Medicago truncatula PHO2 genes have distinct roles in phosphorus homeostasis and symbiotic nitrogen fixation

Three PHO2-like genes encoding putative ubiquitin-conjugating E2 enzymes of Medicago truncatula were characterized for potential roles in phosphorous (P) homeostasis and symbiotic nitrogen fixation (SNF). All three genes, MtPHO2A, B and C, contain miR399-binding sites characteristic of PHO2 genes in other plant species. Distinct spatiotemporal expression patterns and responsiveness of gene expression to P- and N-deprivation in roots and shoots indicated potential roles, especially for MtPHO2B, in P and N homeostasis. Phenotypic analysis of pho2 mutants revealed that MtPHO2B is integral to Pi homeostasis, affecting Pi allocation during plant growth under nutrient-replete conditions, while MtPHO2C had a limited role in controlling Pi homeostasis. Genetic analysis also revealed a connection between Pi allocation, plant growth and SNF performance. Under N-limited, SNF conditions, Pi allocation to different organs was dependent on MtPHO2B and, to a lesser extent, MtPHO2C and MtPHO2A. MtPHO2A also affected Pi homeostasis associated with nodule formation. Thus, MtPHO2 genes play roles in systemic and localized, i.e., nodule, P homeostasis affecting SNF

Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe Context-Dependent Assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations

DRB2 Is Required for MicroRNA Biogenesis in Arabidopsis thaliana

Background The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants). Principal Findings Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants. Conclusions/Significance Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue

Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance

Abstract Medicago truncatula, model legume and alfalfa relative, has served as an essential resource for advancing our understanding of legume physiology, functional genetics, and crop improvement traits. Necrotrophic fungus, Ascochyta medicaginicola, the causal agent of spring black stem (SBS) and leaf spot is a devasting foliar disease of alfalfa affecting stand survival, yield, and forage quality. Host resistance to SBS disease is poorly understood, and control methods rely on cultural practices. Resistance has been observed in M. truncatula accession SA27063 (HM078) with two recessively inherited quantitative-trait loci (QTL), rnpm1 and rnpm2, previously reported. To shed light on host resistance, we carried out a de novo genome assembly of HM078. The genome, referred to as MtHM078 v1.0, is comprised of 23 contigs totaling 481.19 Mbp. Notably, this assembly contains a substantial amount of novel centromere-related repeat sequences due to deep long-read sequencing. Genome annotation resulted in 98.4% of BUSCO fabales proteins being complete. The assembly enabled sequence-level analysis of rnpm1 and rnpm2 for gene content, synteny, and structural variation between SBS-resistant accession SA27063 (HM078) and SBS-susceptible accession A17 (HM101). Fourteen candidate genes were identified, and some have been implicated in resistance to necrotrophic fungi. Especially interesting candidates include loss-of-function events in HM078 because they fit the inverse gene-for-gene model, where resistance is recessively inherited. In rnpm1, these include a loss-of-function in a disease resistance gene due to a premature stop codon, and a 10.85 kbp retrotransposon-like insertion disrupting a ubiquitin conjugating E2. In rnpm2, we identified a frameshift mutation causing a loss-of-function in a glycosidase, as well as a missense and frameshift mutation altering an F-box family protein. This study generated a high-quality genome of HM078 and has identified promising candidates, that once validated, could be further studied in alfalfa to enhance disease resistance

