2968, R2 = 0.8940). Relative abundance of CYP736B transcript with a specific transcription initiation site at the -140 position in the TSS2-containing region also was increased as the distance of each transcription termination/polyadenylation site increased from poly(A)1 to poly(A)2, but not poly(A)3 (P = 0.0010 ~ 0.2828, R2 = 0.8412). Relative abundance of CYP736B transcript with TSS2 sites was much higher than that with TSS1 sites in leaf tissues of PD- resistant plants. Relative abundance of CYP736B transcripts with the TSS1-containing region and TSS2-containing region in leaf tissues of susceptible plants showed similar patterns of change (P = 0.0000 ~ 0.4054, R2 = 0.8450 within TSS1-containing region, P = 0.0030 ~ 0.3039, R2 = 0.8936 within TSS2-containing region, and P = 0.0002 ~ 0.0018 between the TSS1-containing region and TSS2-containing region, respectively). However, abundance of each CYP736B transcript in susceptible plants was lower than in resistant plants (P = 0.0015 ~ 0.0875 as compared within TSS1-containing region; and P = 0.0082 ~ 0.3142 as compared within TSS2-containing region, respectively).
¶ Student T-Test: P1 (within group) indicates the significance between two 5'TSS-3'Poly(A) types of transcripts within same assay group; P2 (between groups) indicates the significance between two 5'TSS-3'Poly(A) types of transcripts between groups.
§ R2 shows correlation efficiency between each group of transcripts with fixed 5'TSS and various lengths of 3'-Poly(A) ends and their relative abundance (fold difference) within each assay group.
We also cloned and sequenced representative cDNA sequences with different 5'TSS and 3'poly(A) sites of CYP736B transcripts from PD-resistant and -susceptible leaf tissues 6 weeks after Xf inoculation. The genomic DNA and cDNA sequence alignment indicated that pre-mRNA splicing patterns of CYP736B transcripts from PD-resistant and -susceptible leaf tissues 6 weeks after Xf inoculation remained the same as that in control plants even though various CYP736B transcripts contained different 5'TSS and 3'poly(A) sites (data not shown). Quantitative analysis of relative frequencies of each cDNA clone with or without intron sequence showed that the frequency of correct CYP736B pre-mRNA splicing was correlated with distance from transcription initiation sites to the translation start codon (ATG) or from transcription termination sites to the stop codon (TGA); the closer the distance, the higher the frequency of correctly spliced CYP736B pre-mRNA (Figure 3b). As distance from the poly(A)-containing region to the stop codon (TGA) increased, frequency of the spliced CYP736B RNA decreased. Statistical analysis showed that correlation of 5'UTRs to mRNA splicing was as high as R2 = 0.8302 in resistant plants and R2 = 0.9808 in susceptible plants. The correlation efficiency of 3'UTRs to mRNA splicing was as high as R2 = 0.8922 in resistant plants and R2 = 0.8678 in susceptible plants.
Cytochrome P450 monooxygenase genes encode for a superfamily of enzymes in plants that seem to be involved in response to abiotic and biotic stresses [8, 15]. Although several P450 genes play crucial roles in biosynthesis of a variety of endogenous lipophilic and antioxidative compounds, little is known about cytochrome P450 monooxygenase gene induction and regulation in response to pathogens. A previous study using cDNA microarray screening revealed that expression of several cytochrome P450 monooxygenase genes was tightly regulated during growth and development of grapevines in response to X. fastidiosa infection [unpublished data]. The current study identified a cluster of three cytochrome P450 monooxygenase genes (CYP736A, CYP736B, and CYP736C) that are organized as tandem repeats and flanked upstream with a RE-LTR sequence on chromosome 7. This organization is likely the result of gene duplication [16–18]. We found that CYP736B was expressed at very low levels in stem tissues but at higher levels in leaf tissues of both PD-resistant and -susceptible grapevines with or without Xf infection. While the level of CYP736B expression was quite different between leaf and stem tissues, patterns of gene expression in leaf and stem tissues were similar in both PD- resistant and susceptible grapevines. Differential expression in response to Xf infection between genotypes suggested that CYP736B may be involved in the host response to Xf infection. It is known that the relative density of Xf populations in stem tissues are much lower in PD-resistant compared to -susceptible grapevines . This phenomenon may indicate that defense response genes, such as cytochrome P450 monooxygenase genes, may contribute to resistance against Xf infection in PD- resistant grapevines. In fact, a wide range of cytochrome P450 monooxygenases mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds. Various biochemical pathways are involved in this process: the DIMBOA biosynthesis pathway that is initiated in response to wounding and naphthalic anhydride treatment ; the camalexin biosynthesis pathway that is coordinately induced and strictly localized to the site of pathogen infection , and the lignin biosynthesis pathway that is induced to ward off passive microbial and fungal invaders or to lignify tissues to limit extent of damage caused by active invaders . Cytochrome P450 monooxygenases have other functions, including versatile biocatalytic reactions that mediate primary detoxification of natural and synthetic toxins [20, 11, 21]. However, relationships between these biochemical pathways and genes regulating disease resistance in plants are largely unknown.
In this study, transcription and pre-mRNA splicing patterns of CYP736B changed differently, depending on growth stage and Xf infection status. Induced expression levels of CYP736B were correlated positively with resistance and negatively with susceptibility in both stem and leaf tissues 6 weeks post-inoculation. These results support the conclusion that CYP736B is involved in the defense response against Xf infection in grapevines. We further demonstrated that expression of CYP736B was post-transcriptionally regulated via pre-mRNA splicing. The very low frequency of unspliced CYP736B transcripts in leaf tissues of PD-resistant grapevines and the very high frequency of unspliced CYP736B transcripts in leaf tissues of PD- susceptible grapevines (especially at 6 weeks post-Xf inoculation) may reveal an important aspect of the grapevine Xf interaction; correctly spliced CYP736B transcripts would be functional in PD-resistant grapevines, whereas unspliced CYP736B transcripts would not be functional in PD- susceptible grapevines. Differences in CYP736B expression levels between stems and leaves of grapevines would suggest that the levels of monooxygenase biosynthesis also vary. It seems that CYP736B expression involves dynamic regulatory mechanisms at both transcription initiation and the post-transcription modification levels via a developmental-dependent splicing pathway in PD- resistant grapevines. This result provides the first evidence that CYP736B is involved in the defense response at a specific stage of pathogenesis in grapevines.
