- Research article
- Open Access
Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection
© Cheng et al; licensee BioMed Central Ltd. 2010
- Received: 27 October 2009
- Accepted: 1 July 2010
- Published: 1 July 2010
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.
- Transcription Initiation
- Stem Tissue
- Transcription Initiation Site
- Polyadenylation Site
- Cytochrome P450 Monooxygenase
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 organization and structure of CYP736 genes in grape
PCR Primers used in this study
Sequences (5' - 3')
Gene and cDNA cloning
Gene and cDNA cloning
Expression detection in gel
Expression detection in gel
Internal primer for DNA sequencing
Internal primer for DNA sequencing
5'UTR cloning (1000 bp)
5'UTR cloning (1000 bp)
3'UTR cloning (600 bp)
3'UTR cloning (600 bp)
Real-time qPCR for gene expression
Real-time qPCR for gene expression
Real-time qPCR for pre-mRNA splicing
Real-time qPCR for pre-mRNA splicing
Real-time qPCR for 5'RACE
Real-time qPCR for 5'RACE
Real-time qPCR for 3'RACE
Real-time qPCR for 3'RACE
5' primer extension
3' primer extension
PCR for pre-mRNA splicing at -300 TSS
PCR for pre-mRNA splicing at -140 TSS
PCR for pre-mRNA splicing at -60 TSS
PCR for pre-mRNA splicing at ++70 poly(A) site
PCR for pre-mRNA splicing at ++200 poly(A) site
PCR for pre-mRNA splicing at ++260 poly(A) site
Temporal and spatial expression of CYP736B genes in grape
Quantitative analyses of CYP736B gene expression in stem and leaf tissues of grapevines at different stages of growth (1week, 6 week and 10 week after inoculation) with Xf infection (T) and without Xf infection (C)
Fold Difference in Expression Relative Folds for
Fold Difference in Expression
Growth Stages (p value)
XfInfection (p value)
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.
Pre-mRNA splicing patterns of CYP736B genes
Relative frequencies of unspliced CYP736B transcripts in leaf tissues of PD-resistant (9621-67) and -susceptible (9621-94) grapevines at different stages of growth (weeks after inoculation) with or without Xf infection
Fold Difference for Growth Stage
Fold Difference for Xf Infection
Determination of candidate 5' termini of grape CYP736B transcripts
Relative abundance of transcription start site usage in TSS2 and TSS3 regions of CYP736B gene in leaf tissues of both PD-resistant (9621-67) and -susceptible (9621-94) plants after 1 and 6 weeks of inoculation with Xf
Determination of candidate 3' termini of grape CYP736B transcripts
Relative abundance of poly(A)2 site in the 3' termini of CYP736B Transcripts in leaf tissues of both PD-resistant (9621-67) and -susceptible (9621-94) plants after 1 and 6 weeks of inoculation with Xf
Correlation among transcription initiation, pre-mRNA splicing and polyadenylation of CYP736B genes
Relative abundance of coordinated 5' TSS and 3' Poly(A) site usage for CYP736B gene transcription in leaf tissues of PD-resistant (9621-67) and -susceptible (9621-94) plants 6 weeks after Xf infection (fold difference)
From 5'TSS to 3'poly(A)
(-300) to (++70)
(-300) to (++200)
(-300) to (++260)
(-140) to (++70)
(-140) to (++200)
(-140) to (++260)
(-300) to (++70)
(-300) to (++200)
(-300) to (++260)
(-140) to (++70)
(-140) to (++200)
(-140) to (++260)
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.
CYP736B expression is regulated by Xf infection and development
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.
Multiple transcription initiations contribute to the regulation of CYP736B gene expression
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.
Polyadenylation sites determine transcriptional termination of CYP736B
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.
Coordination of transcription initiation and polyadenylation play a role in CYP736B splicing
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.
Plant growth and treatment with Xf
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.
Sample collection and evaluation
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.
Gene identification and primer design
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 isolation, cDNA synthesis and RT-PCR analysis of gene expression
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).
Northern blot and hybridization
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).
5'RACE and 3'RACE Analyses
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).
Primer Extension Analysis
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).
Quantification and cloning of representative full-length cDNA
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.
DNA Cloning and Sequencing
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).
5'- and 3'-UTR DNA cis-acting regulatory element analysis
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/.
Protein Block analysis
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.
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