A plant RNA virus hijacks endocytic proteins to establish its infection in plants.

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor-mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin-related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co-opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV-infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1-dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2ß) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2ß impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2ß are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Dynamin-Like Proteins of Endocytosis in Plants Are Coopted by Potyviruses To Enhance Virus Infection.

Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes, including responses to pathogen attack. Here, we report the identification of Glycine max (soybean) endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a bottom-up proteomics approach. Knockdown of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B in Arabidopsis thaliana (AtDRP2A, AtDRP2B), homologs of GmSDL5A, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both A. thalianadrp2a (atdrp2a) and atdrp2b knockout mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI, and 6K2. Of these viral proteins, VPg, CP, and CI are essential for viral intercellular movement, and 6K2, VPg, and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together, these data suggest that dynamin-like proteins are novel host factors of potyviruses and that endocytic processes are involved in potyvirus infection.IMPORTANCE It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.

Genetic mapping and validation of the loci controlling 7S α' and 11S A-type storage protein subunits in soybean [Glycine max (L.) Merr.].

KEY MESSAGE: Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F2 population, which were validated with an F5 RIL population. The storage protein globulins ß-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F2 mapping population (n = 448) and an F5 RIL population (n = 180) were developed by crossing high protein cultivar 'Harovinton' with the breeding line SQ97-0263_3-1a, which lacks the 7S α', 11S A1, 11S A2, 11S A3 and 11S A4 subunits. The storage protein composition of each individual in the F2 and F5 populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F2 plants to identify genomic regions controlling the 7S α' and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A1), Chromosome 10 (7S α' and 11S A4), and Chromosome 13 (11S A3), which were also validated in the F5 RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.

Cadmium treatment alters the expression of five genes at the Cda1 locus in two soybean cultivars [Glycine max (L.) Merr].

Westag 97 has larger capacity of Cd accumulation in roots which prevents Cd from translocating into stems and leaves; conversely, AC Hime has smaller capacity of Cd accumulation in roots; more Cd is transported into stems and leaves. The different capacity of Cd in roots between Westag 97 and AC Hime causes the different Cd concentration in seeds. Meanwhile, according to the different expression levels of RSTK, ISCP, and H(+)-ATPase between Westag 97 and AC Hime, RSTK may be involved in transporting Cd into stems and leaves; H(+)-ATPase may be correlated to the capacity of Cd accumulation in roots; and Cd caused some changes of fundamental life process which leaded to the different expression patterns of ISCP between Westag 97 and AC Hime.

Molecular analysis of glycinin genes in soybean mutants for development of gene-specific markers.

Soybean mutant lines that differ in 11S glycinin and 7S ß-conglycinin seed storage protein subunit compositions were developed. These proteins have significant influence on tofu quality. The molecular mechanisms underlying the mutant lines are unknown. In this study, gene-specific markers for five of the glycinin genes (Gy1 to Gy5) were developed using three 11S null lines, two A(4) null Japanese cultivars, Enrei and Raiden, and a control cultivar, Harovinton. Whereas gene-specific primers produced the appropriate products in the control cultivar for the Gy1, Gy2, Gy3 and Gy5 genes, they did not amplify in mutants missing the A(1a)B(2), A(2)B(1a), A(1b) B(1b), and A(3)B(4) subunits. However, ecotype targeting induced local lesions in genomes (EcoTILLING) and sequencing analysis revealed that the absence of the A(4) peptide in the mutants is due to the same point mutation as that in Enrei and Raiden. Selection efficiency of the gene-specific primer pairs was tested using a number of breeding lines segregating for the different subunits. Primer pairs specific to each of the Gy1, Gy2, Gy3, and Gy5 genes can be used to detect the presence or absence of amplification in normal or mutant lines. The Gy4 null allele can be selected for by temperature-switch PCR (TS-PCR) for identification of the A(4) (G4) null genotypes. In comparison to protein analysis by SDS-PAGE, gene-specific markers are easier, faster and more accurate for analysis, they do not have to use seed, and can be analyzed at any plant growth stage for marker-assisted selection.

Development of candidate gene markers associated to common bacterial blight resistance in common bean.

