Genome-Wide Identification of WRKY Gene Family in Pitaya Reveals the Involvement of HmoWRKY42 in Betalain Biosynthesis

Abstract

The <i>WRKY</i> gene family is a plant-specific transcription factor (TF) that regulates many physiological processes and (a) biotic stress responses. Despite this, little is known about the molecular properties and roles of <i>WRKY</i> TFs in pitaya betalain biosynthesis. Here we report the identification of 70 <i>WRKY</i> in <i>Hylocereus undatus,</i> their gene structure, locations on each chromosome, systematic phylogenetic analysis, conserved motif analysis, and synteny of <i>HuWRKY</i> genes. HmoWRKY42 is a Group IIb WRKY protein and contains a coiled-coil motif, a WRKY domain and a C₂H₂ zinc-finger motif (CX₅CX₂₃HXH). Results from yeast one-hybrid and transient dual-luciferase assays showed that HmoWRKY42 was a transcriptional repressor and could repress <i>HmocDOPA5GT1</i> expression by binding to its promoter. Yeast two-hybrid assays showed that HmoWRKY42 could interact with itself to form homodimers. Knocking out the coiled-coil motif of HmoWRKY42 prevented its self-interaction and prevented it from binding to the <i>HmocDOPA5GT1</i> promoter. Knocking out the WRKY domain and C₂H₂ zinc-finger motif sequence of HmoWRKY42 also prevented it from binding to the <i>HmocDOPA5GT1</i> promoter. The coiled-coil motif, the WRKY domain and the C₂H₂ zinc finger motif are key motifs for the binding of HmoWRKY42 to the <i>HmocDOPA5GT1</i> promoter. HmoWRKY42 is localized in the nucleus and possesses trans-activation ability responsible for pitaya betalain biosynthesis by repressing the transcription of <i>HmocDOPA5GT1</i>. As far as we know, no reports are available on the role of HmoWRKY42 in pitaya betalain biosynthesis. The results provide an important foundation for future analyses of the regulation and functions of the <i>HuWRKY</i> gene family.