The root meristem growth factor BrRGF6 positively regulates Chinese cabbage to infection of clubroot disease caused by Plasmodiophora Brassicae.

Chinese cabbage has a high annual demand in China. However, clubroot disease caused by the infection of Plasmodiophora brassicae seriously affects its yield. Transcriptome analysis identified a root meristem growth factor 6 (BrRGF6) as significantly up-regulated in Chinese cabbage roots infected with Plasmodiophora brassicae. Quantitative reverse-transcription polymerase chain reaction and in situ hybridization analysis showed higher BrRGF6 expression in susceptible materials than in resistant materials. After Plasmodiophora brassicae infection, BrRGF6 expression was significantly up-regulated, especially in susceptible materials. Gene function analysis showed that the roots of Arabidopsis mutant rgf6 grew faster than the wild-type, and delayed the infection progress of Plasmodiophora brassicae. A Protein, nuclear transcription factor Y subunit C (BrNF-YC), was screened from yeast two-hybrid library of Chinese cabbage induced by Plasmodiophora brassicae, and verified to interact with BrRGF6 by yeast two-hybrid co-transfer. Yeast one-hybrid and ß-Glucuronidase activity analysis showed that BrNF-YC could directly bind to and strongly activate the promoter of BrRGF6. Transgenic verification showed that BrRGF6 or BrNF-YC silenced Chinese cabbage significantly decreased the expression of BrRGF6, accelerated root development, and reduced incidence of clubroot disease. However, after overexpression of BrRGF6 or BrNF-YC, the phenotype showed a reverse trend. Therefore, BrRGF6 silencing accelerated root growth and enhanced resistance to clubroot disease, which was regulated by BrNF-YC.

BrUFO positively regulates the infection of Chinese cabbage by Plasmodiophora brassicae.

Introduction: Chinese cabbage is one of the most important vegetable crops in China. However, the clubroot disease caused by the infection of Plasmodiophora brassicae (P. brassicae) has seriously affected the yield and quality of Chinese cabbage. In our previous study, BrUFO gene was found to be significantly up-regulated in diseased roots of Chinese cabbage after inoculation with P. brassicae. UFO (UNUSUAL FLORAL ORGANS) have the properties of substrate recognition during ubiquitin-mediated proteolysis. A variety of plant can activate immunity response through the ubiquitination pathway. Therefore, it is very important to study the function of UFO in response to P. brassicae. Methods: In this study, The expression pattern of BrUFO Gene was measured by qRT-PCR and In situ Hybridization (ISH). The expression location of BrUFO in cells was determined by subcellular localization. The function of BrUFO was verified by Virus-induced Gene Silencing (VIGS). proteins interacting with BrUFO protein were screened by yeast two-hybrid. Results: Quantitative real-time polymerase chain reactions (qRT-PCR) and in situ hybridization analysis showed that expression of BrUFO gene in the resistant plants was lower than that in susceptible plants. Subcellular localization analysis showed that BrUFO gene was expressed in the nucleus. Virus-induced gene silencing (VIGS) analysis showed that silencing of BrUFO gene reduced the incidence of clubroot disease. Six proteins interacting with BrUFO protein were screened by Y2H assay. Two of them (Bra038955, a B-cell receptor-associated 31-like protein and Bra021273, a GDSL-motif esterase/acyltransferase/lipase Enzyme) were confirmed to strongly interact with BrUFO protein. Discussion: BrUFO gene should be a key gene of chinese cabbage against the infection of P. brassicae. BrUFO gene silencing improves the resistance of plants to clubroot disease. BrUFO protein may interact with CUS2 to induce ubiquitination in PRR-mediated PTI reaction through GDSL lipases, so as to achieve the effect of Chinese cabbage against the infection of P. brassicae.

Analysis of the role of BrRPP1 gene in Chinese cabbage infected by Plasmodiophora brassicae.

