ABSTRACT Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-fucose). A nucleocytoplasmic O-fucosyltransferase occurs in the pathogen Toxoplasma gondii, the social amoeba Dictyostelium, and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium, is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that are involved in stress and nutritional responses. O-fucose was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity-purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium, and Arabidopsis, as well as O-fucose occurring on secretory proteins of Dictyostelium and mammalian cells except when blocked by further glycosylation. The antibodies label Toxoplasma, Acanthamoeba, and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-fucose, document the conservation of the O-fucome among unrelated protists, and enable the study of the O-fucomes of other organisms possessing O-fucosyltransferase-like genes.IMPORTANCEO-fucose (O-Fuc), a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet, the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii, and an unrelated protist, the social amoeba Dictyostelium discoideum. Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms, AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii.

Pigments provide a simple means to rapidly visually ascertain the quantities or presence of specific microbes in a complex community. The selection of pigment-producing colonies that are simple to differentiate from common colony phenotypes provides a high degree of certainty for the identity of pigment-tagged strains. Successful employment of pigment production is dependent on various intrinsic factors related to proper levels of gene expression and pigment production that are not always easy to predict and vary within each microbe. We have constructed a simple transposon system that incorporates the genes for the production of deoxyviolacein, a pigment produced from intracellular reserves of the amino acid tryptophan, to randomly insert these genes throughout the genome. This tool allows the user to select from many thousands of potential sites throughout a bacterial genome for an ideal location to generate the desired amount of pigment. We have applied this system to a small selection of endophytes and other model bacteria to differentiate these strains from complex communities and confirm their presence after several weeks in natural environments. We provide two examples of applications using the pigments to trace strains following introduction into plant tissues or to produce a reporter strain for extracellular nitrogen compound sensing. We recognize that this tool could have far broader utility in other applications and microbes, and describe the methodology for use by the greater scientific community.

The Arabidopsis thaliana glycosyl transferases SPINDLY (SPY) and SECRET AGENT (SEC) modify nuclear and cytosolic proteins with O-linked fucose or O-linked N-acetylglucosamine (O-GlcNAc), respectively. O-fucose and O-GlcNAc modifications can occur at the same sites. SPY interacts physically and genetically with GIGANTEA (GI), suggesting that it could be modified by both enzymes. Previously, we found that, when co-expressed in Escherichia coli, SEC modifies GI; however, the modification site was not determined. By analyzing the overlapping sub-fragments of GI, we identified a region that was modified by SEC in E. coli. Modification was undetectable when threonine 829 (T829) was mutated to alanine, while the T834A and T837A mutations reduced the modification, suggesting that T829 was the primary or the only modification site. Mapping using mass spectrometry detected only the modification of T829. Previous studies have shown that the positions modified by SEC in E. coli are modified in planta, suggesting that T829 is O-GlcNAc modified in planta.

ABSTRACT The plant growth-promoting bacterium Gluconacetobacter diazotrophicus was originally discovered in association with sugarcane plants as an endophyte. As a member of the small class of organisms defined as diazotrophs, G. diazotrophicus is capable of fixing nitrogen from the atmosphere and could serve an important role in minimizing the requirements for nitrogen from industrial-derived fertilizers. In addition to sugarcane, G. diazotrophicus is capable of forming endophyte associations with a variety of other important crops. It has been reported that this microbe requires micro-aerobic conditions to effectively fix nitrogen gas from the atmosphere through the enzyme nitrogenase, making it slightly more difficult to study the diazotrophic lifestyle in the laboratory. The ability of the strain to reside within the plant during growth means that any extracellular nitrogen released by this microbe would immediately become available to the plant host. For this reason, it is an ideal target for development as an improved biofertilizer strain. In this work, we constructed strains of G. diazotrophicus that result in enhanced ammonium release, as measured by growing with a closely associated algal strain under micro-aerobic conditions, and by further quantifying ammonium concentrations accumulated under micro-aerobic and aerobic growth. IMPORTANCE Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus. Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe.

Arabidopsis thaliana has two glycosyl transferases, SPINDLY (SPY) and SECRET AGENT (SEC), that modify nuclear and cytosolic protein with O-linked fucose and N-acetylglucosamine (GlcNAc), respectively. SPY interacts physically and genetically with GIGANTEA (GI). Previously, we reported that SEC substrates are O-GlcNAc modified when they are co-expressed in E. coli . By analyzing overlapping sub-fragments of GI, we found a region that was modified by SEC. Mutational mapping of the modified region was then performed. Modification was undetectable when threonine 829 was mutated to alanine (T829A) while the T834A and T837A mutations reduced modification suggesting that T829 was the primary or only modification site. Mapping using several enrichment and mass spectrometry methods all detected only modification of T829.

