Expression of Nitrilases in Brassica juncea var. tumida Tsen in Root Galls Caused by Plasmodiophora brassicae

Five commonly-used reference genes： ACT （actin）, UBE （ubiquitin-conjugating enzyme）, RPL2 （ribosomal protein L2）, BRP II （RNA polymerase II subunit）, and NADH （nicotinamide adenine dinucleotide） were examined using geNorm software as reference genes for RT-qPCR. Among the tested reference genes, ACT and UBE were the most stable in all samples. In parallel, expression analysis of nitrilases in Brassica juncea var. tumida, was performed to preliminarily investigate the molecular interactions between nitrilase and clubroot development at 10, 15, 20, 25, 30, and 40 d postinoculation （dpi） with a suspension of resting spores of Plasmodiophora brassicae. The results showed that different gene expressions of nitrilases were regulated during the initial periods of clubroot development. The expression level of BjNIT1 increased sharply from 20 to 40 dpi in infected roots while there were no remarkable changes in healthy roots. From 15 to 30 dpi, the expression levels of BjNIT2 and BjNIT4 in infected roots were lower than those in non-infected roots. Finally, BjNIT2 in treatment was down approximately to control at 40 dpi. Our results suggest that BjNIT1, which promoted overproductions of auxin, might be involved in P. brassicae infection of B. juncea.

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.

Genome-wide Investigation of micro RNAs and Their Targets in Brassica rapa ssp. pekinensis Root with Plasmodiophora brassicae Infection

Increasing evidence has revealed that micro RNAs play a pivotal role in the post transcriptional regulation of gene expression in response to pathogens in plants. However, there is little information available about the expression patterns of mi RNAs and their targets in Chinese cabbage(Brassica rapa ssp. pekinensis) under Plasmodiophora brassicae stress. In the present study, using deep sequencing and degradome analysis, a genome-wide identification of mi RNAs and their targets during P. brassicae stress was performed. A total of 221 known and 93 potentially novel mi RNAs were successfully identified from two root libraries of one control(635-10CK) and P. brassicae-treated Chinese cabbage samples(635-10T). Of these, 14 known and 10 potentially novel mi RNAs were found to be differentially expressed after P. brassicae treatment. Degradome analysis revealed that the 223 target genes of the 75 mi RNAs could be potentially cleaved. KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis suggested that the putative target genes of the mi RNAs were predominately involved in selenocompound metabolism and plant hormone signal transduction. Then the expression of 12 mi RNAs was validated by quantitative real-time PCR(q RT-PCR). These results provide insights into the mi RNA-mediated regulatory networks underlying the stress response to the plant pathogen P. brassicae.

WeiTsing: a new face of Ca^(2+)-permeable channels in plant immunity

Plants employ pattern-and effector-triggered immunity(PTI and ETI)to synergistically defend invading pathogens and insect herbivores.Both PTI and ETI can induce cytosolic Ca^(2+)spikes,despite in different spatiotemporal patterns,to activate downstream Ca^(2+)-dependent immune signaling cascades.While multiple families of Ca^(2+)-permeable channels at the plasma membrane have been uncovered,the counterparts responsible for Ca^(2+)release from intracellular stores remain poorly understood.In a groundbreaking paper published recently by Cell,the authors reported that WeiTsing,an Arabidopsis endoplasmic reticulum(ER)-resident protein that was specifically expressed in the pericycle upon Plasmodiophora brassicae(Pb)infection,could form resistosome-like Ca^(2+)-conducting channel and protect the stele of Brassica crops from Pb colonization.As the channel activity of WeiTsing was indispensable for its immune function,the findings highlight a previously underappreciated role of Ca^(2+)release from intracellular repertoire in promoting plant disease resistance.