Genome editing in alfalfa (Medicago sativa) to hyper-accumulate phosphate

Poster presentation at the International Forage and Turf Breeding Conference, March 2019.Rock phosphate, the main source of phosphate (P) for crop fertilizers, is a finite resource that is predicted to be depleted in 50-100 years. P is a critical nutrient in agriculture and its application can dramatically improve plant productivity. However, many soils have excess amounts of P from application of animal manures and runoff of phosphate from agricultural lands is the major source of nonpoint water pollution in the Midwestern US. The goal of this project is to create mutations by gene editing in the ubiquitin E2 conjugating enzyme PHO2, involved in P signaling and P homeostasis in alfalfa so that plants hyper-accumulate phosphate. Such plants could be used to reduce soil P levels and reclaim P for use as a fertilizer. From a draft diploid Medicago sativa genome scaffold sequence and the alfalfa transcriptome database (AGED), three PHO2 genes were identified. The genes, two of which are >99% homologous (a/b), each have seven exons interspersed by six introns. The open reading frames are 912 amino acids except when an alternate splice site is used in a/b gene transcript resulting in a 902 amino acid sequence. Alfalfa plants grown under P limiting conditions expressed low levels of the a/b transcripts with higher levels seen for PHO2c, while application of higher P induced increased expression mainly of the a/b transcripts. Under high P conditions, roots and shoots accumulated 4.1x and 2.5x more P than in low P conditions, respectively. An initial CRISPR/Cas9/Cys4 reagent targeting all three genes was generated and used to transform alfalfa cv. RegenSY. A total of 67 verified transgenic plants were screened by acrylamide gel shift assays, cloning, and sequencing to identify plants with mutations. Mutations ranging from a 1 bp insertion to a 25 bp deletion were identified in a total of 10 plants and some plants had multiple targets hit. Recently, a second attempt at CRISPR/Cas9 mutation utilized a cassette vector system with either the tRNA or Cys4 splicing system and exonuclease components. Initial screening results indicate that the tRNA splicing system may have yielded greater numbers of mutations. TaqMan probes were designed to identify plants with changes in the target sites and were verified by restriction digestions, cloning, and sequencing. Data on inheritance of mutations and phosphate accumulation in edited plants will be presented. The results of these experiments demonstrate that editing of multiple targets can be accomplished in alfalfa, although the tetraploid inheritance of genes complicates analysis

Further Disruption of the TAS3 Pathway via the Addition of the AGO7 Mutation to the DRB1, DRB2 or DRB4 Mutations Severely Impairs the Reproductive Competence of Arabidopsis thaliana

The previous assignment of functional roles for AGO7, and the DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2, and DRB4, in either microRNA (miRNA) or trans-acting small-interfering RNA (tasiRNA) production allowed for use of the loss-of-function mutant lines, drb1, drb2, drb4, and ago7, to further functionally characterize the TAS3 pathway in Arabidopsis thaliana (Arabidopsis). Towards achieving this goal, we also describe the developmental and molecular phenotypes expressed by three newly generated Arabidopsis lines, the drb1ago7, drb2ago7, and drb4ago7 double mutants. We show that the previously reported developmental abnormalities displayed by the drb1, drb2, drb4, and ago7 single mutants, are further exacerbated in the drb1ago7, drb2ago7, and drb4ago7 double mutants, with rosette area, silique length, and seed set all impaired to a greater degree in the double mutants. Molecular assessment of the TAS3 pathway in the floral tissues of the seven analyzed mutants revealed that DRB1 is the sole DRB required for miR390 sRNA production. However, DRB2 and DRB4 appear to play secondary roles at this stage of the TAS3 pathway to ensure that miR390 sRNA levels are tightly maintained. We further show that the expression of the TAS3-derived tasiARF target genes, AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4, was altered in drb1ago7, drb2ago7, and drb4ago7 flowers. Altered ARF2, ARF3, and ARF4 expression was in turn demonstrated to lead to changes in the level of expression of KAN1, KAN3, and KAN4, three KANADI transcription factor genes known to be transcriptionally regulated by ARF2, ARF3, and ARF4. Taken together, the demonstrated relationship between altered ARF and KAN gene expression in drb1ago7, drb2ago7 and drb4ago7 flowers, could, in part, explain the more severe developmental defects displayed by the double mutants, compared to milder impact that loss of only a single piece of TAS3 pathway protein machinery was demonstrated to have on drb1, drb2, drb4 and ago7 reproductive development

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 5′ thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms

Additional file 1 of Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance

Supplementary Material 1

RNA silencing and its application in functional genomics

\ud \ud \ud \ud \ud \ud \u

MiRNA accumulation in the SAM region of <i>drb235</i> plants.

<p>Fold changes of the five <i>MIR</i> gene families with the most highly elevated or reduced accumulation in the SAM region of <i>drb235</i> plants. Five <i>MIR</i> gene families with wild-type accumulation are also listed. <i>MIR</i> gene family accumulation was classed as being elevated or reduced in <i>drb235</i> plants if the fold change was greater than ±2.0.</p>1<p>elevated miRNA class representative.</p>2<p>unchanged miRNA class representative.</p>3<p>reduced miRNA class representative.</p