The complete genome sequences of two isolates of spiraea yellow leafspot virus (SYLSV) were determined. Spiraea (Spiraea x bumalda) 'Anthony Waterer' plants showing virus-like symptoms including yellow spotting and leaf deformation were used for sequencing. The viral genome of SYLSV-MN (Minnesota) and SYLSV-MD (Maryland) is 8,017bp in length. The sequences share 95% identity at the nucleotide level. Both isolates have the same genome organization containing three open reading frames (ORFs), with ORF3 being the largest, encoding a putative polyprotein of 232 kDa with conserved domains including a zinc finger, pepsin-like aspartate protease, reverse transcriptase (RT), and RNase H. Pairwise comparisons between members of the genus Badnavirus showed that gooseberry vein banding associated virus GB1 (HQ852248) and rubus yellow net virus isolate Baumforth's Seedling A (KM078034) were the closest related virus sequences to SYLSV, sharing 73% identity at the nucleotide level. Bacilliform virions with dimensions of 150 nm × 30 nm were observed in virus preparations from symptomatic, but not asymptomatic, plants.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.

Ninety-six symptomatic orchids representing 36 genera from seven orchid collections, including two conservatories, in Minnesota, USA were tested for cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV) using dipsticks (CymMV and ORSV), RT-PCR (OFV), and transmission electron microscopy (all viruses). CymMV was identified in 22% of the samples, OFV was identified in 17% of the samples, and ORSV was detected in 6% of the samples. Five percent of samples were infected with both CymMV and ORSV and 1% of samples were infected with both OFV and ORSV. Characteristic orchid virus symptoms of chlorotic and necrotic patterns were observed for the majority of infected orchids. Polyclonal antibodies were produced against Escherichia coli expressed OFV phosphoprotein (OFV P) and evaluated for use in plate trapped antigen-enzyme linked immunosorbent assay (PTA-ELISA). After an overnight incubation with PNPP the OFV P polyclonal antisera diluted from 1:1000–1:20,000 ( v /v) readily differentiated between fresh healthy and OFV infected orchid ( Phalaenopsis hybrid) tissue diluted from 1:5–1:20 ( w / v ). The high incidence of viruses detected in orchids (50%) suggests limited use of certified virus-free propagation stock and highlights the importance of sanitation to prevent transmission between plants.

Filamentous structures were observed in purified extracts from chrysanthemum, gerbera, sunflower and zinnia. When purified filament proteins were subjected to SDS-PAGE, the major protein associated with filaments from all three species has an apparent molecular mass of ≈25 kDa. Protein bands from chrysanthemum, gerbera, and zinnia were subjected to N-terminal protein sequencing while proteins from sunflower were sequenced by CID MS/MS. All of the sequences shared highest similarity to the kunitz trypsin inhibitor family. The sequencing results indicated that the proteins lacked the signal sequences. We tested the gerbera filament protein for glycosylation and found that it was a glycoprotein. Together these results indicate that the filaments are composed of mature KTI protein. This is the first report of a KTI assembling into filaments and the first report of a filament forming Asteraceae enzyme.

Citrus tristeza virus (CTV) is a plant pathogenic virus first reported infecting citrus plants in the 1930’s in Brazil and Argentina and is now widely spread through all citrus growing areas. A CTV isolate from an infected Pakistani sweet orange tree was selected for sequencing to subsequently produce recombinant CTV coat protein. The purified recombinant CTV coat protein was used as an antigen for the production of polyclonal antisera in rabbits. The antisera were tested in direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA) against various CTV isolates from Pakistan and T-30 from the USA. The antisera reacted strongly in DAC-ELISA with both homologous and heterologous isolates of CTV from infected citrus leaf tissue. These antisera were compared with a commercially available ELISA kit and were more sensitive to Pakistani isolates of CTV than the commercial ELISA kit. An inexpensive virus-free testing and certification program using the antisera produced here would provide farmers with the knowledge to eliminate CTV infected and unproductive trees, which could then be replaced with healthy young trees. The implementation of such a scheme would reduce the incidence of CTV in citrus orchards, reduce yield losses caused by CTV, and could lead to the elimination of CTV from Pakistan