Transcriptional initiation of mRNAs is preceded by formation of a pre-initiation complex in eukaryotic cells. Diverse transcriptional initiation sites have been discovered by large scale mapping of mRNA start sites in the human genome . Use of alternative transcription initiation sites is not uncommon in many animal and plant species [23, 24]. Utilization of multiple transcription initiation sites may contribute to genetic flexibility in which expression may be temporally and spatially regulated. Furthermore, expression may be affected post-transcriptionally as demonstrated by differential expression and subcellular targeting of glutathione S-transferase F8 gene in Arabidopsis . At present, regulatory mechanisms of cytochrome P450 monooxygenase genes in plants remain largely unknown. A previous study of cytochrome P450 expression and crosstalk in Arabidopsis revealed that most cytochrome P450 promoters contain the recognition sites MYB and MYC, an ACGT-core sequence, and TGA and W-boxes for WRKY transcription factors . Upon identification of genomic organization and expression patterns in grapevines, further determination of transcriptional initiation sites became a key step towards understanding transcriptional regulatory mechanisms. This study identified three major transcription start sites. Usage frequencies of each transcription start site varied depending on plant genotypes, developmental stages, tissues, and pathogen infection. Transcription start sites TSS2 and TSS3 were the major targets of transcriptional regulation during grapevine growth and development. Our study revealed that two TATA boxes, one P-box and one I-box were located at the far upstream region of CYP736B transcripts, whereas two CAAT boxes and a G-box were located at the TSS1-containing region, and one W-box was located at the TSS2-containing region. Previous studies indicated that TATA boxes are the most common regulatory elements found in promoters of eukaryotic genes because they are associated with basal transcription initiation by RNA polymerase II, especially with cis-acting elements that enhance or repress transcription . I-box (core sequence GATAA) and P-box (core sequence TGTAAAG) were associated with light and ABA responsiveness in tobacco and Arabidopsis respectively [27, 28]. CAAT-box and G-box (core sequence CACGTG) were associated with tissue-specific regulation and heat shock induction in pea [29, 30]. The W-box (core sequence CTGACT) is probably involved in elicitor- and wounding- responsive transcription of defense genes in tobacco [31, 32]. If these cis-elements located within each CYP736B transcript are involved in physical interaction of the transcript with corresponding transcription factors, variation in frequencies of each transcription initiation site could reflect differences in CYP736B regulatory mechanisms triggered by Xf. If so, selective use of transcription initiation sites would play an important role in regulation of CYP736B expression in response to Xf infection.
Transcriptional termination by RNA polymerase II is known to be controlled by various regulatory elements located in 3'UTRs. Termination of transcription requires a functional polyadenylation site and the addition of a 3' poly(A) tail by poly(A) polymerase immediately after transcription [33, 34]. The 3' end processing machinery functions to select an optimal poly(A) site between the poly(A) signal and a U-rich downstream element . The sequence of the poly(A) signal and the number of uridine residues are known to affect polyadenylation efficiency [35, 36]. However, the poly(A) site does not follow a strict consensus , even though a GC dinucleotide functions less efficiently than CA in vitro .
The research presented here revealed multiple poly(A) signals and multiple polyadenylation sites at the 3' UTR of CYP736B transcripts in both PD-resistant and -susceptible grapevines. There were 1 to 3 major polyadenylation sites with A or U as the terminal acceptor for polyadenylation. This result suggests that A and U are targets by poly(A) polymerase to synthesize poly(A) tails of CYP736B transcripts. Although there are examples of polyadenylation sites located between the poly(A) signal and a potential U-rich downstream element, a poly(A) signal could determine the optimal cleavage sites for CYP736B pre-mRNA polyadenylation. One possibility could be that the poly(A) polymerase-centered polyadenylation complex functions within the branched 3'UTR structure of CYP736B transcripts with some degree of flexibility after co-activation by poly(A) signals in grapevines. Given that total usage frequency of poly(A)2 and poly(A)3 sites changed differently between PD- resistant and susceptible grapevines after Xf inoculation, it is possible that bacterial virulence factors are involved directly or indirectly in modulation of CYP736B transcript polyadenylation in grapevines.
Transcription initiation, capping, cleavage/polyadenylation and pre-mRNA splicing are complex processes and tightly coupled to RNA polymerase II transcription. Transcription by RNA polymerase II and pre-mRNA processing are coordinated within the nucleus . In general, capping at the 5' ends minimizes mRNA degradation and most importantly permits interaction with ribosomes. The 3' end is completed by the addition of a polyA tail, resulting in increased mRNA stability, and introns are removed at some steps during this process . Bucheli et al.  found that the balanced competition of transcription factors with mRNA processing factors may promote recognition of proper polyadenylation sites while suppressing cryptic sites. Valencia et al.  suggested that a functional coupling is usually not essential for gene expression, but enhances the rate and/or efficiency of reactions that may serve to increase fidelity of gene expression in higher eukaryotes. Xin et al.  studied relationships between alternative promoters and pre-mRNA splicing and found that transcripts from different alternative promoters tended to splice differently. In the study presented here, variation in 5' transcription initiation sites or 3' polyadenylation sites affected CYP736B splicing. The combination of different transcription initiation sites and different 3' polyadenylation sites determined efficiency of CYP736B splicing in both PD- resistant and susceptible grapevines. For instance, transcription initiation from the TSS1-containing region always produced unspliced CYP736B RNAs in either PD- resistant or susceptible leaf tissues 6 weeks after Xf inoculation. As distance from the TSS-containing region to the start codon ATG decreased, frequency of spliced CYP736B RNA increased. Similarly, transcription termination at the poly(A)1 region of the 3'UTR always produced the highest frequency of spliced CYP736B RNAs. More importantly, frequency of correct CYP736B pre-mRNA splicing was correlated with both transcription initiation and termination sites distant to translation start and stop codons. The closer the distance from transcription initiation site to translation start codon and the distance from poly(A) site to translation stop codon, the higher the frequency of correctly spliced CYP736B transcripts. These relationships seem to be determined by changes in free energy within both 5'UTR and 3'UTR sequence-confined 2D structure constraints. Thus, our study revealed that both transcription initiation and termination have significant effects not only on relative abundance of CYP736B transcripts, but also on pre-mRNA splicing. Grapevine genotype/Xf interactions and their effect on CYP736B expression could be regulated by selective usage of transcription initiation and polyadenylation sites. The detailed regulatory mechanism of how Xf infection and grapevine/bacterial interactions affect selection of transcription initiation sites, polyadenylation sites, and pre-mRNA splicing remains to be understood. This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinated and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth, development and response to Xf infection.
