Identification and characterization of a temperature sensitive chlorotic soybean mutant.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "vir1" phenotype results in smaller stature, weaker stems, and a smaller root system with smaller nodules. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells and RNA sequencing data indicates it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice homolog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean vir1 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild type Glyma.07G102300 in the knockout mutant of the Arabidopsis homolog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the vir1 phenotype.

The GUS Reporter System in Flower Development Studies.

The ß-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development in a broad range of species. Transcriptional and translational GUS fusions are used to characterize gene and protein expression patterns, respectively, during reproductive development. Additionally, GUS reporters can be used to map cis-regulatory elements within promoter sequences and to investigate whether genes are regulated post-transcriptionally. Gene trap/enhancer trap GUS constructs can be used to identify novel genes involved in flower development and marker lines useful in mutant characterization. Flower development studies primarily have used the histochemical assay in which inflorescence tissue from transgenic plants containing GUS reporter genes are stained for GUS activity and examined as whole-mounts or subsequently embedded into wax and examined as tissue sections. In addition, quantitative GUS activity assays can be performed on either floral extracts or intact flowers using a fluorogenic GUS substrate. Another use of GUS reporters is as a screenable marker for plant transformation. A simplified histochemical GUS assay can be used to quickly identify transgenic tissues.

Pol V produced RNA facilitates transposable element excision site repair in Arabidopsis.

The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing : GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways ( MET1 ), in the repair of double-strand DNA breaks.

Transposase expression, element abundance, element size, and DNA repair determine the mobility and heritability of PIF/Pong/Harbinger transposable elements.

Introduction: Class II DNA transposable elements account for significant portions of eukaryotic genomes and contribute to genome evolution through their mobilization. To escape inactivating mutations and persist in the host genome over evolutionary time, these elements must be mobilized enough to result in additional copies. These elements utilize a "cut and paste" transposition mechanism that does not intrinsically include replication. However, elements such as the rice derived mPing element have been observed to increase in copy number over time. Methods: We used yeast transposition assays to test several parameters that could affect the excision and insertion of mPing and its related elements. This included development of novel strategies for measuring element insertion and sequencing insertion sites. Results: Increased transposase protein expression increased the mobilization frequency of a small (430 bp) element, while overexpression inhibition was observed for a larger (7,126 bp) element. Smaller element size increased both the frequency of excision and insertion of these elements. The effect of yeast ploidy on element excision, insertion, and copy number provided evidence that homology dependent repair allows for replicative transposition. These elements were found to preferentially insert into yeast rDNA repeat sequences. Discussion: Identifying the parameters that influence transposition of these elements will facilitate their use for gene discovery and genome editing. These insights in to the behavior of these elements also provide important clues into how class II transposable elements have shaped eukaryotic genomes.

Mobility of mPing and its associated elements is regulated by both internal and terminal sequences.

BACKGROUND: DNA transposable elements are mobilized by a "cut and paste" mechanism catalyzed by the binding of one or more transposase proteins to terminal inverted repeats (TIRs) to form a transpositional complex. Study of the rice genome indicates that the mPing element has experienced a recent burst in transposition compared to the closely related Ping and Pong elements. A previously developed yeast transposition assay allowed us to probe the role of both internal and terminal sequences in the mobilization of these elements. RESULTS: We observed that mPing and a synthetic mPong element have significantly higher transposition efficiency than the related autonomous Ping and Pong elements. Systematic mutation of the internal sequences of both mPing and mPong identified multiple regions that promote or inhibit transposition. Simultaneous alteration of single bases on both mPing TIRs resulted in a significant reduction in transposition frequency, indicating that each base plays a role in efficient transposase binding. Testing chimeric mPing and mPong elements verified the important role of both the TIRs and internal regulatory regions. Previous experiments showed that the G at position 16, adjacent to the 5' TIR, allows mPing to have higher mobility. Alteration of the 16th and 17th base from mPing's 3' end or replacement of the 3' end with Pong 3' sequences significantly increased transposition frequency. CONCLUSIONS: As the transposase proteins were consistent throughout this study, we conclude that the observed transposition differences are due to the element sequences. The presence of sub-optimal internal regions and TIR bases supports a model in which transposable elements self-limit their activity to prevent host damage and detection by host regulatory mechanisms. Knowing the role of the TIRs, adjacent sub-TIRs, and internal regulatory sequences allows for the creation of hyperactive elements.

Critical role for uricase and xanthine dehydrogenase in soybean nitrogen fixation and nodule development.

De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes-mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish-white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly-ß-hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen-fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root-associated gene traits when coupled with hairy root transformation.