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1-9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Aglaonema bacilliform virus (ABV), a member of the genus Badnavirus in the family Caulimoviridae, is associated with leaf deformation and chlorosis in Aglaonema modestum. The complete genome sequence of a Minnesota isolate of ABV was determined. The ABV genome is 7,178 bp in length and similar in size and organization to those of the members of the genus Badnavirus, containing three open reading frames (ORFs) with the potential to encode three proteins of 14.92, 13.33 and 207.95 kDa, respectively. The last ORF (ORF3) encodes a putative polyprotein with conserved domains, including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H domains, in that order. Phylogenetic analysis using the amino acid sequence of the ORF3 polyprotein showed that ABV clusters with several isolates of taro bacilliform CH virus (TaBCHV). Pairwise alignment using the highly conserved RT/RNase H region reveals that ABV has the highest level of identity (71%) at the nucleotide level to a Hawaiian isolate of TaBCHV.

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

The tomato PROCERA gene encodes a DELLA protein, and loss-of-function mutations derepress growth. We used CRISPR/Cas9 and a single guide RNAs (sgRNA) to target mutations to the PROCERA DELLA domain, and recovered several loss-of-function mutations and a dominant dwarf mutation that carries a deletion of one amino acid in the DELLA domain. This is the first report of a dominant dwarf PROCERA allele. This allele retains partial responsiveness to exogenously applied gibberellin. Heterozygotes show an intermediate phenotype at the seedling stage, but adult heterozygotes are as dwarfed as homozygotes.

