[Show abstract][Hide abstract] ABSTRACT: Fusarium oxysporum f. sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change >=2 fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and ten of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.
Preview · Article · Feb 2016 · Frontiers in Plant Science
[Show abstract][Hide abstract] ABSTRACT: Low erucic acid (LEA) rapeseed, which has accumulated mutant fatty acid elongase genes at the BnFAE1.1 and BnFAE1.2 loci of the A- and C-genome, respectively, is an important oilseed crop. Short growing turnip rape (B. rapa) is also important as a catch crop in the continuous cropping of rice in Asia but there is no LEA B. rapa cultivar for cultivation in South Asia. In order to develop LEA turnip rape cultivars, high erucic acid turnip rape cultivars were interspecifically crossed as recurrent parents to a canola quality rapeseed. In the meantime, we monitored incorporation of the mutant bnfae1.1 (e1) gene into A-genome of turnip rape, using a dCAPS primer pair, which can amplify PCR fragment only for the mutant e1 gene from A-genome. The early backcross progenies showed poor seed set, but which was improved in advanced progenies. Finally, homozygous e1e1 genotypes were established in the selfed progenies of BC2–BC3, and their LEA content was confirmed by gas-chromatography analysis. Our results and promising lines will contribute to LEA-trait selection in turnip rape and rapeseed breeding.
[Show abstract][Hide abstract] ABSTRACT: The methylation patterns of plants are unique in that besides the methylation at CG dinucleotides, which occurs in mammals as well, methylation also occurs at non-CG sites. Genes are methylated at CG sites, but transposable elements (TEs) are methylated at both CG and non-CG sites. The role of non-CG methylation in the transcriptional silencing of TEs is being extensively studied, but only very rare transpositions have been reported when the non-CG methylation machineries were compromised. To understand the role of non-CG methylation in the suppression of TEs and in plant development, we characterized rice mutants of the chromomethylase gene, OsCMT3a. oscmt3a mutants exhibited a dramatic decrease in CHG methylation, changes in expression of a number of genes and TEs, and pleiotropic developmental abnormalities. Genome resequencing identified eight TE families mobilized in oscmt3a during normal propagation. These TEs include tissue-culture-activated copia retrotransposons Tos17 and Tos19 (Lullaby), a pericentromeric clustered high-copy-number non-autonomous gypsy retrotransposon Dasheng, two copia retrotransposons Osr4 and Osr13, a hAT-tip100 transposon DaiZ, a MITE transposon mPing, and a LINE element LINE1-6_OS. We confirmed the transposition of these TEs by PCR and/or Southern analysis, and showed that the transposition depends on the oscmt3a mutation. These results demonstrated that OsCMT3a-mediated non-CG DNA methylations play a critical role in development and in the suppression of a wide spectrum of TEs. These in planta mobile TEs are significant for studying the interaction between TEs and the host genome, and for rice functional genomics. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
No preview · Article · Aug 2015 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: Key message:
We identified the candidate gene conferring yellow wilt resistance (YR) in B. oleracea . This work will facilitate YR breeding programs for B. oleracea and its closely related species. Yellow wilt disease is one of the most serious diseases of cabbage worldwide. Type A resistance to the disease is controlled by a single dominant gene that is used in cabbage breeding. Our previous QTL study identified the FocBo1 locus controlling type A resistance. In this study, the FocBo1 locus was fine-mapped by using 139 recombinant F2 plants derived from resistant cabbage (AnjuP01) and susceptible broccoli (GCP04) DH lines. As a result, we successfully delimited the location of FocBo1 within 1.00 cM between markers, BoInd 2 and BoInd 11. Analysis of BAC and cosmid sequences corresponding to the FocBo1 locus identified an orthologous gene of Bra012688 that was recently identified as an candidate gene that confers yellows resistance in Chinese cabbage. The candidate gene-specific DNA markers and phenotypes in F1 cabbage cultivars and their selfed F2 populations showed a perfect correlation. Our identification of the candidate gene for FocBo1 will assist introduction of fusarium resistance into B. oleracea cultivars and contribute further understanding of interaction between Brassica plants and fusarium.
Preview · Article · Oct 2014 · Theoretical and Applied Genetics
[Show abstract][Hide abstract] ABSTRACT: Long terminal repeat retrotransposons occupy a large portion of genomes in flowering plants. In spite of their abundance, the majority are silenced and rarely transpose. One of the examples of a highly active retrotransposon is Lotus Retrotransposon 1 (LORE1), of the model legume Lotus japonicus (Regel) K. Larsen (syn. Lotus corniculatus L. var. japonicus Regel). LORE1 has several unusual characteristics that are interesting both for studying evolutional genomics and for the use of LORE1 as a genetic tool. In this review, we present the characteristics of LORE1 and discuss the biological significance of LORE1 as a member of chromovirus, a chromodomain containing clade of the Gypsy superfamily. Then we discuss possibilities and methodologies for using endogenous transposable elements as mutagens to generate gene tagging populations in plants.
[Show abstract][Hide abstract] ABSTRACT: Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor (SNARE) proteins are crucial for signal transduction and development in plants. Here, we investigate a Lotus japonicus symbiotic mutant defective in one of the SNARE proteins. When in symbiosis with rhizobia, the growth of the mutant was retarded compared with that of the wild-type plant. Although the mutant formed nodules, these exhibited lower nitrogen fixation activity than the wild type. The rhizobia were able to invade nodule cells, but enlarged symbiosomes were observed in the infected cells. The causal gene, designated LjSYP71 (for L. japonicus syntaxin of plants71), was identified by map-based cloning and shown to encode a Qc-SNARE protein homologous to Arabidopsis (Arabidopsis thaliana) SYP71. LjSYP71 was expressed ubiquitously in shoot, roots, and nodules, and transcripts were detected in the vascular tissues. In the mutant, no other visible defects in plant morphology were observed. Furthermore, in the presence of combined nitrogen, the mutant plant grew almost as well as the wild type. These results suggest that the vascular tissues expressing LjSYP71 play a pivotal role in symbiotic nitrogen fixation in L. japonicus nodules.
[Show abstract][Hide abstract] ABSTRACT: We established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population was composed of 2450 plant lines, from which a total of 4532 flanking sequence tags of LORE1 were recovered by pyrosequencing. The two-dimensional arrangement of the plant population, together with the use of multiple identifier sequences in the primers used to amplify the flanking regions, made it possible to trace insertions back to the original plant lines. The large-scale detection of new LORE1 insertion sites revealed a preference for genic regions, especially in exons of protein-coding genes, which is an interesting feature to consider in the interaction between host genomes and chromoviruses, to which LORE1 belongs, a class of retrotransposon widely distributed among plants. Forward screening of the symbiotic mutants from the population succeeded to identify five symbiotic mutants of known genes. These data suggest that LORE1 is robust as a genetic tool.
[Show abstract][Hide abstract] ABSTRACT: The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total
length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets.
They accounted for ∼95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete
and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated
that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny
was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total
of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic
sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying
information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research
with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic
sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.
[Show abstract][Hide abstract] ABSTRACT: Alignment of chromodomains. Chromodomains of two cellular proteins and five chromoviruses were aligned using CLUSTAL W (available at the DDBJ web site: http://clustalw.ddbj.nig.ac.jp/top-j.html). Arrowheads indicate the three amino acid residues in the chromodomain of HP1a that interact with methylated lysine residues on histone H3; these are highly conserved in authentic cellular chromodomains and group I chromodomains in chromoviruses ,. Chromodomain sequences in LORE1 and LORE2 were predicted using Pfam (http://pfam.sanger.ac.uk/) based on their nucleotide sequences. These chromodomains are also classified in group II, according to previous work ,. Other sequences were obtained from . Dm HP1a, Drosophila melanogaster HP1a; AT LHP1∶TFL2, Arabidopsis thaliana Terminal Flower 2; Mg MAGGY, Magnaporthe oryzae MAGGY; Lj LORE2, Lotus japonicus LORE2; Os CHDII, Oryza sativa RIRE3-like element; Lj LORE1a, Lotus japonicus LORE1a; At Tma, Arabidopsis thaliana TMA.
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[Show abstract][Hide abstract] ABSTRACT: Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposable elements in plants. Recent studies have identified chromoviruses to be a widely distributed lineage of Gypsy elements. These elements contain chromodomains in their integrases, which suggests a preference for insertion into heterochromatin. In turn, this preference might have contributed to the patterning of heterochromatin observed in host genomes. Despite their potential importance for our understanding of plant genome dynamics and evolution, the regulatory mechanisms governing the behavior of chromoviruses and their activities remain largely uncharacterized. Here, we report a detailed analysis of the spatio-temporal activity of a plant chromovirus in the endogenous host. We examined LORE1a, a member of the endogenous chromovirus LORE1 family from the model legume Lotus japonicus. We found that this chromovirus is stochastically de-repressed in plant populations regenerated from de-differentiated cells and that LORE1a transposes in the male germline. Bisulfite sequencing of the 5' LTR and its surrounding region suggests that tissue culture induces a loss of epigenetic silencing of LORE1a. Since LTR promoter activity is pollen specific, as shown by the analysis of transgenic plants containing an LTR::GUS fusion, we conclude that male germline-specific LORE1a transposition in pollen grains is controlled transcriptionally by its own cis-elements. New insertion sites of LORE1a copies were frequently found in genic regions and show no strong insertional preferences. These distinctive novel features of LORE1 indicate that this chromovirus has considerable potential for generating genetic and epigenetic diversity in the host plant population. Our results also define conditions for the use of LORE1a as a genetic tool.
[Show abstract][Hide abstract] ABSTRACT: SSAP analysis for detecting new LORE1 insertions. T0 and T1 plants used in Figure 1B were analyzed by 5′ and 3′ SSAP to detect new LORE1 insertions. Bands marked with red asterisks were confirmed by PCR to have originated from new insertions in the T1 plant.
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[Show abstract][Hide abstract] ABSTRACT: LORE1 activity over generations. The results from Southern blots of genomic DNA digested with Hind III and hybridized with the probe indicated in Figure 1A are shown. (A) Continuing LORE1 transposition in T1 plants that already possess an increased number of LORE1 elements. LORE1 copy number was analyzed by genomic Southern blot analysis in one T1 plant and five T2 progeny from each of three plant lines (A–C). New bands were detected in T2 progeny, suggesting that LORE1is still active in T1. (B) LORE1 is inactivated in the nup133-3 mutant line. Genomic Southern blot detected an additional band in the mutant plants; however, the absence of polymorphic bands among the nup133-3 plants indicates no transposition after the initial activation giving rise to the nup133-3 allele. These data indicate that LORE1 has been repressed, at least in the progeny analyzed.
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[Show abstract][Hide abstract] ABSTRACT: Promoter activity of the LORE1a LTR is demonstrated in Arabidopsis. Histochemical analysis of GUS expression in Arabidopsis plants transformed with a LORE1a LTR::GUS fusion. (A) Inflorescence assayed for 48 h. The long incubation revealed that the LTR exhibits promoter activity in the young developing anthers of flower buds marked with black arrowheads. GUS activity in the pollinated flower, marked with a white arrowhead, was more visible after this prolonged incubation than after the 12 h incubation shown in Figure 5F. (B) Close-up of the ovary of the pollinated flower marked by the white arrowhead in (A). Blue stained pollen tubes running to the ovules (O) and a bundle of pollen tubes in the transmitting tract were observed. (C and E) Dark field images of cross-sections of the youngest (C) and oldest (E) GUS-positive anthers shown in (A). Anthers were embedded in Technovit 7100 (Heraeus Kulzer) and sectioned. GUS activity was visualized as red signals. (D,F) DIC images of the same samples shown in (C,E), respectively. Higher GUS activity was detected in the surrounding cell layers than in young, developing pollen grains (pg), expected to be undergoing meiosis (C,D) and mitosis I (E,F) stages, respectively.
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[Show abstract][Hide abstract] ABSTRACT: Cytosine methylation status at three Alu I sites in the 5′ LTR of LORE1a is compared between leaves and flowers. Genomic Southern blot detected fragments containing 5′ flanking DNA from LORE1a. DNA samples of control Gifu and four T0 plants (nos. 3, 11, 30, and 45), extracted from leaves (left) and flowers (right) respectively, were digested with Hind III alone or double digested with Hind III and Alu I. Plants marked with + show transpositional activity of LORE1 and those marked with - do not. Molecular sizes of the DNA makers and the bands detected are indicated on the left. The banding patterns observed in leaves and flowers were consistent with each other.
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