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap), is a major yield-limiting factor of common bean (Phaseolus vulgaris L.) production around the world. Two major CBB-resistant quantitative trait loci (QTL), linked to the sequence characterized amplified region markers BC420 and SU91, are located at chromosomes 6 and 8, respectively. Using map-based cloning approach, four bacterial artificial chromosome (BAC) clones from the BC420-QTL locus and one BAC clone containing SU91 were sequenced by Roche 454 technique and subsequently assembled using merged assemblies from three different programs. Based on the quality of the assembly, only the sequences of BAC 32H6 and 4K7 were used for candidate gene marker (CGM) development and candidate gene (CG) selection. For the BC420-QTL locus, 21 novel genes were predicted in silico by FGENESH using Medicago gene model, whereas 16 genes were identified in the SU91-QTL locus. For each putative gene, one or more primer pairs were designed and tested in the contrasting near isogenic lines. Overall, six and nine polymorphic markers were found in the SU91- and BC420-QTL loci, respectively. Afterwards, association mapping was conducted in a breeding population of 395 dry bean lines to discover marker-trait associations. Two CGMs per each locus showed better association with CBB resistance than the BC420 and SU91 markers, which include BC420-CG10B and BC420-CG14 for BC420_QTL locus, and SU91-CG10 and SU91-CG11 for SU91_QTL locus. The strong associations between CBB resistance and the CGs 10 and 14 from BC420_QTL locus and the CGs 10 and 11 from SU91_QTL locus indicate that the genes 10 and 14 from the BC420 locus are potential CGs underlying the BC420_QTL locus, whereas the genes 10 and 11 from the SU91 locus are potential CGs underlying the SU91_QTL locus. The superiority of SU91-CG11 was further validated in a recombinant inbred line population Sanilac × OAC 09-3. Thus, co-dominant CGMs, BC420-CG14 and SU91-CG11, are recommended to replace BC420 and SU91 for marker-assisted selection of common bean with resistance to CBB.

Bacterial artificial chromosome libraries of pulse crops: characteristics and applications.

Pulse crops are considered minor on a global scale despite their nutritional value for human consumption. Therefore, they are relatively less extensively studied in comparison with the major crops. The need to improve pulse crop production and quality will increase with the increasing global demand for food security and people's awareness of nutritious food. The improvement of pulse crops will require fully utilizing all their genetic resources. Bacterial artificial chromosome (BAC) libraries of pulse crops are essential genomic resources that have the potential to accelerate gene discovery and enhance molecular breeding in these crops. Here, we review the availability, characteristics, applications, and potential applications of the BAC libraries of pulse crops.

Selection of reference genes for normalization of qRT-PCR analysis of differentially expressed genes in soybean exposed to cadmium.

Accurate normalization of gene expression with qRT-PCR depends on the use of appropriate reference genes (RGs) for the species under a given set of experimental conditions. Multiple RGs for gene expression analysis of soybean exposed to heavy metal stress treatment have not been reported in the literature. In this study, we evaluated the expression stability of ten candidate RGs in leaves, roots and stems of two soybean cultivars exposed to cadmium (Cd). Based on the geNorm and NormFinder analysis, ACT3, PP2A, ELF1B and F-box were the most stable RGs in these gene expression studies. In contrast, G6PD, UBC2, TUB, and ELF1A were the most variable ones and should not be used as RGs in these experimental conditions.

Application of image analysis in studies of quantitative disease resistance, exemplified using common bacterial blight-common bean pathosystem.

The effectiveness of image analysis (IA) compared with an ordinal visual scale, for quantitative measurement of disease severity, its application in quantitative genetic studies, and its effect on the estimates of genetic parameters were investigated. Studies were performed using eight backcross-derived families of common bean (Phaseolus vulgaris) (n = 172) segregating for the molecular marker SU91, known to be associated with a quantitative trait locus (QTL) for resistance to common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans. Even though both IA and visual assessments were highly repeatable, IA was more sensitive in detecting quantitative differences between bean genotypes. The CBB phenotypic difference between the two SU91 genotypic groups was consistently more than fivefold for IA assessments but generally only two- to threefold for visual assessments. Results suggest that the visual assessment results in overestimation of the effect of QTL in genetic studies. This may have been caused by lack of additivity and uneven intervals of the visual scale. Although visual assessment of disease severity is a useful tool for general selection in breeding programs, assessments using IA may be more suitable for phenotypic evaluations in quantitative genetic studies involving CBB resistance as well as other foliar diseases.

Novel QTL for Low Seed Cadmium Accumulation in Soybean.