Oxytetracycline and Streptomycin Resistance Genes in Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot in Peach.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017-2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTC R ) Xap strains were found in 2017 and 2020 from four orchards about 20-270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 µg/mL OTC, while one strain, R1, grew on ≤250 µg/mL OTC. Genome sequence analysis of four representative OTC R strains revealed a 14-20 kb plasmid carrying tetC, tetR, and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTC R Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 µg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 µg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 µg/mL OTC. However, in the R1 strain resistant to ≤250 µg/mL OTC, tetR was not expressed, possibly due to the presence of Tn3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC. To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.

Development of mPing-based activation tags for crop insertional mutagenesis.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

Tracking the origin of two genetic components associated with transposable element bursts in domesticated rice.

Transposable elements (TEs) shape genome evolution through periodic bursts of amplification. In this study prior knowledge of the mPing/Ping/Pong TE family is exploited to track their copy numbers and distribution in genome sequences from 3,000 accessions of domesticated Oryza sativa (rice) and the wild progenitor Oryza rufipogon. We find that mPing bursts are restricted to recent domestication and is likely due to the accumulation of two TE components, Ping16A and Ping16A_Stow, that appear to be critical for mPing hyperactivity. Ping16A is a variant of the autonomous element with reduced activity as shown in a yeast transposition assay. Transposition of Ping16A into a Stowaway element generated Ping16A_Stow, the only Ping locus shared by all bursting accessions, and shown here to correlate with high mPing copies. Finally, we show that sustained activity of the mPing/Ping family in domesticated rice produced the components necessary for mPing bursts, not the loss of epigenetic regulation.

A transgenic, visual screenable marker for soybean seeds.

Most soybean cultivars produce buff colored seeds due to a seed coat specific siRNA mechanism. This phenomenon is specifically limited to the seed coat and produces a strong visual effect, thus, a strategy to evade the silencing was used to produce a maternal transgenic marker for soybeans. Expression of a rice chalcone synthase transgene with little DNA sequence homology to the soybean siRNAs resulted in dark colored seed coats. This phenotype is the result of anthocyanin pigment production and does not appear to affect other tissues. This novel approach for producing an easily scored transgenic marker for soybean will facilitate high-throughput screening and analysis of transgenic soybean.

Precise repair of mPing excision sites is facilitated by target site duplication derived microhomology.

BACKGROUND: A key difference between the Tourist and Stowaway families of miniature inverted repeat transposable elements (MITEs) is the manner in which their excision alters the genome. Upon excision, Stowaway-like MITEs and the associated Mariner elements usually leave behind a small duplication and short sequences from the end of the element. These small insertions or deletions known as "footprints" can potentially disrupt coding or regulatory sequences. In contrast, Tourist-like MITEs and the associated PIF/Pong/Harbinger elements generally excise precisely, returning the genome to its original state. The purpose of this study was to determine the mechanisms underlying these excision differences, including the role of the host DNA repair mechanisms. RESULTS: The transposition of the Tourist-like element, mPing, and the Stowaway-like element, 14T32, were evaluated using yeast transposition assays. Assays performed in yeast strains lacking non-homologous end joining (NHEJ) enzymes indicated that the excision sites of both elements were primarily repaired by NHEJ. Altering the target site duplication (TSD) sequences that flank these elements reduced the transposition frequency. Using yeast strains with the ability to repair the excision site by homologous repair showed that some TSD changes disrupt excision of the element. Changing the ends of mPing to produce non-matching TSDs drastically reduced repair of the excision site and resulted in increased generation of footprints. CONCLUSIONS: Together these results indicate that the difference in Tourist and Stowaway excision sites results from transposition mechanism characteristics. The TSDs of both elements play a role in element excision, but only the mPing TSDs actively participate in excision site repair. Our data suggests that Tourist-like elements excise with staggered cleavage of the TSDs, which provides microhomology that facilitates precise repair. This slight modification in the transposition mechanism results in more efficient repair of the double stranded break, and thus, may be less harmful to host genomes by disrupting fewer genes.

Tnt1 retrotransposon mutagenesis: a tool for soybean functional genomics.

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean.

The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean.

Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.

Transposition of the Tourist-MITE mPing in yeast: an assay that retains key features of catalysis by the class 2 PIF/Harbinger superfamily.