The PD-resistant 9621-67 and -susceptible 9621-94 lines were selected from the 9621 population (D8909-15 (V. rupestris 'A. de Serres × V. arizonica b42-26) × F8909-17 (V. rupestris 'A. de Serres' × V. arizonica/candicans b43-17)) . A total of sixty plants from each genotype were propagated and grown at 24 to 32°C in a greenhouse with day lengths adjusted to 18 hours. After one month of growth, plants were cut back to two buds and re-grown for about 6 weeks before inoculation. Plants were inoculated with 10 μl of the "Stag's Leap" strain of Xf (1 × 108 cfu/ml) as treatment groups or inoculated with 10 μl of Xf-free PW3 liquid media as control groups on the stem 10 cm above the pot surface following the procedure of Krivanek et al . Each experimental unit consisted of three plants and was repeated three times in both treatment groups at each sampling date. Plants were arranged in a completely randomized design on greenhouse benches.
Leaf and stem tissues of three plants per treatment were collected at week 1, 6 and 10 after inoculation with or without Xf, respectively. Collected tissues were immediately placed in liquid nitrogen and stored at - 80°C until use. Symptoms were recorded as they appeared over a 12 week period on a separate set of 3 inoculated and 3 non-inoculated plants for each genotype grown under the same conditions.
Based on the previous report , a genomic DNA sequence coding for a specific cytochrome P450 gene was identified from a grape genomic database (GenBank Accessions: AM475392.1, CAAP02000243.1, CAAP02005006.1) using the BLAST http://www.ncbi.nlm.nih.gov/ and FGENESH http://www.softberry.com/berry.phtml programs. A pair of primers, P450F1 and P450R1 (Table 1), was designed using Primer 3 Program http://biotools.umassmed.edu/bioapps/primer3_www.cgi to clone this gene. Another pair of primers, P450F2 and P450R2, for quantitative Real-time PCR analysis of this cytochrome P450 gene expression, was designed using GeneFisher Program http://bibiserv.techfak.uni-bielefeld.de/genefisher2/. Two pairs of primers, 5'UTRF1 and 5'UTRR1, 3'UTRF1 and 3'UTRR1 (Table 1), were designed using the Primer 3 Program to amplify and clone the 1,000 bp upstream and 600 bp downstream of the cytochrome P450 gene, respectively (Table 1).
Total RNA was isolated using Trizol RNA Isolation Kit (Invitrogen, Carlsbad, CA). First strand cDNA was synthesized using oligo(dT)20 primers with the cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). RT-PCR analysis of gene expression was completed in a 25 μl of reaction that consisted of 1× reaction buffer (with 1.5 mM MgCl2), 2 mM dNTPs, 100 ng of cDNA template, 40 ng of primers, and 0.2 μl of Ampli Taq Gold DNA polymerase (5U/μl) (Applied Biosystems, Foster City, CA). The RT-PCR program consisted of preheating at 94°C for 5 min, 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 3 min followed by a 72°C extension for 10 min. RT-PCR products were analyzed on a 1.2% of agarose gel in 1× TBE buffer with ethidium bromide staining. Real-time quantitative PCR analysis of gene expression was completed in a 25 μl of reaction that consisted of 1× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 100 ng of template cDNA and 0.3 μM of each primers SYBR-P450F1 and SYBR-P450R1 (Table 1). The Real-time quantitative PCR was performed on the Bio-Rad iQ5 Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA) with the PCR program of 95°C 10 min, 40 cycles of 95°C 10 sec and 60°C 1 min, The Real-time PCR results were analyzed using a PCR product conversion and accumulation law-based delta Ct method [43, 44]. The same Real-time quantitative PCR also was used for quantitative analysis of pre-mRNA splicing with a pair of primers (SYBR-P450F2 and SYBR-P450R2) under the same experimental and analytic conditions (Table 1).
The Northern Max Formaldehyde-Based System for Northern Blots (Ambion, Texas) was used in this study according to the manufacturer's instruction. A full-length cDNA of grape cytochrome P450 monooxygenase gene CYP736B was labeled with biotin-dUTP at room temperature and used as probe for hybridization at 42°C for 18 hrs. Hybridization signals were detected using the BrightStar BioDetect Nonisotopic Detection Kit (Ambion, Texas).
Determination of 5' upstream transcription start regions and 3' poly(A) termination signal regions were done with the 5'RACE and 3'RACE Kits (Invitrogen, Carlsbad, CA). The two gene specific primers used for upstream 5'RACE cDNA cloning were P450-5'RACE GSP2 and P450-5'RACE GSP3 (Table 1). The two gene specific primers used for downstream 3'RACE cDNA cloning were P450-3'RACE GSP1 and P450-3'RACE GSP2 (Table 1). Further quantitative measurements of the major 5'RACE and 3'RACE cDNAs were done using Real-time PCR analysis with primers SYBR-P450B 5'UTRF1 and SYBR-P450B 5'UTRR1, and SYBR-P450B 3'UTRF1 and SYBR-P450B 3'UTRR1, respectively, as described above (Table 1).
Two gene specific primers, P450-5'RACE GSP3 for 5' primer extension analysis and P450-3'RACE GSP2 for 3'primer extension analysis, were designed to determine upstream transcriptional initiation regions and downstream transcriptional termination regions of grape P450B transcripts, respectively (Table 1). Total RNA-based or the 5'RACE and 3'RACE cDNA-based primer extension was completed in a 25 μl reaction using a ThermoScript RT-PCR Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Primer extension products were separated in a 4% NuSieve® 3:1 Agarose gel (Lonza Rockland, Inc., Rockland, ME), transferred to a Nylon membrane, and detected by the BrightStar BioDetect Nonisotopic Detection Kit (Ambion, Texas).