DELLA proteins were first identified as inhibitors of gibberellin (GA) signalling but have since been shown to be involved in regulation of many growth responses. GA causes responses by triggering the destruction of DELLA proteins (Willige et al., 2007). These proteins contain a conserved DELLA domain, which is followed by a GRAS domain. The GRAS domain which defines a family of proteins, including the DELLA proteins, is a conserved domain named after the first three family members: GAI (gibberellic-acid insensitive), RGA (Repressor of ga1-3), and SCR (Scarecrow). The DELLA domain encompasses the DELLA, LExLE and TVHYNP motifs. These motifs are important for binding to the GA-bound GA receptor GID1, which is one of the initial steps in targeting the DELLA proteins for destruction by the 26S proteasome (Dill et al., 2001). The GA-GID1-DELLA complex interacts with the SLY1/GID2 SCF complex, which marks it for destruction by modifying it with ubiquitin. In this way, the suppression of GA signalling by DELLA is released and GA responses are activated. A number of mutants with mutations affecting the DELLA domain that increase the abundance of DELLA by weakening the interaction with the GA-GID1 complex are known (Ueguchi-Tanaka et al., 2007; Willige et al., 2007). Since these mutant DELLA proteins retain the ability to inhibit GA responses, the mutants are dwarfed. Tomato has one DELLA protein called PROCERA (PRO). There is a well-studied partial loss-of-function mutant, pro, which has a single amino acid substitution, valine to glutamate, in the VHVID motif of its GRAS domain (Bassel et al., 2008; Jupe et al., 1988). Strong recessive pro loss-of-function alleles, proTALEN and pro∆GRAS, were generated using transcription activator-like effector nucleases (TALENs) and an Ac/Ds system, respectively (Livne et al., 2015; Lor et al., 2014). Consistent with these mutations fully activating GA signalling, homozygous mutants are extremely tall, have light green leaves with smoother leaf margins, and do not respond to the GA biosynthesis inhibitor paclobutrazol (PAC) or GA treatment. Recently, a gain-of-function allele affected in the DELLA motif that causes mild dwarfing and reduced GA sensitivity was reported (Tomlinson et al., 2018) but strong gain-of-function alleles have not been reported. Here, we report the generation and characterization of strong dwarfing alleles produced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) system to induce intragenic suppressor mutations of proTALEN alleles. In the course of experiments to identify regenerating shoots in which the proTALEN loss-of-function mutation has been repaired due to CRISPR/cas9 stimulated homologous recombination, we recovered several dwarf plants with dark green serrated leaves (Figure 1a, b). The regenerated T0 plants were dwarfs and the dwarfing alleles PROGF8 and PROGF9 from plants #8 and #9, respectively, were heritable. Sequencing confirmed that the PROGF8 and PROGF9 are intragenic suppressors of proTALEN1 and proTALEN7 respectively and that each encodes a full-length mutant protein (Figure 1c, d). The protein encoded by PROGF8 is predicted to have a 12 amino acid insertion and 2 amino acid substitution affecting the LExLE motif, while the protein encoded by PROGF9 has a three amino acid deletion and an E to R substitution that affects the LExLE motif. PROGF8 was characterized further. F1 seedlings from a cross between plant #8 and wild-type (M82) were either extreme dwarfs with dark green leaves or wild-type in appearance (Figure 1e). Only the dwarf plants had inherited the PROGF8 allele indicating that it was dominant or semidominant. We tested the effect of PROGF8 on GA responsiveness. In contrast to wild-type M82 plants, which grew more rapidly when sprayed every 2 days with 50 μM GA3, PRO/PROGF8 were unaffected (Figure 1f, g). Because DELLA proteins promote GA synthesis, endogenous GA levels are highly elevated in plants carrying DELLA gain-of-function alleles (Talón et al., 1990). To determine if the apparent GA insensitivity of PRO/PROGF8 plants is because they contain saturating levels of endogenous bioactive GA, we treated PRO/PROGF8 (Figure 1h, i) and wild-type (not shown) plants with the GA biosynthesis inhibitor paclobutrazol (PAC) or a combination of PAC and GA3. PAC treatment reduced the height of both PRO/PROGF8 and wild-type plants and the GA3 treatment reversed this effect. The effects of the treatments were much smaller for PRO/PROGF8, indicating that, while PRO/PROGF8 responds to GA, it is nearly insensitive to it. While germination tests found that initially only one half of the newly harvested F1 seeds from a cross between the T0 plant #8 and M82 germinated, the non-germinated seeds germinated after they were scarified. The seedlings from seeds that did not require scarification were tall and did not carry the PROGF allele while all of the seedlings from seeds that required scarification were PRO/PROGF8 and dwarf (not shown). When we examined the germination kinetics after 4 months of storage, seeds that germinated by day 3 gave rise to tall plants whereas all seeds that germinated after day 3 produced dwarf seedlings (Figure 1j). After 10 days, 30% of the seeds had not germinated. Following scarification, these ungerminated seeds germinated and gave rise to dwarf seedlings. We also tested germination of fresh seeds harvested from selfed PRO/PROGF8 (Figure 1k). After 3 days, all of the seeds that produced wild-type stature seedlings had germinated (26%). The seeds that had not germinated could be divided into two groups. One group comprising 44% of the seeds germinated by day 10 and produced dwarf seedlings. The remaining seed required scarification and also produced dwarf seedlings. The observed ratios for the tall seedlings, dwarf seedlings from seed that germinated without scarification, and dwarf from seeds requiring scarification [Tall vs. Dwarf (non-scarified) vs. Dwarf (scarified)] fit a 1 : 2 : 1 ratio (three trials Chi-square range from 0.458 to 3.471, P value from 0.176 to 0.795), which suggested that the PROGF8 allele is semidominant. Consistent with this hypothesis, when genotyped, 16 out of 16 dwarf plants from seeds that germinated without scarification were heterozygous and 10 of 11 dwarf seedlings from seeds requiring scarification were homozygous for PROGF8 and the remaining plant was heterozygous. Plants from seeds that require scarification to germinate were shorter than the dwarf plants from seeds that germinated without scarification (not shown) indicating that proGF8 is also semidominant with respect to plant stature. Molecular modelling of PRO and PROGF8 predicts that PROGF8 protein has a longer disordered region between the LExLE and TVHYNP motifs and the intervening alpha helix structure(s) are also affected (Figure 1l–o). The changes in PROGF8 are predicted to weaken its interaction with the GA-bound GID1 because the stabilizing interaction between the second glutamic acid in LExLE motif and arginine and lysine in GID1 are disrupted. Weakening the interaction with GID1 likely increases the abundance of PROGF8. We thank Kathryn Fajardo for assistance with growing and maintain plants and Kristin Grandt for comments on the manuscript. This work was supported by a grant from the US Israel Binational Agriculture Research and Development fund to N.O. and D.W. (grant no. US-4813-15C). Zhiguo Zhu was supported by a State Scholarship Fund from China Scholarship Council (no. 201606050088). The authors declare that there is no conflict of interest.

Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae and fungi (1, 2).….

Plant development requires coordination among complex signaling networks to enhance the plant's adaptation to changing environments. DELLAs, transcription regulators originally identified as repressors of phytohormone gibberellin signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we found that DELLA is mono-O-fucosylated by the novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3) and PIF4. Moreover, spy mutants displayed elevated responses to gibberellin and brassinosteroid, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding may have broader importance because SPY orthologs are conserved in prokaryotes and eukaryotes, thus suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms.