BRAD V3.0: an upgraded Brassicaceae database.

The Brassicaceae Database (BRAD version 3.0, BRAD V3.0; http://brassicadb.cn) has evolved from the former Brassica Database (BRAD V2.0), and represents an important community portal hosting genome information for multiple Brassica and related Brassicaceae plant species. Since the last update in 2015, the complex genomes of numerous Brassicaceae species have been decoded, accompanied by many omics datasets. To provide an up-to-date service, we report here a major upgrade of the portal. The Model-View-ViewModel (MVVM) framework of BRAD has been re-engineered to enable easy and sustainable maintenance of the database. The collection of genomes has been increased to 26 species, along with optimization of the user interface. Features of the previous version have been retained, with additional new tools for exploring syntenic genes, gene expression and variation data. In the 'Syntenic Gene @ Subgenome' module, we added features to view the sequence alignment and phylogenetic relationships of syntenic genes. New modules include 'MicroSynteny' for viewing synteny of selected fragment pairs, and 'Polymorph' for retrieval of variation data. The updated BRAD provides a substantial expansion of genomic data and a comprehensive improvement of the service available to the Brassicaceae research community.

Conserved noncoding sequences correlate with distant gene contacts in Arabidopsis and Brassica.

Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions. To investigate the potential importance of distant contacts, we performed high-throughput chromatin conformation capture (Hi-C) experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea. We then combined our results with published Hi-C data from Arabidopsis thaliana. We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene. Genes with higher numbers of conserved noncoding sequences (CNSs) nearby were more likely to have contact with distant genes. With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity. In addition, for the genes we studied, distant contacting genes with CNSs were more likely to be transcriptionally coordinated. These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors, correlating with distant contacts to ensure coordinated expression. This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.

Transposable element insertion: a hidden major source of domesticated phenotypic variation in Brassica rapa.

Transposable element (TE) is prevalent in plant genomes. However, studies on their impact on phenotypic evolution in crop plants are relatively rare, because systematically identifying TE insertions within a species has been a challenge. Here, we present a novel approach for uncovering TE insertion polymorphisms (TIPs) using pan-genome analysis combined with population-scale resequencing, and we adopt this pipeline to retrieve TIPs in a Brassica rapa germplasm collection. We found that 23% of genes within the reference Chiifu-401-42 genome harbored TIPs. TIPs tended to have large transcriptional effects, including modifying gene expression levels and altering gene structure by introducing new introns. Among 524 diverse accessions, TIPs broadly influenced genes related to traits and acted a crucial role in the domestication of B. rapa morphotypes. As examples, four specific TIP-containing genes were found to be candidates that potentially involved in various climatic conditions, promoting the formation of diverse vegetable crops in B. rapa. Our work reveals the hitherto hidden TIPs implicated in agronomic traits and highlights their widespread utility in studies of crop domestication.

Roles of Species-Specific Legumains in Pathogenicity of the Pinewood Nematode Bursaphelenchus xylophilus.

Peptidases are very important to parasites, which have central roles in parasite biology and pathogenesis. In this study, by comparative genome analysis, genome-wide peptidase diversities among plant-parasitic nematodes are estimated. We find that genes encoding cysteine peptidases in family C13 (legumain) are significantly abundant in pine wood nematodes Bursaphelenchus genomes, compared to those in other plant-parasitic nematodes. By phylogenetic analysis, a clade of B. xylophilus-specific legumain is identified. RT-qPCR detection shows that these genes are highly expressed at early stage during the nematode infection process. Utilizing transgene technology, cDNAs of three species-specific legumain were introduced into the Arabidopsis γvpe mutant. Functional complementation assay shows that these B. xylophilus legumains can fully complement the activity of Arabidopsis γVPE to mediate plant cell death triggered by the fungal toxin FB1. Secretory activities of these legumains are experimentally validated. By comparative transcriptome analysis, genes involved in plant cell death mediated by legumains are identified, which enrich in GO terms related to ubiquitin protein transferase activity in category molecular function, and response to stimuli in category biological process. Our results suggest that B. xylophilu-specific legumains have potential as effectors to be involved in nematode-plant interaction and can be related to host cell death.

Improved Brassica oleracea JZS assembly reveals significant changing of LTR-RT dynamics in different morphotypes.

Brassica oleracea is an important vegetable crop that has provided ancestor genomes of the two most important Brassica oil crops, Brassica napus and Brassica carinata. The current B. oleracea reference genome (JZS, also named 02-12) displays problems of large mis-assemblies, low sequence continuity, and low assembly integrity, thus limiting genomic analysis. We reported an updated assembly of the B. oleracea reference genome (JZS v2) obtained through single-molecule sequencing and chromosome conformation capture technologies. We assembled an additional 83.16 Mb of genomic sequences, and the updated genome features a contig N50 size of 2.37 Mb, representing an ~ 88-fold improvement. We detected a new round of long terminal repeat retrotransposon (LTR-RT) burst in the new assembly. Comparative analysis with the reported genome sequences of two other genomes of B. oleracea (TO1000 and HDEM) identified extensive gene order and gene structural variation. In addition, we found that the genome-specific amplification of Gypsy-like LTR-RTs occurred around 0-1 million years ago (MYA). In particular, the athila, tat, and Del families were extensively amplified in JZS around 0-1 MYA. Moreover, we identified that the syntenic genes were modified due to the insertion of genome-specific LTR-RTs. These results indicated that the genome-specific LTR-RT dynamics were associated with genome diversification in B. oleracea.

Genome sequence of Isaria javanica and comparative genome analysis insights into family S53 peptidase evolution in fungal entomopathogens.

The fungus Isaria javanica is an important entomopathogen that parasitizes various insects and is effective for pest control. In this study, we sequenced and assembled the genomes (IJ1G and IJ2G) of two I. javanica strains isolated from different insects. The genomes were approximately 35 Mb in size with 11,441 and 11,143 protein-coding genes, respectively. Using a phylogenomic approach, we evaluated genome evolution across five entomopathogenic fungi in Cordycipitaceae. By comparative genome analysis, it was found that family S53 serine peptidases were expanded in Cordycipitaceae entomopathogens, particularly in I. javanica. Gene duplication events were identified based on phylogenetic relationships inferred from 82 S53 peptidases within six entomopathogenic fungal genomes. Moreover, we found that carbohydrate-active enzymes and proteinases were the largest secretory protein groups encoded in the I. javanica genome, especially chitinases (GH18), serine and aspartic peptidases (S53, S08, S10, A01). Pathogenesis-related genes and genes for bacterial-like toxins and secondary metabolites were also identified. By comparative transcriptome analysis, differentially expressed genes in response to insect nutrients (in vitro) were identified. Moreover, most S53 peptidases were detected to be significantly upregulated during the initial fungal infection process in insects (in vivo) by RT-qPCR. Our results provide new clues about understanding evolution of pathogenic proteases and may suggest that abundant S53 peptidases in the I. javanica genome may contribute to its effective parasitism on various insects.

Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus Purpureocillium lilacinum and Their Inhibition on Phytophthora Revealed by Genome Mining.

Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent.

The sequence and de novo assembly of the giant panda genome.

Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.

Analysis of the complete mitochondrial genome of Pochonia chlamydosporia suggests a close relationship to the invertebrate-pathogenic fungi in Hypocreales.

BACKGROUND: The fungus Pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents (BCAs) for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. The complete mitochondrial (mt) genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in Hypocreales. RESULTS: The complete mitogenome sequence of P. chlamydosporia is 25,615 bp in size, containing the 14 typical protein-coding genes, two ribosomal RNA genes, an intronic ORF coding for a putative ribosomal protein (rps3) and a set of 23 transfer RNA genes (trn) which recognize codons for all amino acids. Sequence similarity studies and syntenic gene analyses show that 87.02% and 58.72% of P. chlamydosporia mitogenome sequences match 90.50% of Metarhizium anisopliae sequences and 61.33% of Lecanicillium muscarium sequences with 92.38% and 86.04% identities, respectively. A phylogenetic tree inferred from 14 mt proteins in Pezizomycotina fungi supports that P. chlamydosporia is most closely related to the entomopathogenic fungus M. anisopliae. The invertebrate-pathogenic fungi in Hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi (Fusarium spp.) and Hypocrea jecorina. A comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in Sordariomycetes. Evolutionary analysis shows that rps3 is under positive selection, leading to the display of unique evolutionary characteristics in Hypocreales. Moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes. Gene rearrangement analysis shows that operation of transposition drives the rearrangement events in Pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in Clavicipitaceae. CONCLUSIONS: We present the complete annotated mitogenome sequence of P. chlamydosporia. Based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in Hypocreales. The invertebrate-pathogenic fungi in Hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. Our rps3 and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in Hypocreales.

Global protein-protein interaction network of rice sheath blight pathogen.

Rhizoctonia solani is the major pathogenic fungi of rice sheath blight. It is responsible for the most serious disease of rice (Oryza sativa L.) and causes significant yield losses in rice-growing countries. Identifying the protein-protein interaction (PPI) maps of R. solani can provide insights into the potential pathogenic mechanisms and assign putative functions to unknown genes. Here, we exploited a PPI map of R. solani anastomosis group 1 IA (AG-1 IA) based on the interolog and domain-domain interaction methods. We constructed a core subset of high-confidence protein networks consisting of 6705 interactions among 1773 proteins. The high quality of the network was revealed by comprehensive methods, including yeast two-hybrid experiments. Pathogenic interaction subnetwork, secreted proteins subnetwork, and mitogen-activated protein kinase (MAPK) cascade subnetwork and their interacting partners were constructed and analyzed. Moreover, to exactly predict the pathogenic factors, the expression levels of the interaction proteins were investigated by analyzing RNA sequences that consisted of samples from the entire infection progress. The PPIs offer an exceptionally rich source of data that can be used to understand the gene functions and biological processes of this serious disease at the system level.

Parasitism of Hirsutella rhossiliensis on Different Nematodes and Its Endophytism Promoting Plant Growth and Resistance against Root-Knot Nematodes.

The endoparasitic fungus Hirsutella rhossiliensis is an important biocontrol agent of cyst nematodes in nature. To determine the potential parasitism of the fungus on a non-natural host, the pinewood nematode (Bursaphelenchus xylophilus) living in pine trees and the endophytic ability of the fungus on plants, in this paper, we first constructed and utilized a green fluorescent protein (GFP)-tagged H. rhossiliensis HR02 transformant to observe the fungal infection process on B. xylophilus and its colonization on Arabidopsis roots. Then, we compared the fungal parasitism on three species of nematodes with different lifestyles, and we found that the fungal parasitism is correlated with nematode species and stages. The parasitic effect of H. rhossiliensis on adults of B. xylophilus is similar to that on second-stage juveniles (J2) of the root-knot nematode Meloidogyne incognita after 24 h of inoculation, although the virulence of the fungus to second-stage juveniles of M. incognita is stronger than that to those of B. xylophilus and Caenorhabditis elegans. Moreover, the endophytism of H. rhossiliensis was confirmed. By applying an appropriate concentration of H. rhossiliensis conidial suspension (5 × 106 spores/mL) in rhizosphere soil, it was found that the endophytic fungus can promote A. thaliana growth and reproduction, as well as improve host resistance against M. incognita. Our results provide a deeper understanding of the fungus H. rhossiliensis as a promising biocontrol agent against plant-parasitic nematodes.

Mitochondrial-targeting effector RsIA_CtaG/Cox11 in Rhizoctonia solani AG-1 IA has two functions: plant immunity suppression and cell death induction mediated by a rice cytochrome c oxidase subunit.

Rhizoctonia solani AG-1 IA causes a necrotrophic rice disease and is a serious threat to rice production. To date, only a few effectors have been characterized in AG-1 IA. We previously identified RsIA_CtaG/Cox11 and showed that infiltration of the recombinant protein into rice leaves caused disease-like symptoms. In the present study, we further characterized the functionality of RsIA_CtaG/Cox11. RsIA_CtaG/Cox11 is an alternative transcript of cytochrome c oxidase copper chaperone Cox11 that starts from the second AUG codon, but contains a functional secretion signal peptide. RNA interference with RsIA_CtaG/Cox11 reduced the pathogenicity of AG-1 IA towards rice and Nicotiana benthamiana without affecting its fitness or mycelial morphology. Transient expression of the RsIA_CtaG/Cox11-GFP fusion protein demonstrated the localization of RsIA_CtaG/Cox11 to mitochondria. Agro-infiltration of RsIA_CtaG/Cox11 into N. benthamiana leaves inhibited cell death by BAX and INF1. In contrast to rice, agro-infiltration of RsIA_CtaG/Cox11 did not induce cell death in N. benthamiana. However, cell death was observed when it was coinfiltrated with Os_CoxVIIa, which encodes a subunit of cytochrome c oxidase. Os_CoxVIIa appeared to interact with RsIA_CtaG/Cox11. The cell death triggered by coexpression of RsIA_CtaG/Cox11 and Os_CoxVIIa is independent of the leucine-rich repeat receptor kinases BAK1/SOBIR1 and enhanced the susceptibility of N. benthamiana to AG-1 IA. Two of the three evolutionarily conserved cysteine residues at positions 25 and 126 of RsIA_CtaG/Cox11 were essential for its immunosuppressive activity, but not for cell death induction. This report suggests that RsIA_CtaG/Cox11 appears to have a dual role in immunosuppression and cell death induction during pathogenesis.

URA3 as a Selectable Marker for Disruption and Functional Assessment of PacC Gene in the Entomopathogenic Fungus Isaria javanica.

An effective selection marker is necessary for genetic engineering and functional genomics research in the post-genomic era. Isaria javanica is an important entomopathogenic fungus with a broad host range and prospective biocontrol potentials. Given that no antibiotic marker is available currently in this fungus, developing an effective selection marker is necessary. In this study, by applying overlap PCR and split-marker deletion strategy, combining PEG-mediated protoplasm transformation method, the uridine auxotrophy gene (ura3) in the I. javanica genome was knocked out. Then, using this transformation system, the pH response transcription factor gene (IjpacC) was disrupted successfully. Loss of IjpacC gene results in an obvious decrease in conidial production, but little impact on mycelial growth. The virulence of the ΔIjpacC mutant on caterpillars is similar to that of the wild-type strain. RT-qPCR detection shows that expression level of an acidic-expressed S53 gene (IF1G_06234) in ΔIjpacC mutant is more significantly upregulated than in the wild-type strain during the fungal infection on caterpillars. Our results indicate that a markerless transformation system based upon complementation of uridine auxotrophy is successfully developed in I. javanica, which is useful for exploring gene function and for genetic engineering to enhance biological control potential of the fungus.

A New Insight into 6-Pentyl-2H-pyran-2-one against Peronophythora litchii via TOR Pathway.

The litchi downy blight disease of litchi caused by Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.

An Optimized Protocol for Detecting Guard Cell-specific Gene Expression by in situ RT-PCR in Brassica rapa.

Since the genetic transformation of Chinese cabbage (Brassica rapa) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell-specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.

Expanding the genetic variation of Brassica juncea by introgression of the Brassica rapa Genome.

Brassica juncea is an important vegetable and oil crop cultivated worldwide. To increase its genetic variation, we introgressed the A-genome of Brassica rapa into B. juncea. We used three each of heading and semi-heading B. juncea accessions as recipient parents and a B. rapa line, B9008, as the donor parent. We obtained 101 BC1S1 lines in total with expanded phenotypic variations such as leafy head shapes. We developed 132 SNP markers, which could distinguish the A genome of B. juncea from B. rapa genome, and tracked the introgression of B. rapa segments in the new B. juncea germplasm. On average, 59.2% of the B. juncea A genome in the B. juncea introgression lines was covered by the donor segments. We also identified three markers whose donor genotype frequencies were significantly lower than the theoretical value, suggesting strong selection of the recipient genotype during the introgression process. We provide an effective strategy to evaluate the diversity of the new germplasm based on the combination of parental re-sequencing data and marker genotyping results. Further genetic analysis of 1642 SNPs showed that the genetic diversity of the new B. juncea germplasm with the introgressed B. rapa genome was significantly increased. This study illustrated the potential for expanding the genetic diversity of B. juncea through the introgression of the B. rapa genome.

Improved Reference Genome Annotation of Brassica rapa by Pacific Biosciences RNA Sequencing.

The species Brassica rapa includes several important vegetable crops. The draft reference genome of B. rapa ssp. pekinensis was completed in 2011, and it has since been updated twice. The pangenome with structural variations of 18 B. rapa accessions was published in 2021. Although extensive genomic analysis has been conducted on B. rapa, a comprehensive genome annotation including gene structure, alternative splicing (AS) events, and non-coding genes is still lacking. Therefore, we used the Pacific Biosciences (PacBio) single-molecular long-read technology to improve gene models and produced the annotated genome version 3.5. In total, we obtained 753,041 full-length non-chimeric (FLNC) reads and collapsed these into 92,810 non-redundant consensus isoforms, capturing 48% of the genes annotated in the B. rapa reference genome annotation v3.1. Based on the isoform data, we identified 830 novel protein-coding genes that were missed in previous genome annotations, defined the untranslated regions (UTRs) of 20,340 annotated genes and corrected 886 wrongly spliced genes. We also identified 28,564 AS events and 1,480 long non-coding RNAs (lncRNAs). We produced a relatively complete and high-quality reference transcriptome for B. rapa that can facilitate further functional genomic research.