Identification and Characterization of Circular RNAs in Brassica rapa in Response to \u3ci\u3ePlasmodiophora brassicae\u3c/i\u3e

Plasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants and causes clubroot disease. CircRNAs are noncoding RNAs, widely existing in plant and animal species. Although knowledge of circRNAs has been updated continuously and rapidly, information about circRNAs in the regulation of clubroot disease resistance is extremely limited in Brassica rapa. Here, Chinese cabbage (BJN 222) containing clubroot resistance genes (CRa) against P. brassicae Pb4 was susceptible to PbE. To investigate the mechanism of cicRNAs responsible for clubroot disease resistance in B. rapa, circRNA-seq was performed with roots of ‘BJN 222’ at 0, 8, and 23 days postinoculated (dpi) with Pb4 and PbE. A total of 231 differentially expressed circRNAs were identified between the groups. Based on the differentially expressed circRNAs, the circRNA–miRNA–mRNA network was constructed using the target genes directly or indirectly related to plant resistance. Upregulated novel_circ_000495 suppressed the expression of miR5656-y, leading to the upregulation of Bra026508, which might cause plant resistance. Our results provide new insights into clubroot resistance mechanisms and lay a foundation for further studies exploring complex gene regulation networks in B. rapa

Fungus Pichia kudriavzevii XTY1 and heterotrophic nitrifying bacterium Enterobacter asburiae GS2 cannot efficiently transform organic nitrogen via hydroxylamine and nitrite

Heterotrophic nitrification is a process of organic nitrogen degradation completed by the participation of heterotrophic nitrifying microorganisms, which can accelerate the nitrogen transformation process. However, the current research mainly focuses on heterotrophic nitrifying bacteria and their ammonium degradation capacities. And there is little accumulation of research on fungi, the main force of heterotrophic nitrification, and their capacities to transform organic nitrogen. In this study, novel heterotrophic nitrifying fungus (XTY1) and bacterium (GS2) were screened and isolated from upland soil, and the strains were identified and registered through GenBank comparison. After 24 h single nitrogen source tests and 15N labeling tests, we compared and preliminarily determined the heterotrophic nitrification capacities and pathways of the two strains. The results showed that XTY1 and GS2 had different transformation capacities to different nitrogen substrates and could efficiently transform organic nitrogen. However, the transformation capacity of XTY1 to ammonium was much lower than that of GS2. The two strains did not pass through NH2OH and NO2− during the heterotrophic nitrification of organic nitrogen, and mainly generated intracellular nitrogen and low N2O. Other novel organic nitrogen metabolism pathways may be existed, but they remain to be further validated

Omics Meets Phytonutrients in Vegetable Brassicas: For Nutritional Quality Breeding

Consumers understand the health benefits of eating vegetables nowadays and thus there is currently a high demand for phytonutrient products. Recent advances in genomics, transcriptomics, proteomics and metabolomics have allowed the investigation of the genetic mechanism involved in Brassica phytonutrient metabolism. We discuss the application and opportunity of an omics approach to reveal the underlying genetics of the accumulation and regulation of various phytonutrients, such as well-known glucosinolates, carotenoids, anthocyanin and vitamins, which may assist in molecular breeding and metabolic engineering designed for nutritional quality enhancement of vegetable Brassica crops

Effects of Exogenous Ergothioneine on <i>Brassica rapa</i> Clubroot Development Revealed by Transcriptomic Analysis

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae that leads to a serious yield reduction in cruciferous plants. In this study, ergothioneine (EGT) was used to culture P. brassicae resting spores, the germination of which was significantly inhibited. Further exogenous application of EGT and P. brassicae inoculation in Chinese cabbage showed that EGT promoted root growth and significantly reduced the incidence rate and disease index. To further explore the mechanism by which EGT improves the resistance of Chinese cabbage to clubroot, a Chinese cabbage inbred line BJN3-2 susceptible to clubroot treated with EGT was inoculated, and a transcriptome analysis was conducted. The transcriptome sequencing analysis showed that the differentially expressed genes induced by EGT were significantly enriched in the phenylpropanoid biosynthetic pathway, and the genes encoding related enzymes involved in lignin synthesis were upregulated. qRT-PCR, peroxidase activity, lignin and flavonoid content determination showed that EGT promoted the lignin and flavonoid synthesis of Chinese cabbage and improved its resistance to clubroot. This study provides a new insight for the comprehensive prevention and control of cruciferous clubroot and for further study of the effects of EGT on clubroot disease

Sugar Transporters in Plasmodiophora brassicae: Genome-Wide Identification and Functional Verification

Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host&rsquo;s carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host

Identification and Characterization of Circular RNAs in <i>Brassica rapa</i> in Response to <i>Plasmodiophora brassicae</i>

Plasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants and causes clubroot disease. CircRNAs are noncoding RNAs, widely existing in plant and animal species. Although knowledge of circRNAs has been updated continuously and rapidly, information about circRNAs in the regulation of clubroot disease resistance is extremely limited in Brassica rapa. Here, Chinese cabbage (BJN 222) containing clubroot resistance genes (CRa) against P. brassicae Pb4 was susceptible to PbE. To investigate the mechanism of cicRNAs responsible for clubroot disease resistance in B. rapa, circRNA-seq was performed with roots of ‘BJN 222’ at 0, 8, and 23 days post-inoculated (dpi) with Pb4 and PbE. A total of 231 differentially expressed circRNAs were identified between the groups. Based on the differentially expressed circRNAs, the circRNA–miRNA–mRNA network was constructed using the target genes directly or indirectly related to plant resistance. Upregulated novel_circ_000495 suppressed the expression of miR5656-y, leading to the upregulation of Bra026508, which might cause plant resistance. Our results provide new insights into clubroot resistance mechanisms and lay a foundation for further studies exploring complex gene regulation networks in B. rapa

Transcriptome Arofile of Brassica rapa L. Reveals the Involvement of Jasmonic Acid, Ethylene, and Brassinosteroid Signaling Pathways in Clubroot Resistance

Plasmodiophora brassicae is a protozoan pathogen that causes clubroot disease in cruciferous plants, particularly Chinese cabbage (Brassica rapa). A previous study identified a clubroot resistance gene (CRd) conferring race-specific resistance to P. brassicae. However, the defense mechanisms of B. rapa against virulent vs. avirulent P. brassicae are poorly understood. In this study, we carried out a global transcriptional analysis in the clubroot-resistant Chinese cabbage inbred line &ldquo;85&ndash;74&rdquo; carrying the CRd gene and inoculated with avirulent (LAB-4) or virulent (SCCD-52) P. brassicae. RNA sequencing showed that &ldquo;85&ndash;74&rdquo; responded most rapidly to SCCD-52 infection, and the number of differentially expressed genes was much higher in SCCD-52-treated as compared to LAB-4-treated plants (5552 vs. 304). Transcriptome profiling revealed that plant hormone signal transduction and plant&ndash;pathogen interaction pathways played key roles in the late stages of P. brassicae infection. Genes relating to the salicyclic acid (SA), jasmonic acid (JA)/ethylene (ET), and brassinosteroid (BR) signaling pathways were up-regulated relative to untreated plants in response to LAB-4 infection at 8, 16, and 32 days post-inoculation (dpi) whereas JA, ET, and BR signaling-related genes were not activated in response to SCCD-52, and SA signaling-related genes were up-regulated in both LAB-4 and SCCD-52, suggesting that SA signaling is not the key factor in host resistance to avirulent P. brassicae. In addition, genes associated with phosphorylation and Ca2+ signaling pathways were down-regulated to a greater degree following LAB-4 as compared to SCCD-52 infection at 8 dpi. These results indicate that effector-triggered immunity in &ldquo;85&ndash;74&rdquo; is more potently activated in response to infection with avirulent P. brassicae and that JA, ET, and BR signaling are important for the host response at the late stage of infection. These findings provide insight into P. brassicae pathotype-specific defense mechanisms in cruciferous crops

Identification and Mapping of the Clubroot Resistance Gene CRd in Chinese Cabbage (Brassica rapa ssp. pekinensis)

