JANUS, a spliceosome-associated protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are important regulators of genes expression. Their levels are precisely controlled through modulating the activity of the microprocesser complex (MC). Here, we report that JANUS, a homology of the conserved U2 snRNP assembly factor in yeast and human, is required for miRNA accumulation. JANUS associates with MC components Dicer-like 1 (DCL1) and SERRATE (SE) and directly binds the stem-loop of pri-miRNAs. In a hypomorphic janus mutant, the activity of DCL1, the numbers of MC, and the interaction of primary miRNA transcript (pri-miRNAs) with MC are reduced. These data suggest that JANUS promotes the assembly and activity of MC through its interaction with MC and/or pri-miRNAs. In addition, JANUS modulates the transcription of some pri-miRNAs as it binds the promoter of pri-miRNAs and facilitates Pol II occupancy of at their promoters. Moreover, global splicing defects are detected in janus. Taken together, our study reveals a novel role of a conserved splicing factor in miRNA biogenesis.

Serrate-Associated Protein 1, a splicing-related protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are essential regulators of gene expression in metazoans and plants. In plants, most miRNAs are generated from primary miRNA transcripts (pri-miRNAs), which are processed by the Dicer-like 1 (DCL1) complex along with accessory proteins. Serrate-Associated Protein 1 (SEAP1), a conserved splicing-related protein, has been studied in human and yeast. However, the functions of SEAP1 in plants remain elusive. Lack of SEAP1 results in embryo lethality and knockdown of SEAP1 by an artificial miRNA (amiRSEAP1 ) causes pleiotropic developmental defects and reduction in miRNA accumulation. SEAP1 associates with the DCL1 complex, and may promote the interaction of the DCL1 complexes with pri-miRNAs. SEAP1 also enhances pri-miRNA accumulation, but does not affect pri-miRNA transcription, suggesting it may indirectly or directly stabilize pri-miRNAs. In addition, SEAP1 affects the splicing of some pri-miRNAs and intron retention of messenger RNAs at global levels. Our findings uncover both conserved and novel functions of SEAP1 in plants. Besides the role as a splicing factor, SEPA1 may promote miRNA biogenesis by positively modulating pri-miRNA splicing, processing and/or stability.

MAC3A and MAC3B, Two Core Subunits of the MOS4-Associated Complex, Positively Influence miRNA Biogenesis.

MAC3A and MAC3B are conserved U-box-containing proteins in eukaryotes. They are subunits of the MOS4-associated complex (MAC) that plays essential roles in plant immunity and development in Arabidopsis thaliana However, their functional mechanisms remain elusive. Here, we show that Arabidopsis MAC3A and MAC3B act redundantly in microRNA (miRNA) biogenesis. Lack of both MAC3A and MAC3B in the mac3b mac3b double mutant reduces the accumulation of miRNAs, causing elevated transcript levels of miRNA targets. mac3a mac3b also decreases the levels of primary miRNA transcripts (pri-miRNAs). However, MAC3A and MAC3B do not affect the promoter activity of genes encoding miRNAs (MIR genes), suggesting that they may not affect MIR transcription. This result, together with the fact that MAC3A associates with pri-miRNAs in vivo, indicates that MAC3A and MAC3B may stabilize pri-miRNAs. Furthermore, we find that MAC3A and MAC3B interact with the DCL1 complex that catalyzes miRNA maturation, promote DCL1 activity, and are required for the localization of HYL1, a component of the DCL1 complex. Besides MAC3A and MAC3B, two other MAC subunits, CDC5 and PRL1, also function in miRNA biogenesis. Based on these results, we propose that MAC functions as a complex to control miRNA levels through modulating pri-miRNA transcription, processing, and stability.

Genome-wide analysis of genes encoding core components of the ubiquitin system in soybean (Glycine max) reveals a potential role for ubiquitination in host immunity against soybean cyst nematode.

BACKGROUND: Ubiquitination is a major post-translational protein modification that regulates essentially all cellular and physiological pathways in eukaryotes. The ubiquitination process typically involves three distinct classes of enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). To date, a comprehensive identification and analysis of core components comprising of the whole soybean (Glycine max) ubiquitin system (UBS) has not been reported. RESULTS: We performed a systematic, genome-wide analysis of genes that encode core members of the soybean UBS in this study. A total of 1431 genes were identified with high confidence to encode putative soybean UBS components, including 4 genes encoding E1s, 71 genes that encode the E2s, and 1356 genes encoding the E3-related components. Among the E3-encoding genes, 760 encode RING-type E3s, 124 encode U-box domain-containing E3s, and 472 encode F-box proteins. To find out whether the identified soybean UBS genes encode active enzymes, a set of genes were randomly selected and the enzymatic activities of their recombinant proteins were tested. Thioester assays indicated proteins encoded by the soybean E1 gene GmUBA1 and the majority of selected E2 genes are active E1 or E2 enzymes, respectively. Meanwhile, most of the purified RING and U-box domain-containing proteins displayed E3 activity in the in vitro ubiquitination assay. In addition, 1034 of the identified soybean UBS genes were found to express in at least one of 14 soybean tissues examined and the transcript level of 338 soybean USB genes were significantly changed after abiotic or biotic (Fusarium oxysporum and Rhizobium strains) stress treatment. Finally, the expression level of a large number of the identified soybean UBS-related genes was found significantly altered after soybean cyst nematode (SCN) treatment, suggesting the soybean UBS potentially plays an important role in soybean immunity against SCN. CONCLUSIONS: Our findings indicate the presence of a large and diverse number of core UBS proteins in the soybean genome, which suggests that target-specific modification by ubiquitin is a complex and important part of cellular and physiological regulation in soybean. We also revealed certain members of the soybean UBS may be involved in immunity against soybean cyst nematode (SCN). This study sets up an essential foundation for further functional characterization of the soybean UBS in various physiological processes, such as host immunity against SCN.