To determine relationships among different transcription initiation sites, pre-mRNA splicing, and polyadenylation signals, a modified 5'UTR-defined long 3'RACE method was designed in combination of the representative specific 5'UTR primers with the standard 3'RACE technology (Invitrogen, Carlsbad, CA). These specific 5'UTR primers were -300UF, -140UF, and -60UF (Table 1). The 5'UTR-defined long 3'RACE cDNA products were electrophoresed in a 4% agarose gel with TBE Buffer. Similarly, three representative specific 3'UTR reverse primers also were designed from the standard 3'RACE sequences: ++70UR, ++200UR, and ++260UR (Table 1). The PCR system consisted of 1× LA Polymerase Buffer (with 1.5 mM MgCl2), 2 mM dNTPs, 100 ng of cDNA template, 40 ng of primers, and 0.125 μl of TAKARA Taq polymerase (1U/μl) in a 25 μl reaction. The PCR program consisted of preheating at 98°C for 10 sec, 30 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 5 min, and post-PCR extension at 72°C for 10 min. The relative abundance of each specific 5'UTR and 3'UTR primer-defined P450 transcript was measured using Real-time Quantitative PCR as described above.
Genomic DNA, cDNA and RT-PCR product bands were excised from agarose gels, purified with the PCR Gel DNA Purification Kit (Invitrogen, Carlsbad, CA), and cloned into pGEM-T easy vector (Promega, Madison, WI). Cloned DNA fragments were sequenced with M13F, M13R and a pair of internal P450 primers, P450F3 and P450R3 (Table 1), using BigDye Terminator V3.1 Sequencing and Clean-Up Kits (Applied Biosystems, Foster City, CA).
A PLACE database http://www.dna.affrc.go.jp/PLACE/signalscan.html was scanned to search key cis-acting regulatory elements located at the 5'- and 3'-UTRs of grape P450 genes. A PromScan program was used for grape P450 gene promoter analysis http://molbiol-tools.ca/promscan/.
The amino acid sequences of in silico translated P450 proteins were subjected to protein block analysis using the on-line program http://blocks.fhcrc.org/blocks/blocks_search.html.
Gene expression experiments in this study were repeated three times with three plants of each genotype at each time of sample collection. The statistic significance of experimental variation was calculated using ANOVA at confidence levels of 99% (p < 0.01) and 95% (p < 0.05). The Student T-test was used to analyze significance between treatments for gene expression at the same confidence levels of 99% (p < 0.01) and 95% (p < 0.05).
We gratefully acknowledge financial support from the California Department of Food and Agriculture's Pierce's Disease and Glassy-winged Sharpshooter Board and USDA, Agricultural Research Service.
DC, HL, YT and AW designed and conducted research experiments. DC was involved most expression analyses and the manuscript preparation. HL, AW, EC and DS were involved in organization of the work, writing and editing of the manuscript. HL was PI and initiator of the project. All authors took part in drafting, reviewing and approval of the final manuscript.

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Murray C.J., Geoghegan J., Fennelly J., Macken J., Finnegan J.
Murray C.J., Geoghegan J., Macken J., Finnegan J.
JUDGMENT of Mr. Justice Fennelly delivered the 31st day of July, 2009.
1.	Where, as in this case, fundamental rights are invoked as a restraint on the exercise of statutory powers, the courts are increasingly called upon to strike a balance. In the present case, the Planning Tribunal wants to investigate a “leak” of confidential information to the Irish Times and is met with a countervailing claim based on confidentiality of journalists’ sources.
2.	The appeal is taken against an Order made by the High Court pursuant to section 4 of the Tribunal of Inquiries (Evidence) (Amendment) Act, 1997 directing a journalist and the editor of that newspaper to answer questions posed by the Tribunal intended to unveil the source of documents published in the Irish Times.
4.	The first-named appellant has been a journalist for over twenty years. He is the Public Affairs Correspondent of the Irish Times. I will refer to him as Mr Keena. The second-named appellant is the editor of The Irish Times. I will refer to her as Ms Kennedy.
5.	On 29th June, 2006, as part of its private investigations the Tribunal wrote to Mr. David McKenna seeking information in relation to certain payments said to have been made to Mr. Bertie Ahern, T.D. The letter asked Mr McKenna for a detailed narrative statement setting out the circumstances in which he had made any such payment. The letter specified a number of the matters to be included in the statement.
The Tribunal regarded this phase of its investigations as private and confidential. It claimed the power to impose an obligation of confidentiality on the recipient of the letter and did not consent to its publication.
7.	On 19th September 2006, Mr. Keena received anonymously an unsolicited copy of the letter from the Tribunal to Mr McKenna. It contained information relating to payments alleged to have been made to An Taoiseach, Bertie Ahern T.D., in 1993 when he was the Minister for Finance.
“A wealthy businessman, David McKenna, has been contacted by the Mahon Tribunal about payments to the Taoiseach, Bertie Ahern.
The Tribunal is investigating a number of payments to Mr. Ahern in or around December, 1993 including cash payments, the Irish Times has learned.
The Tribunal requested a detailed statement from Mr McKenna. He was asked to name the person who requested the payment and his understanding as to why it was required. He was also asked who the payment was given to and whether it was in cash or another form.
It is understood a solicitor who was an associate and personal friend of Mr Ahern's, the late Gerry Brennan, may have played a role in the matters being inquired into. Mr Brennan, a former director of Telecom Éireann, died in 1997.
Mr McKenna, a friend of Mr Ahern's and a known supporter of both him and his party, was estimated to be worth more than €60 million a number of years ago. However, his publicly-listed recruitment firm, Marlborough Recruitment, collapsed in 2002.
Mr McKenna is also a friend and business associate of Des Richardson, the businessman appointed by Mr Ahern in 1993 as full-time fundraiser for Fianna Fáil and who also fundraises for Mr Ahern's constituency operation. The Tribunal was told in private that Mr McKenna was one of the people who made a payment to Mr Ahern.
Special adviser to the Taoiseach Gerry Howlin was not available for comment as he was on leave.
When contacted by The Irish Times the Government press secretary, Mandy Johnston, passed the query on to the Fianna Fáil press officer Olivia Buckley, who said Mr Ahern did not comment on Tribunal matters.
Mr McKenna, when asked about the matter, said: ‘Contact my solicitor.’ His solicitor said he had no comment.
The Mahon Tribunal and Mr Ahern are scheduled to make representations about the matter before the President of the High Court, Mr Justice Joseph Finnegan, next month.
In December 1993, Mr Ahern was Minister for Finance and Fianna Fáil treasurer.