Introduction: The clubroot disease caused by Plasmodiophora brassicae (P. brassicae) poses a serious threat to the economic value of cruciferous crops, which is a serious problem to be solved worldwide. Some resistance genes to clubroot disease in Brassica rapa L. ssp pekinensis cause by P. brassicae have been located on different chromosomes. Among them, Rcr1 and Rcr2 were mapped to the common candidate gene Bra019410, but its resistance mechanism is not clear yet. Methods: In this experiment, the differences of BrRPP1 between the resistant and susceptible material of Chinese cabbage were analyzed by gene cloning and qRT-PCR. The gene function was verified by Arabidopsis homologous mutants. The expression site of BrRPP1 gene in cells was analyzed by subcellular localization. Finally, the candidate interaction protein of BrRPP1 was screened by yeast two-hybrid library. Results: The results showed that the cDNA sequence, upstream promoter sequence and expression level of BrRPP1 were quite different between the resistant and susceptible material. The resistance investigation found that the Arabidopsis mutant rpp1 was more susceptible to clubroot disease than the wild type, which suggested that the deletion of rpp1 reduces resistance of plant to clubroot disease. Subcellular location analysis confirmed that BrRPP1 was located in the nucleus. The interaction proteins of BrRPP1 screened from cDNA Yeast Library by yeast two-hybrid are mainly related to photosynthesis, cell wall modification, jasmonic acid signal transduction and programmed cell death. Discussion: BrRPP1 gene contains TIR-NBS-LRR domain and belongs to R gene. The cDNA and promoter sequence of BrRPP1 in resistant varieties was different from that in susceptible varieties led to the significant difference of the gene expression of BrRPP1 between the resistant varieties and the susceptible varieties. The high expression of BrRPP1 gene in resistant varieties enhanced the resistance of Chinese cabbage to P. brassicae, and the interaction proteins of BrRPP1 are mainly related to photosynthesis, cell wall modification, jasmonic acid signal transduction and programmed cell death. These results provide important clues for understanding the mechanism of BrRPP1 in the resistance of B. rapa to P. brassicae.

Fine Mapping of BoVl Conferring the Variegated Leaf in Ornamental Kale (Brassica oleracea var. acephala)

Ornamental kale, as a burgeoning landscaping plant, is gaining popularity for its rich color patterns in leaf and cold tolerance. Leaf variegation endows ornamental kale with unique ornamental characters, and the mutants are ideal materials for exploring the formation mechanisms of variegated phenotype. Herein, we identified a novel variegated leaf kale mutant 'JC007-2B' with green margins and white centers. Morphological observations and physiological determinations of the green leaf stage (S1), albino stage (S2) and variegated leaf stage (S3) demonstrated that the chloroplast structure and photosynthetic pigment content in the white sectors (S3_C) of variegated leaves were abnormal. Genetic analysis revealed that a single dominant nuclear gene (BoVl) controlled the variegated leaf trait of 'JC007-2B', and three candidate genes for BoVl were fine-mapped to a 6.74 Kb interval on chromosome C03. Multiple sequence alignment among the green-leaf mapping parent 'BS', recombinant individuals, mutant parent 'JC007-2B' and its same originated DH line population established that the mutation sites in Bo3g002080 exhibited a complete consensus. Bo3g002080, homologous to Arabidopsis MED4, was identified as the candidate gene for BoVl. Expression analysis showed that Bo3g002080 displayed a 2158.85-fold higher expression at albino stage than that in green leaf stage. Transcriptome analysis showed that related pathways of photosynthesis and chloroplast development were significantly enriched in the white sectors, and relevant DEGs involved in these pathways were almost down-regulated. Overall, our study provides a new gene resource for cultivar breeding in ornamental kale and contributes to uncovering the molecular genetic mechanism underlying the variegated leaf formation.

An Improved Technique for Isolation and Characterization of Single-Spore Isolates of Plasmodiophora brassicae.

