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.