The inquiries into Mr Ahern's finances are understood to have begun after allegations were made about supposed payments to him by property developer Owen O'Callaghan in relation to the Quarryvale development in west Dublin.
This article quoted from the contents of the letter of 29th June, 2006 from the Tribunal to Mr. McKenna.
8.	The Tribunal wrote to Ms Kennedy on the day of publication of Mr Keenas’s article stating that it quoted “the content of a letter written to Mr David McKenna by a solicitor acting on behalf of the Tribunal” and asserting that “such communication was confidential to the Tribunal and was expressed to be so.” The Tribunal letter also claimed that the publication of this material was in breach of an injunction granted by the Supreme Court on 7th October, 2005.
“… the circumstances of this matter are straightforward. The Irish Times received an unsolicited and anonymous communication that I considered an important matter in the public interest for this newspaper to verify and publish. The vital issue of public interest which I considered I had a duty to publish was that the Taoiseach, Mr. Ahern, whilst a serving minister was in receipt of certain payments of money. The fact of these payments is a matter that this newspaper has a proper interest in publishing.
Ms Kennedy’s letter also denied that there had been a breach of any court order. That is not an issue in this case.
9.	On 25th September, 2006 the Tribunal made an order, expressed to be made pursuant to its powers under the Tribunals of Inquiry (Evidence) Acts 1921 to 2004 in particular section 1(i)(b) of the Tribunals of Inquiry (Evidence) Act 1921 as amended by section 3 Tribunals of Inquiry (Evidence) (Amendment) Act 1979 requiring Ms Kennedy and Mr Keena to produce to the Tribunal at its offices all documents which comprised the communication received by the Irish Times which led to the publication of the article of 21st September, 2006. The reference to section 3 (which deals with the power of the High Court) of the Act of 1979 should presumably be a reference to section 4. No point has ever been taken in that respect.
10.	By a reply of the same date Ms Kennedy stated that the Irish Times was not in a position to comply with this order as the material sought had been destroyed. She also disputed the right of the Tribunal to make the order. She said that “the production of any such material would run the risk of identifying sources.” It would, she said, “put in jeopardy the primary obligation of every editor and journalist to protect their sources of information.” Finally, she asserted that it was in the public interest that this obligation and right be protected, in the production of a story, which in itself was published in the public interest.
13.	Ms Kennedy, in her written statement, said that it was a matter of legitimate public interest that the Taoiseach of the day had received monies from businessmen while he was Minister for Finance in 1993.” She said that “in making the decision to publish she was conscious of the possibility that the Tribunal could find that the matter was outside its terms of reference and that it might never enter the public domain.” She said that she had instructed Mr Keena to destroy the documents and that she had also destroyed any copies in her own possession.
14.	On the same day the appellants appeared before the Tribunal. They were unable to produce the documents sought by the Tribunal, because they had destroyed them. They declined to answer any questions which in their view would give any assistance in identifying the source of the anonymous communication. Mr Keena, in particular, refused to say whether the version of the letter from the Tribunal which he had seen and which was referred to in the article was an original (with letter heading) or a copy.
15.	The Tribunal considered that the behaviour of Mr Keena and Ms Kennedy amounted to breaches of its orders and of the Tribunals of Inquiry (Evidence) Acts, 1921 to 2004. It decided to exercise the powers conferred upon it by section 4 of the Tribunals of Inquiry (Evidence) (Amendment) Act, 1997 and to seek orders from the High Court compelling Ms. Kennedy and Mr. Keena to comply with the Tribunal’s orders.
·	to answer all further questions to which the Tribunal may require answers.
“The key issue of journalism is that people who approach reporters in confidence with information of legitimate public interest, do not subsequently have their identities revealed by the reporters. If this occurs, it serves to make future disclosures less likely, as people would be more wary of coming forward. This makes it less effective journalism. It is also detrimental to the public good."
18.	The proceedings were heard in the High Court by a Divisional Court composed of the Johnson P, Kelly J and O’Neill J. Judgment was delivered on 23rd October 2007. The Court made orders pursuant to section 4 of the Tribunals of Inquiry (Evidence) (Amendment) Act, 1997 in accordance with the Special Summons. Essentially, the appellants are required to attend before the Tribunal and “to answer all questions to which the Tribunal may require answers in relation to the source and present whereabouts of the documents mentioned in the witness summons” dated 26th September 2006.
3. Assuming positive answers to the first two questions, how that right of the Tribunal is to be balanced against the defendants’ right to freedom of expression as guaranteed in Article 40, section 6,1º.i of the Constitution, and Article 10 of the European Convention on Human Rights and in particular the public interest in the preservation from disclosure of journalistic sources, as an essential prerequisite of a free press in a democratic society.
“Going hand in hand with this, is the critical importance of a free press as an essential organ in a democratic society. An essential feature of the operation of a free press is the availability of sources of information. Without sources of information journalists will be unable to keep society informed on matters which are or should be of public interest. Thus there is a very great public interest in the cultivation of and protection of journalistic sources of information as an essential feature of a free and effective press. As between the parties in this case there was no dispute whatever concerning these fundamental aspects of the right to freedom of expression as set out in Article 10 of the Convention.
"It need hardly be said, that such a manner of proceeding is anathema to the rule of law and an affront to democratic order. If tolerated it is the surest way to anarchy."
“Thus, the only means the Tribunal has of learning anything about these documents is through the questions which it proposes to put to the defendants. The content of the letter addressed to Mr. McKenna and his reply are of course well known to the Tribunal, therefore nothing is to be revealed in that regard. The only additional information that can be revealed by the defendants is whether or not the version or copy of the letter seen by them had the Tribunal’s letter heading on it or whether it was signed. All that this information would do is to indicate whether or not the copy of the letter furnished to the defendants came from inside the Tribunal or from elsewhere. Insofar as answers to these queries would tend to indicate that the documents furnished to the defendants came from elsewhere, it is clear from the evidence given that Mr. McKenna and his solicitor vehemently deny any role in furnishing these documents to the defendants. Thus, in all probability, having regard to the fact that the documents are now destroyed, the most that can be achieved by way of answers to questions proposed to be asked by the Tribunal of the defendants is to indicate that as a matter of probability the Tribunal was not the source of the leak. Beyond that the source will remain, as of course the source always intended, shrouded in impenetrable mystery with its anonymity safely beyond the reach of forensic inquiry.