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease that occurs in cruciferous crops worldwide. P. brassicae usually exists as a mixture of several pathotypes, which has hampered the research on resistance mechanisms of cruciferous crops against P. brassicae. In this study, clubroot galls were collected from a field in Shenyang, China, as a pathogen source to develop an efficient protocol for a single-spore isolation system of P. brassicae by optimizing the seedling age for inoculation, host inoculation method, and plant culture method. The operational steps of the single-spore isolation method were optimized as follows: the use of 2-day-old seedlings for inoculation, substituting a cryobox (100 × 2.0-ml vials) for culture dishes, the addition of nutrient solution culture, and microscopic observations of single spores. The rate of infection success was substantially improved, and single-spore isolates of four pathotypes (4, 8, 9, and 11) were acquired in this system. Subsequently, the optimized system was used to isolate and characterize the pathotypes of single-spore isolates of P. brassicae collected from five fields in regions in China. Approximately four to nine pathotypes were isolated from each region. Among these, pathotype 4 was the most prevalent. This study provides a source of valuable information that can eventually be used for the genetic analysis of host-P. brassicae interaction.

Genome structural evolution in Brassica crops.

The cultivated Brassica species include numerous vegetable and oil crops of global importance. Three genomes (designated A, B and C) share mesohexapolyploid ancestry and occur both singly and in each pairwise combination to define the Brassica species. With organizational errors (such as misplaced genome segments) corrected, we showed that the fundamental structure of each of the genomes is the same, irrespective of the species in which it occurs. This enabled us to clarify genome evolutionary pathways, including updating the Ancestral Crucifer Karyotype (ACK) block organization and providing support for the Brassica mesohexaploidy having occurred via a two-step process. We then constructed genus-wide pan-genomes, drawing from genes present in any species in which the respective genome occurs, which enabled us to provide a global gene nomenclature system for the cultivated Brassica species and develop a methodology to cost-effectively elucidate the genomic impacts of alien introgressions. Our advances not only underpin knowledge-based approaches to the more efficient breeding of Brassica crops but also provide an exemplar for the study of other polyploids.

Discovery of BrATG6 and its potential role in Brassica rapa L. resistance to infection by Plasmodiophora brassicae.

Clubroot disease, caused by Plasmodiophora brassicae infection, occurs in cruciferous vegetable crops in many areas of the world, sometimes leading to yield loss. In this study, a differentially expressed protein (0305), was found between control and clubroot-diseased Chinese cabbage (Brassica rapa L.) roots through two-dimensional electrophoresis. Mass spectrometry analysis showed that Bra003466 was highly matched to protein 0305. Because the sequence of Bra003466 had 89% percent identity with ATG6 of Arabidopsis thaliana and other Brassica, the gene was named as BrATG6. However, 790 bp sequences were mismatched with the cDNA sequence of the Bra003466 gene from the Brassica database. In this study, we cloned the cDNA of Bra003466 and found the BrATG6 was highly expressed in roots among all organs. When plants were inoculated with P. brassicae Woronin, the expression of BrATG6 was significantly increased in infected roots of Chinese cabbage. This result was verified by reverse transcription-qPCR and in situ hybridization. Examination of disease resistance showed that, compared with wild type plants, A. thaliana ATG6 deletion mutants were more easily infected by P. brassicae than WT. This shows that BrATG6 may play a potential role in the resistance of B. rapa to P. brassicae infection.

iTRAQ-based proteomic analysis of fertile and sterile flower buds from a genetic male sterile line 'AB01' in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