BACKGROUND: PIF/Harbinger is the most recently discovered DNA transposon superfamily and is now known to populate genomes from fungi to plants to animals. Mobilization of superfamily members requires two separate element-encoded proteins (ORF1 and TPase). Members of this superfamily also mobilize Tourist-like miniature inverted repeat transposable elements (MITEs), which are the most abundant transposable elements associated with the genes of plants, especially the cereal grasses. The phylogenetic analysis of many plant genomes indicates that MITEs can amplify rapidly from one or a few elements to hundreds or thousands.The most active DNA transposon identified to date in plants or animals is mPing, a rice Tourist-like MITE that is a deletion derivative of the autonomous Ping element. Ping and the closely related Pong are the only known naturally active PIF/Harbinger elements. Some rice strains accumulate ~40 new mPing insertions per plant per generation. In this study we report the development of a yeast transposition assay as a first step in deciphering the mechanism underlying the amplification of Tourist-MITEs. RESULTS: The ORF1 and TPase proteins encoded by Ping and Pong have been shown to mobilize mPing in rice and in transgenic Arabidopsis. Initial tests of the native proteins in a yeast assay resulted in very low transposition. Significantly higher activities were obtained by mutation of a putative nuclear export signal (NES) in the TPase that increased the amount of TPase in the nucleus. When introduced into Arabidopsis, the NES mutant protein also catalyzed higher frequencies of mPing excision from the gfp reporter gene. Our yeast assay retains key features of excision and insertion of mPing including precise excision, extended insertion sequence preference, and a requirement for two proteins that can come from either Ping or Pong or both elements. CONCLUSIONS: The yeast transposition assay provides a robust platform for analysis of the mechanism underlying transposition catalyzed by the two proteins of PIF/Harbinger elements. It recapitulates all of the features of excision and reinsertion of mPing as seen in plant systems. Furthermore, a mutation of a putative NES in the TPase increased transposition both in yeast and plants.

Unexpected consequences of a sudden and massive transposon amplification on rice gene expression.

High-copy-number transposable elements comprise the majority of eukaryotic genomes where they are major contributors to gene and genome evolution. However, it remains unclear how a host genome can survive a rapid burst of hundreds or thousands of insertions because such bursts are exceedingly rare in nature and therefore difficult to observe in real time. In a previous study we reported that in a few rice strains the DNA transposon mPing was increasing its copy number by approximately 40 per plant per generation. Here we exploit the completely sequenced rice genome to determine 1,664 insertion sites using high-throughput sequencing of 24 individual rice plants and assess the impact of insertion on the expression of 710 genes by comparative microarray analysis. We find that the vast majority of transposable element insertions either upregulate or have no detectable effect on gene transcription. This modest impact reflects a surprising avoidance of exon insertions by mPing and a preference for insertion into 5' flanking sequences of genes. Furthermore, we document the generation of new regulatory networks by a subset of mPing insertions that render adjacent genes stress inducible. As such, this study provides evidence for models first proposed previously for the involvement of transposable elements and other repetitive sequences in genome restructuring and gene regulation.

Tuned for transposition: molecular determinants underlying the hyperactivity of a Stowaway MITE.

Miniature inverted repeat transposable elements (MITEs) are widespread in eukaryotic genomes, where they can attain high copy numbers despite a lack of coding capacity. However, little is known about how they originate and amplify. We performed a genome-wide screen of functional interactions between Stowaway MITEs and potential transposases in the rice genome and identified a transpositionally active MITE that possesses key properties that enhance transposition. Although not directly related to its autonomous element, the MITE has less affinity for the transposase than does the autonomous element but lacks a motif repressing transposition in the autonomous element. The MITE contains internal sequences that enhance transposition. These findings suggest that MITEs achieve high transposition activity by scavenging transposases encoded by distantly related and self-restrained autonomous elements.

Transposition of the rice miniature inverted repeat transposable element mPing in Arabidopsis thaliana.

An active miniature inverted repeat transposable element (MITE), mPing, was discovered by computer-assisted analysis of rice genome sequence. The mPing element is mobile in rice cell culture and in a few rice strains where it has been amplified to >1,000 copies during recent domestication. However, determination of the transposase source and characterization of the mechanism of transposition have been hampered by the high copy number of mPing and the presence of several candidate autonomous elements in the rice genome. Here, we report that mPing is active in Arabidopsis thaliana, where its transposition is catalyzed by three sources of transposase from rice: the autonomous Ping and Pong elements and by a cDNA derived from a Ping transcript. In addition to transposase, the product of a second element-encoded ORF of unknown function is also required for mPing transposition. Excision of mPing in A. thaliana is usually precise, and transposed copies usually insert into unlinked sites in the genome that are preferentially in or near genes. As such, this will be a valuable assay system for the dissection of MITE transposition and a potentially powerful tagging system for gene discovery in eukaryotes.