Candidate promoter-associated factors of BrAGPS1 identified by yeast one-hybrid screening in Chinese cabbage during Plasmodiophora brassicae infection

Abstract

Clubroot disease is caused by the obligate biotrophic protist Plasmodiophora brassicae and severely limits Chinese cabbage (Brassica rapa subsp. pekinensis) production. Infection is accompanied by extensive reprogramming of host carbon metabolism and starch accumulation in enlarged roots. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch biosynthesis, but the upstream regulation of the Chinese cabbage small-subunit gene BrAGPS1 remains poorly understood. We cloned the promoter regions of BrAGPS1a (BraA07g007740.3.5C) and BrAGPS1b (BraA01g014740.3.5C) from the inbred line ‘Chiifu-401-42’. PlantCARE analysis predicted core promoter motifs and putative cis-regulatory elements associated with hormone, stress, and light responses. A root cDNA library prepared from 1, 3, and 5 weeks post-inoculation (wpi) was screened by yeast one-hybrid (Y1H) using selected promoter fragments as baits to recover candidate promoter-associated proteins. The screen yielded 76 nonredundant library hits, 15 of which were annotated as proteins with potential transcriptional, DNA-binding, histone-binding, or chromatin-associated functions. Integration with an RNA-sequencing (RNA-seq) time course at 1, 2, 3, and 5 wpi identified 28 Y1H-derived genes that were differentially expressed at one or more time points. Five candidates—a calcium-dependent protein kinase (CPK), a basic leucine zipper (bZIP) transcription factor, a three-high-mobility-group-box (3×HMG-box) protein, and two Alfin1-like plant-homeodomain (PHD-domain) proteins—were selected on the basis of functional annotation and infection-responsive expression. Quantitative real-time PCR (qRT-PCR) confirmed distinct treatment- and time-dependent expression patterns but did not test DNA binding or regulatory function. This study provides a prioritized set of BrAGPS1 promoter-associated candidates and testable hypotheses for investigating the transcriptional control of starch metabolism during clubroot development. One-to-one Y1H back-assays, electrophoretic mobility shift assays, promoter transactivation tests, and genetic analyses are required to establish direct binding, regulatory direction, and biological function.