“In these circumstances, we are of opinion that because of the destruction of the documents and the consequent deliberate frustration of forensic inquiry thereby brought about, there is little or no risk of the questions proposed to be asked leading to the identification of the source who provided these documents to the defendants. Because of this we are of opinion, therefore, that very slight weight indeed is to be attached to the defendants’ privilege against disclosure of their sources in this case.
(iii)	the [ appellants] to attend before the Tribunal and then to answer all further questions to which the Tribunal may require answers.
29.	The appellants did not contest the power of the Tribunal to make an order pursuant to section 4 of the Tribunals of Inquiry (Evidence) (Amendment) Act, 1979, as interpreted by this court in Kiberd v. Hamilton, cited above. Mr Donal O’Donnell, Senior Counsel, for the appellant's submitted that Clause J (5) of the Terms of Reference of the Tribunal, being expressed in negative form, did not apply. He also argued that the Tribunal was engaging in a "leak inquiry” which would not assist it in enquiring into matter the subject matter of its Terms of Reference. The appellants argued in their written submissions that the documents at issue are not confidential and the Tribunal did not have any authority to create the confidentiality for which it contends. They relied on Mahon v. Post Publications Limited  3 I.R. 338.
30.	The principal, indeed almost the exclusive, focus of Mr O'Donnell's submissions was a critique of the balance struck by the High Court between the rights of the Tribunal and of the appellants. He said that, by virtue of the provisions of the European Convention on Human Rights Act, 2003, Article 10 of the Convention applied, respectively, to the Tribunal as an "organ of the State" and to the High Court when interpreting either the common law or relevant statutes.
31.	Following a comprehensive survey of the case-law of the European Court of Human Rights, he submitted that the balance has almost always been struck by that court in favour of protection of journalists’ sources. Extraordinarily strong countervailing circumstances are required before a journalist can be obliged to disclose sources.
32.	Mr O'Donnell criticised what he called the “fallacious logic" of the High Court judgment. In effect, he said, the judgment requires questions to be asked only in circumstances where they cannot achieve the object of identifying the source. The view of the High Court was that the Tribunal could require questions to be answered for the purpose of exculpating the Tribunal itself and not to enable the actual source to be identified. The High Court, he argued, was, in particular, led into error by reason of its extremely critical views regarding the destruction by the appellants of the documents. For that reason, the High Court had erroneously taken the view that the journalists’ privilege against disclosure had almost no weight.
33.	Mr Michael Collins, Senior Counsel, for the Tribunal, accepted the link between the fundamental right of freedom of expression, guaranteed by Article 10 of the Convention, the privilege of journalists with regard to their sources and the need to recognise the "chilling effect" of an order for disclosure of sources. He referred to the trust and confidence which exists between the journalist and the source but argued that this is significantly weakened and the privilege would be entitled to very slight weight when the source is anonymous.
Mr Collins laid particular emphasis on the right of Tribunals generally to carry on preliminary investigations in private and to protect the confidentiality of persons cooperating with it in its private phase. The Tribunal has a legitimate interest, in Convention terms, in seeking to identify the source of a leak of confidential information.
Mr Collins referred to case-law in the United States, in particular the decision of the United States Supreme Court in Branzburg v Hayes, discussed later, declining First Amendment protection to journalists refusing to give evidence before a grand jury.
34.	The appellants do not seriously contest the power of the Tribunal to inquire into the unauthorised disclosure of its confidential information. The decision of this Court in Kiberd v Hamilton, cited above is conclusive on the point. It concerned an inquiry by Mr Justice Hamilton as Chairman and sole member of the Tribunals of Inquiry into the Beef Processing Industry into an unauthorised disclosure and publication in a newspaper of information confidential to the Tribunal. The newspaper contested the power of the Tribunal. Blayney J delivered the unanimous judgment of this Court. In reliance on the judgment of this Court in State (Lynch) v Cooney  I.R. 337, he held that what had to be considered was, "firstly, whether the Tribunal’s opinion that the making of the order was necessary for the purpose of its functions was bona fide held, secondly, whether that opinion was supported by the facts, and finally, whether it can be said of it that it was not unreasonable.” The learned judge rejected the challenge to the jurisdiction of the Tribunal, noted that it was not contested that its opinion was bona fide held, and concluded that there were grounds to support the view of the Tribunal which was not unreasonable.
35.	It is notable that the central issue of concern for the Tribunal in that case was the fear that, if people invited to cooperate with the Tribunal believed that information furnished confidentially by them would appear in the press, there was a real danger that witnesses would be dissuaded from coming forward with material relevant to the Tribunal’s inquiry. Kiberd v Hamilton, therefore, constitutes sufficient authority for the proposition that the Tribunal has power to investigate unauthorised disclosure of its confidential information. However, I am also satisfied that the High Court was correct to hold that Clause J (5) of the Terms of Reference of the Tribunal adopted by Resolution of Dáil Éireann passed on 17th November, 2004 confirmed the existence of that power.
It is not necessary to consider the English case of Norwich Pharmacal v Customs and Excise Commissioners  AC 133, which was discussed by counsel for the appellants at the hearing of the appeal, although that case and a number of cases which followed confirm the existence of a power in the courts to order disclosure of confidential information which has come into the possession even of innocent parties.
38.	I am satisfied that these concerns are legitimate. The information communicated to Mr McKenna was highly confidential and sensitive. The Tribunal was not only entitled but bound to make every effort to keep it so. It was, of course, quite clear to every person reading the Tribunal’s letter to Mr McKenna that it was and was expressed to be entirely private and confidential.
39.	The High Court was, in my view, perfectly correct to uphold the power of the Tribunal to conduct an inquiry into the source of the unauthorised disclosure and to hold that the documents were confidential.
40.	The appeal, therefore, turns entirely on the third point, namely on the balance struck by the High Court between the power of the Tribunal to investigate and the right of the appellants to refuse to disclose any information about their sources.
41.	I have postponed to this point reference to the Convention and the decisions of the European Court of Human Rights. Both have, of course, been considered in great detail by the High Court in its judgment and have been argued by both parties to the appeal. Those decisions are relevant to the outcome of the present proceedings because of the effect that has been given to the Convention in Irish law.
