Integrated Analysis of Transcriptome and Metabolome Reveals Differential Responses to Alternaria brassicicola Infection in Cabbage (Brassica oleracea var. capitata).

Black spot, caused by Alternaria brassicicola (Ab), poses a serious threat to crucifer production, and knowledge of how plants respond to Ab infection is essential for black spot management. In the current study, combined transcriptomic and metabolic analysis was employed to investigate the response to Ab infection in two cabbage (Brassica oleracea var. capitata) genotypes, Bo257 (resistant to Ab) and Bo190 (susceptible to Ab). A total of 1100 and 7490 differentially expressed genes were identified in Bo257 (R_mock vs. R_Ab) and Bo190 (S_mock vs. S_Ab), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that "metabolic pathways", "biosynthesis of secondary metabolites", and "glucosinolate biosynthesis" were the top three enriched KEGG pathways in Bo257, while "metabolic pathways", "biosynthesis of secondary metabolites", and "carbon metabolism" were the top three enriched KEGG pathways in Bo190. Further analysis showed that genes involved in extracellular reactive oxygen species (ROS) production, jasmonic acid signaling pathway, and indolic glucosinolate biosynthesis pathway were differentially expressed in response to Ab infection. Notably, when infected with Ab, genes involved in extracellular ROS production were largely unchanged in Bo257, whereas most of these genes were upregulated in Bo190. Metabolic profiling revealed 24 and 56 differentially accumulated metabolites in Bo257 and Bo190, respectively, with the majority being primary metabolites. Further analysis revealed that dramatic accumulation of succinate was observed in Bo257 and Bo190, which may provide energy for resistance responses against Ab infection via the tricarboxylic acid cycle pathway. Collectively, this study provides comprehensive insights into the Ab-cabbage interactions and helps uncover targets for breeding Ab-resistant varieties in cabbage.

Correlations between NLR, NHR, and clinicopathological characteristics, and prognosis of acute ischemic stroke.

Neuroinflammation plays an essential role in the process of acute ischemic stroke (AIS) injury repair. The current study seeks to investigate the relationship between the neutrophil/lymphocyte ratio (NLR) and neutrophil/high-density lipoprotein cholesterol ratio (NHR) and AIS disease severity and short-term prognosis. As such, the primary aim of this study is to improve AIS diagnosis and treatment. A total of 136 patients with AIS at the Nantong Third People's Hospital were retrospectively analyzed. The inclusion criteria comprised patients with ischemic stroke admitted to the hospital <24 hours after symptom onset. Baseline, clinical, and laboratory data were collected from all patients within 24 hours of admission. Univariate, multivariate and receiver operating characteristic curve analysis were performed to determine the relationship between NLR, NHR, AIS severity, and short-term prognosis. NLR (odds ratio [OR] = 1.448, 95% confidence interval [CI] 1.116-1.878, P = .005) and NHR (OR = 1.480, 95% CI 1.158-1.892, P = .002) were identified as independent risk factors for stroke severity. Additionally, the correlation between combined NLR and NHR and AIS severity achieved a sensitivity of 81.4% and specificity of 60.4% with a best cutoff value of 6.989. This outcome was superior to that of the single composite inflammatory index. Moreover, NLR (OR = 1.252, 95% CI 1.008-1.554, P = .042) was an independent risk factor for poor short-term prognosis in patients with AIS. When the optimal cutoff value was 2.605, the sensitivity of NLR correlation with the short-term prognosis of AIS was 82.2%, and the specificity was 59.3%. NLR combined with NHR exhibits a strong correlation with disease severity in AIS. Meanwhile, an elevated NLR in patients with AIS can predict a poor short-term prognosis.

Identification of F-box gene family in Brassica oleracea and expression analysis in response to low-temperature stress.

Unfavorable climatic conditions, such as low temperatures, often hinder the growth and production of crops worldwide. The F-box protein-encoding gene family performs an essential role in plant stress resistance. However, a comprehensive analysis of the F-box gene family in cabbage (Brassica oleracea var capitata L.) has not been reported yet. In this study, genome-wide characterization of F-box proteins in cabbage yielded 303 BoFBX genes and 224 BoFBX genes unevenly distributed on 9 chromosomes of cabbage. Phylogenetic analysis of 303 BoFBX genes was classified into nine distinct subfamily groups (GI-GIX). Analysis of the gene structure of BoFBX genes indicated that most genes within the same clade are highly conserved. In addition, tissue-specific expression analysis revealed that six F-box genes in cabbage showed the highest expression in rosette leaves, followed by roots and stems and the lowest expression was observed in the BoFBX156 gene. In contrast, the expression of the other five genes, BoFBX100, BoFBX117, BoFBX136, BoFBX137 and BoFBX213 was observed to be upregulated in response to low-temperature stress. Moreover, we found that the expression level of the BoFBX gene in the cold-tolerant cultivar "ZG" was higher than that in cold-sensitive "YC" with the extension of stress duration, while expression levels of each gene in "ZG" were higher than "YC" at 24 h. Knowledge of the various functions provided by BoFBXs genes and their expression patterns provides a firm theoretical foundation for explaining the functions of BoFBXs, thereby contributing to the molecular breeding process of cabbage.

Comparative genomic analyses reveal genetic characteristics and pathogenic factors of Bacillus pumilus HM-7.

Bacillus pumilus plays an important role in industrial application and biocontrol activities, as well as causing humans and plants disease, leading to economic losses and biosafety concerns. However, until now, the pathogenesis and underlying mechanisms of B. pumilus strains remain unclear. In our previous study, one representative isolate of B. pumilus named HM-7 has been recovered and proved to be the causal agent of fruit rot on muskmelon (Cucumis melo). Herein, we present a complete and annotated genome sequence of HM-7 that contains 4,111 coding genes in a single 3,951,520 bp chromosome with 41.04% GC content. A total of 3,481 genes were functionally annotated with the GO, COG, and KEGG databases. Pan-core genome analysis of HM-7 and 20 representative B. pumilus strains, as well as six closely related Bacillus species, discovered 740 core genes and 15,205 genes in the pan-genome of 21 B. pumilus strains, in which 485 specific-genes were identified in HM-7 genome. The average nucleotide identity (ANI), and whole-genome-based phylogenetic analysis revealed that HM-7 was most closely related to the C4, GR8, MTCC-B6033, TUAT1 and SH-B11 strains, but evolutionarily distinct from other strains in B. pumilus. Collinearity analysis of the six similar B. pumilus strains showed high levels of synteny but also several divergent regions for each strains. In the HM-7 genome, we identified 484 genes in the carbohydrate-active enzymes (CAZyme) class, 650 genes encoding virulence factors, and 1,115 genes associated with pathogen-host interactions. Moreover, three HM-7-specific regions were determined, which contained 424 protein-coding genes. Further investigation of these genes showed that 19 pathogenesis-related genes were mainly associated with flagella formation and secretion of toxic products, which might be involved in the virulence of strain HM-7. Our results provided detailed genomic and taxonomic information for the HM-7 strain, and discovered its potential pathogenic mechanism, which lay a foundation for developing effective prevention and control strategies against this pathogen in the future.

Genome-Wide Study of Hsp90 Gene Family in Cabbage (Brassica oleracea var. capitata L.) and Their Imperative Roles in Response to Cold Stress.

Heat shock protein 90 (Hsp90) plays an important role in plant developmental regulation and defensive reactions. Several plant species have been examined for the Hsp90 family gene. However, the Hsp90 gene family in cabbage has not been well investigated to date. In this study, we have been discovered 12 BoHsp90 genes in cabbage (Brassica oleracea var. capitata L.). These B. oleracea Hsp90 genes were classified into five groups based on phylogenetic analysis. Among the five groups, group one contains five Hsp90 genes, including BoHsp90-1, BoHsp90-2, BoHsp90-6, BoHsp90-10, and BoHsp90-12. Group two contains three Hsp90 genes, including BoHsp90-3, BoHsp90-4, and BoHsp90. Group three only includes one Hsp90 gene, including BoHsp90-9. Group four were consisting of three Hsp90 genes including BoHsp90-5, BoHsp90-7, and BoHsp90-8, and there is no Hsp90 gene from B. oleracea in the fifth group. Synteny analysis showed that a total of 12 BoHsp90 genes have a collinearity relationship with 5 Arabidopsis genes and 10 Brassica rapa genes. The promoter evaluation revealed that the promoters of B. oleracea Hsp90 genes included environmental stress-related and hormone-responsive cis-elements. RNA-seq data analysis indicates that tissue-specific expression of BoHsp90-9 and BoHsp90-5 were highly expressed in stems, leaves, silique, and flowers. Furthermore, the expression pattern of B. oleracea BoHsp90 exhibited that BoHsp90-2, BoHsp90-3, BoHsp90-7, BoHsp90-9, BoHsp90-10, and BoHsp90-11 were induced under cold stress, which indicates these Hsp90 genes perform a vital role in cold acclimation and supports in the continual of normal growth and development process. The cabbage Hsp90 gene family was found to be differentially expressed in response to cold stress, suggesting that these genes play an important role in cabbage growth and development under cold conditions.

Genome-wide analysis of proline-rich extension-like receptor protein kinase (PERK) in Brassica rapa and its association with the pollen development.

BACKGROUND: Proline-rich extension-like receptor protein kinases (PERKs) are an important class of receptor kinases located in the plasma membrane, most of which play a vital role in pollen development. RESULTS: Our study identified 25 putative PERK genes from the whole Brassica rapa genome (AA). Phylogenetic analysis of PERK protein sequences from 16 Brassicaceae species divided them into four subfamilies. The biophysical properties of the BrPERKs were investigated. Gene duplication and synteny analyses and the calculation of Ka/Ks values suggested that all 80 orthologous/paralogous gene pairs between B. rapa and A. thaliana, B. nigra and B. oleracea have experienced strong purifying selection. RNA-Seq data and qRT-PCR analyses showed that several BrPERK genes were expressed in different tissues, while some BrPERKs exhibited high expression levels only in buds. Furthermore, comparative transcriptome analyses from six male-sterile lines of B. rapa indicated that 7 BrPERK genes were downregulated in all six male-sterile lines. Meanwhile, the interaction networks of the BrPERK genes were constructed and 13 PERK coexpressed genes were identified, most of which were downregulated in the male sterile buds. CONCLUSION: Combined with interaction networks, coexpression and qRT-PCR analyses, these results demonstrated that two BrPERK genes, Bra001723.1 and Bra037558.1 (the orthologs of AtPERK6 (AT3G18810)), were downregulated beginning in the meiosis II period of male sterile lines and involved in anther development. Overall, this comprehensive analysis of some BrPERK genes elucidated their roles in male sterility.

Map-based cloning and characterization of BoCCD4, a gene responsible for white/yellow petal color in B. oleracea.

BACKGROUND: Brassica oleracea exhibits extensive phenotypic diversity. As an important trait, petal color varies among different B. oleracea cultivars, enabling the study of the genetic basis of this trait. In a previous study, the gene responsible for petal color in B. oleracea was mapped to a 503-kb region on chromosome 3, but the candidate gene has not yet been identified. RESULTS: In the present study, we report that the candidate gene was further delineated to a 207-kb fragment. BoCCD4, a homolog of the Arabidopsis carotenoid cleavage dioxygenase 4 (CCD4) gene, was selected for evaluation as the candidate gene. Sequence analysis of the YL-1 inbred line revealed three insertions/deletions and 34 single-nucleotide polymorphisms in the coding region of BoCCD4. Functional complementation showed that BoCCD4 from the white-petal inbred line 11-192 can rescue the yellow-petal trait of YL-1. Expression analysis revealed that BoCCD4 is exclusively expressed in petal tissue of white-petal plants, and phylogenetic analysis indicated that CCD4 homologs may share evolutionarily conserved roles in carotenoid metabolism. These findings demonstrate that BoCCD4 is responsible for white/yellow petal color variation in B. oleracea. CONCLUSIONS: This study demonstrated that function loss of BoCCD4, a homolog of Arabidopsis CCD4, is responsible for yellow petal color in B. oleracea.

Insights into the Draft Genome Sequence of the Kiwifruit-Associated Pathogenic Isolate Pseudomonas fluorescens AHK-1.

