Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.