The definition of an “organ of State” in section 1 includes “a tribunal………which is established by law or through which any of the legislative, executive or judicial powers of the State are exercised.” The Tribunal undoubtedly comes within that definition.
and a court shall, when interpreting and applying the Convention provisions, take due account of the principles laid down by those declarations, decisions, advisory opinions, opinions and judgments.
43.	The combined effect of these provisions for the purposes of the present case is that the relevant sections of the Tribunals of Inquiry legislation must be interpreted in “a manner compatible with the State’s obligations under the Convention provisions.” For that purpose, the court must take judicial notice of the Convention provisions themselves and of the various documents mentioned in section 4 of the Act of 2003. Foremost among those are the judgments of the European Court of Human Rights. The requirement that the Court take judicial notice of the Convention and of the various documents referred to means that they can be relied upon by the Court without special proof. The Court must, in addition, as the concluding words of the provision make clear, “take due account” of the principles laid down in those judgments. This is not the same as saying that they constitute binding precedents.
44.	Although no issue arises in the present case of conflict between the Convention provisions and the Constitution, it is important to recall that, in the event of such a conflict, it is the Constitution which must prevail. Both the High Court and this Court on appeal have been concerned with the effect that is to be given in Irish law to the provisions of the Tribunals of Inquiry Acts and the orders made by the Tribunal. It is to state the obvious that a distinction must be made between this exercise of jurisdiction and that performed by the Court at Strasbourg exercising jurisdiction in international law, potentially leading to decisions binding on the State.
47.	The appellants cited from a large number of judgments of the European Court of Human Rights dealing with restrictions or penalties imposed by Member States claiming reliance on Article 10.2. Amongst these decisions were Lingens v. Austria (1986) 87 EHRR 329 Castells v. Spain, (1992) 14 EHRR 445 Fressoz and Roire v. France, (1999) 31 EHRR 28 Tromso v. Norway, (1999) 29 EHRR 12 Radio Twist AS v. Slovakia, (Unreported, European Court of Human Rights, 19 December 2006).
These judgments emphasise not merely the fundamental right to freedom of expression but, in the case of the press, its indispensable contribution to the functioning of a democratic society.
“39. The adjective ‘necessary’, within the meaning of Article 10.2, implies the existence of a ‘pressing social need’. The Contracting States have a certain margin of appreciation in assessing whether such a need exists, but it goes hand in hand with a European supervision, embracing both the legislation and the decisions applying it, even those given by an independent court. The Court is therefore empowered to give the final ruling on whether a ‘restriction’ or ‘penalty’ is reconcilable with freedom of expression as protected by Article 10.
40.	In exercising its supervisory jurisdiction, the Court cannot confine itself to considering the impugned court decisions in isolation; it must look at them in the light of the case as a whole, including the articles held against the applicant and the context in which they were written. The Court must determine whether the interference at issue was ‘proportionate to the legitimate aim pursued’ and whether the reasons adduced by the Austrian courts to justify it are ‘relevant and sufficient’.
41.	In this connection, the Court has to recall that freedom of expression, as secured in paragraph 1 of Article 10, constitutes one of the essential foundations of a democratic society and one of the basic conditions for its progress and for each individual’s self-fulfilment. Subject to paragraph 2, it is applicable not only to ‘information’ or ‘ideas’ that are favourably received or regarded as inoffensive or as a matter of indifference, but also to those that offend, shock or disturb. Such are the demands of that pluralism, tolerance and broadmindedness without which there is no ‘democratic society’.
These principles are of particular importance as far as the press is concerned. Whilst the press must not overstep the bounds set, inter alia, for the ‘protection of the reputation of others’, it is nevertheless incumbent on it to impart information and ideas on political issues just as on those in other areas of public interest. Not only does the press have the task of imparting such information and ideas: the public also has a right to receive them. In this connection, the Court cannot accept the opinion, expressed in the judgment of the Vienna Court of Appeal, to the effect that the task of the press was to impart information, the interpretation of which had to be left primarily to the reader.
42.	Freedom of the press furthermore affords the public one of the best means of discovering and forming an opinion of the ideas and attitudes of political leaders. More generally, freedom of political debate is at the very core of the concept of a democratic society which prevails throughout the Convention.
48.	The European Court has been at pains to emphasise that the right to freedom of expression is not unlimited. It usually states, as in the above passage, that the press must not "overstep certain bounds.” The court has said that "Article 10 does not…… guarantee a wholly unrestricted freedom of expression even with respect to press coverage of matters of serious public concern.” For example, it may be necessary, depending on the circumstances, to balance an individual's right to private and family life guaranteed by Article 8 of the Convention. Member States have a "certain margin of appreciation in assessing whether” there is a need for a restriction.
50.	One of the public interests recognised by Article 10.2 as potentially justifying a restriction on the exercise of freedom of expression is “preventing the disclosure of information received in confidence.” It is cases concerning this precise issue that are the most relevant to the present appeal. Two cases deserve careful consideration.
In the case of Fressoz and Roire, cited above, the applicants were a publisher and a journalist with the French satirical newspaper, Le Canard enchaîné. During a period of industrial unrest involving the motor-car manufacturer, Peugeot, the applicants published an article including details of the personal notices of assessment to tax of the chairman and managing director of the company. The second applicant said that the documents had been sent anonymously in an envelope addressed to him by name. The applicants were prosecuted and ultimately convicted by a Paris court of an offence of handling these documents which had been obtained through a breach of professional confidence by an unidentified tax official. They were fined 10,000 and 5,000FF respectively and ordered to pay the managing director 1FF for non-pecuniary damage and 10,000FF for costs.
The court went on to explain, at paragraph 53, that it “must …determine whether the objective of protecting fiscal confidentiality, which in itself is legitimate, constituted a relevant and sufficient justification for the interference."
It should be noted that the European Court did not doubt that there was a “pressing social need” for restraint of publication of the information.
However, the matter is not as clear as might appear at first sight. The decision was by a 5 to 4 majority. Powell J, though part of the majority, stressed, at page 709, “the limited nature of the Court’s holding.” He argued for recognition of the constitutional rights of journalists “with respect to the gathering of news or in safeguarding their sources.” Moreover, later cases have suggested that Branzburg v Hayes is authority only in the case of a summons to testify before a grand jury. A number of recent high-profile cases in the federal courts show that journalists cannot claim any general privilege against disclosure when required to testify before a grand jury investigating the possible commission of criminal offences.