To investigate the molecular basis of multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis), we performed differential proteomic analysis using iTRAQ to identify differentially abundant proteins between fertile and sterile flower buds from the genetic male sterile line 'AB01'. We identified 5932 high-confidence proteins; 1494 were differentially abundant between the two samples, including 749 up- and 745 down-regulated proteins. The up- and down-regulated proteins that could be essential for anther development and male sterility in sterile buds were mainly involved in (1) carbohydrate and energy metabolism (pyruvate dehydrogenase, glycolysis/gluconeogenesis, TCA cycle, starch and sucrose metabolism), (2) pollen wall synthesis and regulation (pectinesterase, polygalacturonase, pectate lyase, beta-galactosidase, glycosyl hydrolase), (3) protein synthesis and degradation (proteasome subunits, ribosome proteins, ABC transporters, RNA transport, protein processing in endoplasmic reticulum), (4) flavonoid biosynthesis, and (5) plant hormone signal transduction. We identified 10 genes/proteins that were both up-regulated and 122 that were both down-regulated in a conjoint analysis. Multiple reaction monitoring and qRT-PCR validation showed that the iTRAQ results were accurate and reliable. These findings will provide valuable information on proteins involved in anther development, and will contribute to the understanding of the molecular mechanism(s) that underlie male sterility in Chinese cabbage. BIOLOGICAL SIGNIFICANCE: Chinese cabbage is an allogamous plant with bisexual flowers that displays significant heterosis. The application of male sterile lines is a very efficient way to produce hybrid seeds, which can generate stronger plants that develop more rapidly and produce higher yield. However, the molecular mechanism(s) underlying multiple-allele-inherited male sterility in Chinese cabbage is unknown. In this study, we used a quantitative proteomic approach (iTRAQ) to identify DAPs between fertile and sterile buds of the GMS line 'AB01'. Subsequently, we also performed conjoined analysis of the iTRAQ results and our previously reported transcriptomics results. The aim of this research was to obtain the key DAPs and to identify the significantly enriched pathways involved in anther development and male sterility. These results may provide new insights into the molecular mechanism(s) underlying multiple-allele-inherited male sterility in Chinese cabbage.

BrSKS13, a multiple-allele-inherited male sterility-related gene in Chinese cabbage (Brassica rapa L. ssp. pekinensis), affects pollen development and pollination/fertilization process.

Multiple-allele-inherited male sterility (MAMS) is important in Chinese cabbage (Brassica rapa L. ssp. pekinensis) breeding, but the molecular mechanisms leading to male sterility are poorly understood. In this study, we cloned a novel gene, BrSKS13, that is differentially expressed in fertile and sterile flower buds of Chinese cabbage. BrSKS13 is most similar to Arabidopsis thaliana AT3G13400 (SKS13) and encodes a predicted 61.87 kDa protein with three cupredoxin superfamily conserved domains in the multicopper oxidase family. Semi-quantitative reverse-transcription PCR (sqRT-PCR) showed that expression of BrSKS13 is higher in fertile buds than in sterile buds. Quantitative RT-PCR (qRT-PCR) and in situ hybridization showed that BrSKS13 is highly expressed in fertile anthers, peaking at pollen-maturation stage VI, but is weakly expressed in other tissues and floral organs. Expression patterns of BrSKS13 promoter::GUS reporter fusions in Arabidopsis showed that the BrSKS13 promoter drives expression of the GUS gene only in anthers. The relative expression of Brsks13 in fertile buds was higher than in sterile buds for all other MAMS lines of Chinese cabbage examined. These results suggest that BrSKS13 affects pollen development. In situ hybridization analysis of flower stigmas at different times after pollination showed that BrSKS13 expression was first observed in stigmas and immature seeds at 1 h after pollination, and the signal intensity in seeds increased with increasing maturity. Compared to Col-0, A. thaliana sks13 mutant plants have shorter and fewer siliques, shriveled pollen grains, pollen tube abnormalities, and reduced seed number. The phenotype of sks13 mutant was recovered by over-expressing BrSKS13. Our results suggest that BrSKS13 affects pollen development and the pollination/fertilization process, and will enable further study of the genetic mechanisms underlying MAMS in Chinese cabbage.

Comparative transcript profiling of fertile and sterile flower buds from multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