Soybean is a valuable crop, used in animal feed and for human consumption. Selecting soybean cultivars with low seed cadmium (Cd) concentration is important for the purpose of minimizing the transfer of Cd into the human body. To ensure international trade, farmers need to produce soybean that meets the European Union (EU) Cd limit of 0.2 mg kg-1. In this study, we evaluated two populations of recombinant inbred lines (RILs), X5154 and X4050, for seed Cd accumulation. Linkage maps were constructed with 325 and 280 polymorphic simple sequence repeat (SSR) markers, respectively, and used to identify a novel minor quantitative trait locus (QTL) on chromosome 13 in the X4050 population between SSR markers Satt522 and Satt218. Based on a gene ontology search within the QTL region, seven genes were identified as candidates responsible for low seed Cd accumulation, including Glyma.13G308700 and Glyma.13G309100. In addition, we confirmed the known major gene, Cda1, in the X5154 population and developed KASP and CAPS/dCAPS allele-specific markers for efficient marker-assisted breeding for Cda1.

Association mapping of common bacterial blight resistance QTL in Ontario bean breeding populations.

BACKGROUND: Common bacterial blight (CBB), incited by Xanthomonas axonopodis pv. phaseoli (Xap), is a major yield-limiting factor of common bean (Phaseolus vulgaris L.) production around the world. Host resistance is practically the most effective and environmentally-sound approach to control CBB. Unlike conventional QTL discovery strategies, in which bi-parental populations (F2, RIL, or DH) need to be developed, association mapping-based strategies can use plant breeding populations to synchronize QTL discovery and cultivar development. RESULTS: A population of 469 dry bean lines of different market classes representing plant materials routinely developed in a bean breeding program were used. Of them, 395 lines were evaluated for CBB resistance at 14 and 21 DAI (Days After Inoculation) in the summer of 2009 in an artificially inoculated CBB nursery in south-western Ontario. All lines were genotyped using 132 SNPs (Single Nucleotide Polymorphisms) evenly distributed across the genome. Of the 132 SNPs, 26 SNPs had more than 20% missing data, 12 SNPs were monomorphic, and 17 SNPs had a MAF (Minor Allelic Frequency) of less than 0.20, therefore only 75 SNPs were used for association study, based on one SNP per locus. The best possible population structure was to assign 36% and 64% of the lines into Andean and Mesoamerican subgroups, respectively. Kinship analysis also revealed complex familial relationships among all lines, which corresponds with the known pedigree history. MLM (Mixed Linear Model) analysis, including population structure and kinship, was used to discover marker-trait associations. Eighteen and 22 markers were significantly associated with CBB rating at 14 and 21 DAI, respectively. Fourteen markers were significant for both dates and the markers UBC420, SU91, g321, g471, and g796 were highly significant (p ≤ 0.001). Furthermore, 12 significant SNP markers were co-localized with or close to the CBB-QTLs identified previously in bi-parental QTL mapping studies. CONCLUSIONS: This study demonstrated that association mapping using a reasonable number of markers, distributed across the genome and with application of plant materials that are routinely developed in a plant breeding program can detect significant QTLs for traits of interest.

Molecular basis of seed lipoxygenase null traits in soybean line OX948.

The poor stability and off-flavors of soybean oil and protein products can be reduced by eliminating lipoxygenases from soybean seed. Mature seeds of OX948, a lipoxygenase triple null mutant line, do not contain lipoxygenase proteins. The objective of this study was to determine the molecular basis of the seed lipoxygenase null traits in OX948. Comparisons of the sequences for lipoxygenase 1 (Lx1) and lipoxygenase 2 (Lx2) genes in the mutant (OX948) with those in a line with normal lipoxygenase levels (RG10) showed that the mutations in these genes affected a highly conserved group of six histidines necessary for enzymatic activity. The OX948 mutation in Lx1 is a 74 bp deletion in exon 8, which introduces a stop codon that prematurely terminates translation. A single T-A substitution in Lx2 changes histidine H532 (one of the iron-binding ligands essential for L-2 activity) to glutamine. The mutation in the lipoxygenase 3 (Lx3) gene in OX948 is in the promoter region and represents two single base substitutions in a cis-acting AAATAC paired box. All three mutations would result in the loss of lipoxygenase activity in mature seed. The seed lipoxygenase gene mutation-based molecular markers could be used to accelerate and simplify breeding efforts for soybean cultivars with improved flavor.

Identification of candidate genes associated with CBB resistance in common bean HR45 (Phaseolus vulgaris L.) using cDNA-AFLP.