54.	Mr Collins referred us to a comprehensive article by Randall D. Eliason entitled "Leakers, Bloggers and Fourth Estate Inmates: the Misguided pursuit of a reporter's privilege." 2006. 24 Cardozo Arts & Ent. LJ 385. That article shows that multiple distinctions need to be borne in mind between, for example: constitutional and common law; criminal and civil law; statute and judge-made law; state and federal law. Many states have laws protecting journalists’ privilege. It is protected, for example, in the Constitution of the State of California.
55.	There is no federal legislative protection of journalists’ sources. Moreover, it seems fair to say that the existence of a journalistic privilege of nondisclosure is highly contested in the federal courts and that, at the very least, it is certainly not regarded, where it is recognised, as absolute. The author, Mr Eliason, is himself highly critical "of a profession whose members believe that their professional obligations require them to break the law." He observes of journalists "claiming the right to decide for themselves how best to protect an institution: the free press." The American courts display a notable reluctance to allow to journalists a form of immunity from the obligation incumbent on all other citizens to obey the law, in particular, to give evidence when summoned and generally to participate in the judicial process. The federal courts have shown themselves willing, in the very recent past, to imprison journalists for contempt of court for refusal to give evidence before a grand jury, where a source would be revealed.
56.	As already stated, it is clear that the High Court was perfectly correct to hold that the Tribunal had the power, pursuant to section 4 of the Act of 1979, to conduct an inquiry into the source of the unauthorized disclosure of information communicated during its private investigative phase. It is equally clear that the information communicated by the Tribunal in its letter of 29th June 2006 to Mr McKenna had the character of confidential information. The Tribunal had the right and was under a duty to protect confidential information communicated to it and by it during its private investigative phase. The Tribunal is right to investigate unauthorised disclosure of such information and the courts must support its efforts.
57.	It is also clear, on the other hand, and has never been disputed on the part of the Tribunal, that the information in question, relating, as it did, to allegations of the payment of monies to an important political figure, was a matter of public interest, which a newspaper would, in the ordinary way be entitled to print.
59.	The key issue in the appeal is, therefore, whether the High Court was correct in deciding to make its order of 9th November 2007 which requires the appellants to appear before the Tribunal and to answer questions in relation to the source and present whereabouts of the documents which Mr Keena had received anonymously. The answer to that question depends on whether the High Court was correct in the way in which it balanced the right and interest of the Tribunal in seeking to uncover the source of the unauthorised disclosure and the interests of the appellants in protecting their source.
60.	Following an extensive examination of the case-law of the European Court of Human Rights, the High Court expressed the view that the exercise of deciding between competing interests “in a democratic society based on the rule of law is reserved to courts established by law for that purpose.” That, it seems to me, is a correct and unexceptionable principle. I have summarised, as did the High Court, the judgments of the European Court concerning the balance which the courts must strike between the right to freedom of expression, on the one hand, and the interests recognised in article 10.2 of the Convention, on the other. That court accords a margin of appreciation to the Contracting States, and their courts, when they decide those matters. It does not propose that these matters can be decided other than by courts. The High Court correctly said that the appellants had “cast themselves as the adjudicators of the proper balance to be struck between the rights and interests of concerned.” The courts cannot and should not abdicate their responsibility to decide when a journalist is obliged to disclose his or her source. The unilateral decision of a journalist to destroy evidence with intent to deprive the courts of jurisdiction is, as the High Court has held, designed to subvert the rule of law. The Courts cannot shirk their duty to penalise journalists who refuse to answer questions legitimately and lawfully put to them.
61.	Careful consideration needs, of course, to be given to the Goodwin case. The court must “take due account” of the principles it lays down. At this point, I raise the question as to whether it can truly be said to be in accord with the interests of a democratic society based on the rule of law that journalists, as a unique class, have the right to decide for themselves to withhold information from any and every public institution or court regardless of the existence of a compelling need, for example, for the production of evidence of the commission of a serious crime. While the present case does not concern information about the commission of serious criminal offences, it cannot be doubted that such a case could arise. Who would decide whether the journalist’s source had to be protected? There can be only one answer. In the event of conflict, whether in a civil or criminal context, the courts must adjudicate and decide, while allowing all due respect to the principle of journalistic privilege. No citizen has the right to claim immunity from the processes of the law.
65.	If the “leaked” document was not headed and thus, as a matter of probability, came from a Tribunal source, that might possibly assist the Tribunal in its endeavour to identify the source. At the same time, a number of other hypotheses cannot be excluded. One must recall that the person “leaking” the document would, in all probability, have wished to disguise the source. Thus, by the use of a photocopying machine, the heading might be removed from an original letter. Equally, an original might have been photocopied prior to its despatch from the Tribunal.
66.	On the contrary hypothesis that it did not come from a Tribunal source, but was, as the High Court expressed it “shrouded in impenetrable mystery,” it clearly would not be possible to justify the making of the order.
67.	If the apparent anonymity of the source weakens the appellants’ case for resisting the order, it must correspondingly weaken the Tribunal’s case for obtaining it. If, as was the case in Goodwin, the source was a person known to the journalist, it could, at the least, be argued that there was some concrete benefit to be obtained from the making of the order. Where the source is anonymous, the benefit is speculative at best. Mr Collins submitted, on behalf of the Tribunal, that the relationship of trust and confidence between the journalist and the source which is the basis of the journalist’s privilege was absent where the source was anonymous. There has been some discussion in the United States of theories of privilege: is it there to protect the source or the journalist? I do not find any such notion in the European case-law, which seems to proceed on a functional theory: is there a pressing social need for the imposition of the restriction?
68.	Looking at the High Court judgment as a whole, I have come to the conclusion that the great weight which it attached to the reprehensible conduct of the appellants in destroying documents led it to adopt an erroneous approach to the balancing exercise.
69.	According to the reasoning of the European Court in Goodwin, an order compelling the appellants to answer questions for the purpose of identifying their source could only be “justified by an overriding requirement in the public interest.” Once the High Court had devalued the journalistic privilege so severely, the balance was clearly not properly struck. On the other side, I find it very difficult to discern any sufficiently clear benefit to the Tribunal from any answers to the questions they wish to pose to justify the making of the order.
70.	I would, therefore, allow the appeal and substitute an order dismissing the Tribunal’s application.

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