Pseudomonas fluorescens is a physiologically diverse species of bacteria present in many habitats, which possesses multifunctional traits that provide it with the capability to exhibit biological control activities, promote plant health or cause plant disease. Here, we present the draft genome sequence of the kiwifruit-associated pathogenic isolate AHK-1 of P. fluorescens, which was isolated from the diseased leaves of kiwifruit plants. The genome size of AHK-1 was found to be 7,035,786 bp, with a G + C content of 60.88%. It is predicted to contain a total of 6327 genes, of which 3998 were homologous to genes in the other two sequenced P. fluorescens isolates (SBW25 and GcM5-1A) and 946 were unique to AHK-1 based on comparative genomic analysis. Furthermore, we identified several candidate virulence factors in the genome of AHK-1, including the fliA gene encoding flagellar biosynthetic protein for biosynthesis, and the genes for components of type VI, III, and IV secretion systems. This genomic resource will serve as a reference for better understanding the genetics of pathogenic and non-pathogenic strains, and will help to elucidate the pathogenic mechanisms of P. fluorescens associated with plant disease.

BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility.

KEY MESSAGE : BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology. We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes' extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.

Molecular cloning and characterization of a novel pollen predominantly membrane protein gene BcMF12 from Brassica campestris ssp. chinensis.

A novel membrane protein gene, BcMF12, was isolated from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) using rapid amplification of the cDNA ends based on a pollen-specific cDNA fragment (DN237936). The cDNA was 1,155 bp in length with an open reading frame of 894 bp capable of encoding a putative polypeptide of 297 amino acids with an estimated molecular mass of 34.6 kDa and a predicted isoelectric point of 9.6. Comparative and bioinformatics analyses revealed that BcMF12 showed high similarities with some membrane protein sequences previously published in the public database and contained six highly conserved transmembrane domains corresponding to six highly hydrophobic regions. This indicates that BcMF12 may be a putative membrane protein. RNA gel blot analysis indicated that the transcripts of BcMF12 were abundant in the flower bud, flower and anther, but not detected in the root, stem, leaf and pistil. Moreover, the BcMF12 transcripts were detectable at the late stages of pollen development. Morphological investigations of pollen from the BcMF12 antisense transgenic plants showed that most of pollen grains of transgenic plants were abnormal. These results strongly suggest that BcMF12 is a novel pollen-preferentially membrane protein which play an important role during the pollen development in Chinese cabbage.

[Identification of the Burkholderia cepacia complex genomovars and their virulence in an alfalfa infection model].

OBJECTIVE: To confirm Burkholderia cepacia complex (Bcc) genomovars from agricultural niches and clinical samples, and to evaluate their possible virulence to human body on alfalfa infection model in China. METHODS: A total of 57 Bcc strains were isolated and collected from the rhizosphere, soil and clinical samples in China. The genomovars composition of the Bcc strains was analyzed by species-specific PCR tests, and the virulence of the Bce strains was tested on alfalfa seedlings. RESULTS: Four genomovars of the ten genomovars were detected among the Bcc strains, including B. cepacia (genomovar I), B. cenocepacia (genomovar III), B. vietnamiensis (genomovarV) and B. pyrrocinia (genomovar IX). Bcc genomovars I and III A from clinic, and genomovar III B from rhizosphere were the most virulent in the alfalfa infection model, and caused symptoms in 69%, 68% and 55% of seedlings, respectively. There were significant variances in the mean percentage of seedlings with symptoms for genomovars I , III A and III B compared to those for genomovar V and IX. CONCLUSION: There was difference in the ability to cause disease in alfalfa for different genomovar strains from agricultural inches. The strains of Bcc genomovar III B from rhizosphere were more virulent similar to those of Bcc genomovar III A from clinic.

BcMF11, a putative pollen-specific non-coding RNA from Brassica campestris ssp. chinensis.

A full-length cDNA, BcMF11, has been cloned from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) using rapid amplification of the cDNA ends (RACE) based on a pollen-specific cDNA fragment (DN237921). The BcMF11 cDNA has a total length of 828bp with poly (A) tail. Analysis of the sequence demonstrated that BcMF11 is a novel non-coding RNA which has no prominent open reading frame (ORF) or coding capacity. No significant similarities were observed between BcMF11 and previously published sequences in GenBank. Transcription analysis indicated that BcMF11 is a novel pollen-specific ncRNA and may be involved in the pollen development of Chinese cabbage.