We studied the underlying causes of multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis) by identifying differentially expressed genes (DEGs) related to pollen sterility between fertile and sterile flower buds. In this work, we verified the stages of sterility microscopically and then performed transcriptome analysis of mRNA isolated from fertile and sterile buds using Illumina HiSeq 2000 platform sequencing. Approximately 80% of ~229 million high-quality paired-end reads were uniquely mapped to the reference genome. In sterile buds, 699 genes were significantly up-regulated and 4096 genes were down-regulated. Among the DEGs, 28 pollen cell wall-related genes, 54 transcription factor genes, 45 phytohormone-related genes, 20 anther and pollen-related genes, 212 specifically expressed transcripts, and 417 DEGs located on linkage group A07 were identified. Six transcription factor genes BrAMS, BrMS1, BrbHLH089, BrbHLH091, BrAtMYB103, and BrANAC025 were identified as putative sterility-related genes. The weak auxin signal that is regulated by BrABP1 may be one of the key factors causing pollen sterility observed here. Moreover, several significantly enriched GO terms such as "cell wall organization or biogenesis" (GO:0071554), "intrinsic to membrane" (GO:0031224), "integral to membrane" (GO:0016021), "hydrolase activity, acting on ester bonds" (GO:0016788), and one significantly enriched pathway "starch and sucrose metabolism" (ath00500) were identified in this work. qRT-PCR, PCR, and in situ hybridization experiments validated our RNA-seq transcriptome analysis as accurate and reliable. This study will lay the foundation for elucidating the molecular mechanism(s) that underly sterility and provide valuable information for studying multiple-allele-inherited male sterility in the Chinese cabbage line 'AB01'.

SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.).

The genic multiple-allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L.) was identified as a spontaneous mutation. Applying this gene to hybrid seed production, several B. rapa cultivars have been successfully bred in China. A BC(1) population (244 plants) was constructed for mapping the Ms gene. Screening 268 simple sequence repeat (SSR) markers which cover the entire genome of Chinese cabbage was performed with bulked segregant analysis (BSA). On the basis of linkage analysis, the Ms gene was located on linkage group R07. In addition, through the amplified fragment length polymorphism (AFLP) and the sequence-characterized amplified region (SCAR) techniques combining BSA, two SCAR markers which were converted from corresponding AFLP markers flanked the Ms gene. Finally, a genetic map of the Ms gene was constructed covering a total interval of 9.0 cM. Two SCAR markers, syau_scr01 and syau_scr04, flanked the Ms gene at distances of 0.8 and 2.5 cM, respectively. All the SSR markers (cnu_m273, cnu_m030, cnu_m295, and syau_m13) were mapped on the same side of the gene as syau_scr04, the nearest one of which, syau_m13, was mapped at a distance of 3.3 cM. These SSR and SCAR markers may be useful in marker-assisted selection and map-based cloning.

Overexpression of Brassica napus MPK4 enhances resistance to Sclerotinia sclerotiorum in oilseed rape.

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus) resulting in significant economic losses. Studies on the Arabidopsis thaliana MPK4 loss-of-function mutant have implicated that AtMPK4 is involved in plant defense regulation, and its effect on disease resistance varies in different plant-pathogen interactions. In this study, we isolated a B. napus mitogen-activated protein kinase, BnMPK4, and found that BnMPK4 along with PDF1.2 are inducible in resistant line Zhongshuang9 but both are consistently suppressed in susceptible line 84039 after inoculation with S. sclerotiorum. Transgenic oilseed rape overexpressing BnMPK4 markedly enhances resistance to S. sclerotiorum and Botrytis cinerea. Further experiments showed that transgenic plants inhibited growth of S. sclerotiorum and constitutively activated PDF1.2 but decreased H2O2 production and constitutively suppressed PR-1 expression. Treatment of roots of the transgenic plants with H2O2 solution resulted in enhanced susceptibility to the two pathogens. Our results support the idea that MPK4 positively regulates jasmonic acid-mediated defense response, which might play an important role in resistance to S. sclerotiorum in oilseed rape.

[Method for measuring water content in fresh meat using diffusion reflectance near infrared spectroscopy and experiment].

A method was established to determine water content of fresh meat in deep layer based on NIRS diffusion reflectance spectroscopy by using LEDs. The experimental apparatus was established to investigate internal water content with repeated experiments for pork and mince pork; the water concentration and the water content detected by standard method is linear, and the r2 is larger than 0.90. Calculation of coefficient of variability (CV) and intra class correlation (ICC) procedures were applied to assess test-retest reliability of this measuring method. The CV and ICC values were found to be good (CV < or = 5%; ICC = 0.83). The experimental result indicated that the fast non-invasive measurement method for detecting water content in deep layer of fresh meat based on spatially-resolved spectroscopy was feasible.

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus).

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H2O2 levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum.