Role of Brassica rapa SWEET genes in the defense response to Plasmodiophora brassicae.

Abstract

Interactions of plants with biotic stress factors including bacteria, fungi, and viruses have been extensively investigated to date. Plasmodiophora brassicae, a protist pathogen, causes clubroot disease in Cruciferae plants. Infection of Chinese cabbage (Brassica rapa) plants with P. brassica results in the formation of root galls, which inhibits the roots from absorbing soil nutrients and water. Sugar, the major source of carbon for all living organisms including pathogens and host plants, plays an important role in plant growth and development. To explore the roles of BrSWEET2, BrSWEET13, and BrSWEET14 in P. brassicae resistance, Arabidopsis thaliana T-DNA knockout mutants sweet2, sweet13, and sweet14 were employed. To isolate total RNA from the collected root nodules, the root tissues washed several times with running water and frozen tissues with liquid nitrogen. Total RNA was extracted using the Spectrum™ Plant Total RNA Kit (SIGMA) and cDNA was synthesized in a 20μl reaction volume using the ReverTra Ace-α-® kit (TOYOBO). Real-time PCR was performed in a 10μl reaction volume containing 1μl of template DNA, 1μl of forward primer, 1μl of reverse primer, 5μl of 2× iQTM SYBR® Green Supermix (BioRad), and 2μl of sterile distilled water. The SWEET genes were genotyped using BioFACT™ 2× TaqBasic PCR Master Mix 2. Both sweet2 and sweet14 showed strong resistance to P. brassicae compared with wild-type Arabidopsis and Chinese cabbage plants and sweet13 mutant plants. Pathogenicity assays indicated that the SWEET2 gene plays an important role in clubroot disease resistance in higher plants.