The rapid spread of clubroot disease, which is caused by Plasmodiophora brassicae, threatens Brassicaceae crop production worldwide. Breeding plants that have broad-spectrum disease resistance is one of the best ways to prevent clubroot. In the present study, eight Chinese cabbage germplasms were screened using published clubroot-resistant (CR) loci-/gene-linked markers. A CR gene Crr3 potential carrier “85-74” was detected which linked to marker BRSTS61; however, “85-74” shows different responses to local pathogens “LAB-19,” “LNND-2,” and “LAB-10” from “CR-73” which harbors Crr3. We used a next-generation sequencing-based bulked segregant analysis approach combined with genetic mapping to detect CR genes in an F2 segregant population generated from a cross between the Chinese cabbage inbred lines “85-74” (CR) and “BJN3-1” (clubroot susceptible). The “85-74” line showed resistance to a local pathogen “LAB-19” which was identified as race 4; a genetic analysis revealed that the resistance was conferred by a single dominant gene. The CR gene which we named CRd was mapped to a 60 kb (1 cM) region between markers yau389 and yau376 on chromosome A03. CRd is located upstream of Crr3 which was confirmed based on the physical positions of Crr3 linked markers. The identification of CRd linked markers can be applied to marker-assisted selection in the breeding of new CR cultivars of Chinese cabbage and other Brassica crops

Genome Wide Identification and Expression Profiling of SWEET Genes Family Reveals Its Role During Plasmodiophora brassicae-Induced Formation of Clubroot in Brassica rapa

Plasmodiophora brassicae is a soil borne pathogen and the causal agent of clubroot, a devastating disease of Brassica crops. The pathogen lives inside roots, and hijacks nutrients from the host plants. It is suggested that clubroot galls created an additional nutrient sink in infected roots. However, the molecular mechanism underlying P. brassicae infection and sugar transport is unclear. Here, we analyzed sugar contents in leaves and roots before and after P. brassicae infection using a pair of Chinese cabbage near-isogenic lines (NILs), carrying either a clubroot resistant (CR) or susceptible (CS) allele at the CRb locus. P. brassicae infection caused significant increase of glucose and fructose contents in the root of CS-NIL compared to CR-NIL, suggesting that sugar translocation and P. brassicae growth are closely related. Among 32 B. rapa SWEET homologs, several BrSWEETs belonging to Clade I and III were significantly up-regulated, especially in CS-NIL upon P. brassicae infection. Moreover, Arabidopsis sweet11 mutant exhibited slower gall formation compared to the wild-type plants. Our studies suggest that P. brassicae infection probably triggers active sugar translocation between the sugar producing tissues and the clubbed tissues, and the SWEET family genes are involved in this process

Marker-Assisted Pyramiding of Genes for Multilocular Ovaries, Self-Compatibility, and Clubroot Resistance in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Molecular marker-assisted gene pyramiding combined with backcrossing has been widely applied for crop variety improvement. Molecular marker identification could be used in the early stage of breeding to achieve the rapid and effective pyramiding of multiple genes. To create high-quality germplasm for Chinese cabbage breeding, multi-gene pyramiding for self-compatibility, multilocular, and clubroot resistance was performed through molecular marker-assisted selection. The results showed that self-compatibility and multilocular traits were controlled by a pair of recessive genes. Two flanking markers, sau_um190 and cun_246a, and marker Teo-1, based on the gene sequence related to multilocular ovaries, were used for multilocular ovary trait selection. Two flanking markers, SCF-6 and SC-12, and marker Sal-SLGI /PK1+PK4, based on the gene sequence, were used for self-compatibility selection. Two flanking markers, TCR74 and TCR79, closely linked to clubroot resistance gene CRb, were used as foreground selection markers. Based on Chinese cabbage genomic information, 111 SSR markers covering 10 chromosomes were applied for background selection. After multiple generations of selection, a multi-gene pyramided line from a BC4F2 population with self-compatibility, multilocular ovaries, and clubroot resistance was obtained with a high genomic background recovery rate. The improved pyramided line is expected to be utilized as a potential material in further breeding programs