bra-miR167a Targets ARF8 and Negatively Regulates Arabidopsis thaliana Immunity against Plasmodiophora brassicae.

Clubroot is a soil-borne disease caused by Plasmodiophora brassicae, which can seriously affect the growth and production of cruciferous crops, especially Chinese cabbage crops, worldwide. At present, few studies have been conducted on the molecular mechanism of this disease's resistance response. In this experiment, we analyzed the bioinformation of bra-miR167a, constructed a silencing vector (STTM167a) and an overexpression vector (OE-miR167a), and transformed them to Arabidopsis to confirm the role of miR167a in the clubroot resistance mechanism of Arabidopsis. Afterwards, phenotype analysis and expression level analysis of key genes were conducted on transgenic plants. From the result, we found that the length and number of lateral roots of silence transgenic Arabidopsis STTM167a was higher than that of WT and OE-miR167a. In addition, the STTM167a transgenic Arabidopsis induced up-regulation of disease resistance-related genes (PR1, PR5, MPK3, and MPK6) at 3 days after inoculation. On the other hand, the auxin pathway genes (TIR1, AFB2, and AFB3), which are involved in maintaining the balance of auxin/IAA and auxin response factor (ARF), were down-regulated. These results indicate that bra-miR167a is negative to the development of lateral roots and auxins, but positive to the expression of resistance-related genes. This also means that the STTM167a can improve the resistance of clubroot by promoting lateral root development and the level of auxin, and can induce resistance-related genes by regulating its target genes. We found a positive correlation between miR167a and clubroot disease, which is a new clue for the prevention and treatment of clubroot disease.

Integrative Transcriptome, miRNAs, Degradome, and Phytohormone Analysis of Brassica rapa L. in Response to Plasmodiophora brassicae.

Clubroot is an infectious root disease caused by Plasmodiophora brassicae in Brassica crops, which can cause immeasurable losses. We analyzed integrative transcriptome, small RNAs, degradome, and phytohormone comprehensively to explore the infection mechanism of P. brassicae. In this study, root samples of Brassica rapa resistant line material BrT24 (R-line) and susceptible line material Y510-9 (S-line) were collected at four different time points for cytological, transcriptome, miRNA, and degradome analyses. We found the critical period of disease resistance and infection were at 0-3 DAI (days after inoculation) and 9-20 DAI, respectively. Based on our finding, we further analyzed the data of 9 DAI vs. 20 DAI of S-line and predicted the key genes ARF8, NAC1, NAC4, TCP10, SPL14, REV, and AtHB, which were related to clubroot disease development and regulating disease resistance mechanisms. These genes are mainly related to auxin, cytokinin, jasmonic acid, and ethylene cycles. We proposed a regulatory model of plant hormones under the mRNA-miRNA regulation in the critical period of P. brassicae infection by using the present data of the integrative transcriptome, small RNAs, degradome, and phytohormone with our previously published results. Our integrative analysis provided new insights into the regulation relationship of miRNAs and plant hormones during the process of disease infection with P. brassicae.