Common bacterial blight (CBB), incited by Xanthomonas axonopodis pv. phaseoli (Xap), is a serious seed-borne disease of common bean (Phaseolus vulgaris L.) in both temperate and tropical production zones. The line HR45 is highly resistant to Xap infection on leaves and pods in both field and greenhouse. To understand the molecular mechanisms underlying CBB resistance in HR45, cDNA-amplified fragment length polymorphism (AFLP) technique was used to identify the genes that are differentially expressed in the leaves of HR45 at different time-periods after inoculation. Selective amplifications with 34 primer combinations allowed the visualization of 2,448 transcript-derived fragments (TDFs) in infected leaves, and 259 (10.6%) of them were differentially expressed TDFs (DE-TDFs). Seventy-seven of the DE-TDFs were cloned and sequenced. Thirty-nine of the 77 (50.6%) DE-TDFs representing bean transcripts were not previously reported in any EST database. The expression patterns of 10 representative DE-TDFs were further confirmed by real-time RT-PCR. BLAST analysis suggested that 40% (31 of 77) of the DE-TDFs were homologous to the genes related to metabolism, photosynthesis, and cellular transport, whereas 28% (22 of 77) of the DE-TDFs showed homology to the genes involved in defence response, response to stimulus, enzyme regulation, and transcription regulation. Thus, the 22 pathogenesis-related DE-TDFs were selected as potential functional candidate genes (FCGs) in association with CBB resistance. Meanwhile, six of the DE-TDFs (1FCG and five other DE-TDFs) were in silico mapped to the distal region of the bean linkage group B6 (the genomic location containing the major CBB resistance QTL in HR45) and, therefore, were considered as positional candidate genes (PCGs). This study represents a first step towards the discovery of bean genes expressed upon Xap infection. This information will be useful for elucidating the molecular basis of the resistance response process and identifying the genes that underlie the CBB-resistance.

Pyramided QTL underlying tolerance to Phytophthora root rot in mega-environments from soybean cultivars 'Conrad' and 'Hefeng 25'.

Phytophthora root rot (PRR) of soybean (Glycine max (L.) Merr.) is the second most important cause of yield loss by disease in North America, surpassed only by soybean cyst nematode (Wrather et al. in Can J Plant Pathol 23:115-121, 2001). Tolerance can provide economically useful disease control, conditioning partial resistance of soybean to PRR. The aims of this study were to identify new quantitative trait loci (QTL) underlying tolerance to PRR, and to evaluate the effects of pyramided or stacked loci on the level of tolerance. A North American cultivar 'Conrad' (tolerant to PRR) was crossed with a northeastern China cultivar 'Hefeng 25' (tolerant to PRR). Through single-seed descent, 140 F2:5 and F2:6 recombinant inbred lines were advanced. A total of 164 simple sequence repeat (SSR) markers were used to construct a genetic linkage map. The percentage of seedling death was measured over 2 years (2007 and 2008) in the field at four naturally infested locations in Canada and China following additional soil infestation and in the greenhouse following inoculation with Phytophthora sojae isolate. A total of eight QTL underlying tolerance to PRR were identified, located in five linkage groups (F, D1b+w, A2, B1, and C2). The phenotypic variation contributed by the loci ranged from 4.24 to 27.98%. QPRR-1 (anchored in the interval of SSR markers Satt325 and Satt343 of LG F), QPRR-2 (anchored in the interval of Satt005 and Satt600 of LG D1b+w), and QPRR-3 (anchored in the interval of Satt579 and Sat_089 of LG D1b+w) derived their beneficial allele from 'Conrad'. They were located at chromosomal locations known to underlie PRR tolerance in diverse germplasm. Five QTL that derived beneficial alleles from 'Hefeng 25' were identified. The QTL (QPRR-1 to QPRR-7) that were detected across at least three environments were selected for loci stacking and to analyze the relationship between number of tolerance loci and disease loss percentage. The accumulation of tolerance loci was positively correlated with decreases in disease loss percentage. The pyramid of loci underlying tolerance to PRR provided germplasm useful for crop improvement by marker-assisted selection and may provide durable cultivar tolerance against the PRR disease.

Mapping and validation of simple sequence repeat markers linked to a major gene controlling seed cadmium accumulation in soybean [Glycine max (L.) Merr].

Daily consumption of cadmium (Cd) contaminated foods poses a risk to human health. Cultivar selection is an important method to limit Cd uptake and accumulation; however, analyzing grain Cd concentration is costly and time-consuming. Developing markers for low Cd accumulation will facilitate marker assisted selection (MAS). Inheritance studies using a threshold value of 0.2 mg kg(-1) for low and high and an F(2:3) population showed that low Cd accumulation in soybean seed is under the control of a major gene (Cda1, proposed name) with the allele for low accumulation being dominant. A recombinant inbred line (RIL) population (F(6:8)) derived from the cross AC Hime (high Cd accumulation) and Westag-97 (low Cd accumulation) was used to identify the DNA markers linked to Cda gene(s) or quantitative trait loci (QTLs) controlling low Cd accumulation. We screened 171 simple sequence repeat (SSR) primers that showed polymorphism between parents on the 166 RILs. Of these, 40 primers were newly developed from the soybean genomic DNA sequence. Seven SSR markers, SatK138, SatK139, SatK140 (0.5 cM), SatK147, SacK149, SaatK150 and SattK152 (0.3 cM), were linked to Cda1 in soybean seed. All the linked markers were mapped to the same linkage group (LG) K. The closest flanking SSR markers linked to Cda1 were validated using a parallel population (RILs) involving Leo x Westag-97. Linked markers were also validated with diverse soybean genotypes differing in their seed Cd concentration and showed that SSR markers SatK147, SacK149, and SattK152 clearly differentiated the high and low Cd accumulating genotypes tested. To treat Cd uptake as a quantitative trait, QTL analysis using a linkage map constructed with 161 markers identified a major QTL associated with low Cd concentration in the seeds. The QTL was also mapped to the same location as Cda1 on LG-K. This QTL accounted for 57.3% of the phenotypic variation. Potential candidate genes (genes with known or predicted function that could influence the seed Cd concentration) like protein kinase, putative Adagio-like protein, and plasma membrane H(+)-ATPase were found to be located in the locus of interest. Of the four SSR markers located in the region, SattK152 was localized in the plasma membrane H(+)-ATPase gene. SSR markers closely linked to Cda1 in seeds of soybean were identified and have potential to be used for MAS to develop low Cd accumulating cultivars in a breeding program.

Mutagenesis of seed storage protein genes in Soybean using CRISPR/Cas9.

OBJECTIVE: Soybean seeds are an important source of vegetable proteins for both food and industry worldwide. Conglycinins (7S) and glycinins (11S), which are two major families of storage proteins encoded by a small family of genes, account for about 70% of total soy seed protein. Mutant alleles of these genes are often necessary in certain breeding programs, as the relative abundance of these protein subunits affect amino acid composition and soy food properties. In this study, we set out to test the efficiency of the CRISPR/Cas9 system in editing soybean storage protein genes using Agrobacterium rhizogenes-mediated hairy root transformation system. RESULTS: We designed and tested sgRNAs to target nine different major storage protein genes and detected DNA mutations in three storage protein genes in soybean hairy roots, at a ratio ranging from 3.8 to 43.7%. Our work provides a useful resource for future soybean breeders to engineer/develop varieties with mutations in seed storage proteins.

Genome-wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome-wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP-tagged CLF/SWN under their respective native promoters and used them for ChIP-seq analyses to profile the genome-wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild-type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA-like and Telo-box-like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

GmHMA3 sequesters Cd to the root endoplasmic reticulum to limit translocation to the stems in soybean.

A single point-mutation in GmHMA3 (Glycine max heavy metal-associated ATPase; a wild type allele cloned from a low Cd-accumulated soybean) is closely associated with seed cadmium (Cd) concentration. It is linked to Cd transportation in yeast, and is primarily expressed in the roots of plants. We hypothesized that the function of GmHMA3w in soybean would be akin to that of OsHMA3 in rice, which expresses in the root tonoplast and sequestrates Cd into the root vacuole to reduce Cd translocation to the shoots and limit its accumulation in the seeds. In this study, the transient expression of the GmHMA3w-GFP fusion protein in rice mesophyll protoplasts indicated that the subcellular localization of GmHAM3w was in the endoplasmic reticulum (ER). Overexpression of GmHMA3w increased the Cd concentration in the roots, decreased the Cd concentration in the stems, and did not affect the Cd concentration in the leaves. Additionally, its overexpression did not alter the Cd concentration across the whole plant. These findings indicated that GmHMA3w does not influence the Cd uptake, but limits the translocation of Cd from the roots to the stems. GmHMA3w thus acts in metal transportation. Assessment of the subcellular distribution of Cd indicated that GmHMA3w facilitated transport of Cd from the cell wall fraction to the organelle fraction, and then sequestrated Cd into the root ER, thus limiting its translocation to the stems. Additionally, the results also suggested that the ER constitutes a site of particularly high Cd sensitively in plants.

Correction: Genome-Wide Investigation Using sRNA-Seq, Degradome-Seq and Transcriptome-Seq Reveals Regulatory Networks of microRNAs and Their Target Genes in Soybean during Soybean mosaic virus Infection.

[This corrects the article DOI: 10.1371/journal.pone.0150582.].