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity.

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart.

Functional analysis of the seven in absentia ubiquitin ligase family in tomato.

Seven in absentia (SINA) protein is one subgroup of ubiquitin ligases possessing an N-terminal cysteine-rich really interesting new gene (RING) domain, two zinc-finger motifs, and a C-terminal domain responsible for substrate-binding and dimerization. In tomato (Solanum lycopersicum), the SINA gene family has six members, and we characterize in this study all tomato SINA (SlSINA) genes and the gene products. Our results show that SlSINA genes are differentially regulated in leaf, bud, stem, flower, and root. All SlSINA proteins possess RING-dependent E3 ubiquitin ligase activity, exhibiting similar specificity towards the E2 ubiquitin-conjugating enzyme. SlSINA1/3/4/5/6 are localized in both cytoplasm and nucleus, whereas SlSINA2 is exclusively localized in the nucleus. Moreover, all SlSINAs can interact with each other for homo- or hetero-dimerization. The functionality of SlSINA proteins has been investigated. SlSINA4 plays a positive role in defense signalling, as manifested by elicitation of E3-dependent hypersensitive response-like cell death; the other SlSINAs are negative regulator and capable to suppress hypersensitive response cell death. Transgenic tomato plants overexpressing SlSINA2 exhibit pale-green leaf phenotype, suggesting SlSINA2 regulates chlorophyll level in plant cells, whereas transgenic tomato plants overexpressing SlSINA5 have altered floral structure with exserted stigma, implicating SlSINA5 plays a role in flower development.

Suppression of Arabidopsis ARGONAUTE1-mediated slicing, transgene-induced RNA silencing, and DNA methylation by distinct domains of the Cucumber mosaic virus 2b protein.

Unique among the known plant and animal viral suppressors of RNA silencing, the 2b protein interacts directly with both small interfering RNA (siRNA) and ARGONAUTE1 (AGO1) and AGO4 proteins and is targeted to the nucleolus. However, it is largely unknown which regions of the 111-residue 2b protein determine these biochemical properties and how they contribute to its diverse silencing suppressor activities. Here, we identified a functional nucleolar localization signal encoded within the 61-amino acid N-terminal double-stranded RNA (dsRNA) binding domain (dsRBD) that exhibited high affinity for short and long dsRNA. However, physical interaction of 2b with AGOs required an essential 33-residue region C-terminal to the dsRBD and was sufficient to inhibit the in vitro AGO1 Slicer activity independently of its dsRNA binding activities. Furthermore, the direct 2b-AGO interaction was not essential for the 2b suppression of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) in vivo. Lastly, we found that the 2b-AGO interactions in vivo also required the nucleolar targeting of 2b and had the potential to redistribute both the 2b and AGO proteins in nucleus. These findings together suggest that 2b may suppress PTGS and RdDM in vivo by binding and sequestering siRNA and the long dsRNA precursor in a process that is facilitated by its interactions with AGOs in the nucleolus.

The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.

Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae.

Verticillium dahliae Kleb. is a hemibiotrophic, phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. Successful host colonization by hemibiotrophic pathogens requires the induction of plant cell death to provide the saprophytic nutrition for the transition from the biotrophic to the necrotrophic stage. In this study, we identified a necrosis-inducing Phytophthora protein (NPP1) domain-containing protein family containing nine genes in a virulent, defoliating isolate of V. dahliae (V592), named the VdNLP genes. Functional analysis demonstrated that only two of these VdNLP genes, VdNLP1 and VdNLP2, encoded proteins that were capable of inducing necrotic lesions and triggering defense responses in Nicotiana benthamiana, Arabidopsis, and cotton plants. Both VdNLP1 and VdNLP2 induced the wilting of cotton seedling cotyledons. However, gene-deletion mutants targeted by VdNLP1, VdNLP2, or both did not affect the pathogenicity of V. dahliae V592 in cotton infection. Similar expression and induction patterns were found for seven of the nine VdNLP transcripts. Through a comparison of the conserved amino acid residues of VdNLP with different necrosis-inducing activities, combined with mutagenesis-based analyses, we identified several novel conserved amino acid residues, in addition to the known conserved heptapeptide GHRHDWE motif and the cysteine residues of the NPP domain-containing protein, that are indispensable for the necrosis-inducing activity of the VdNLP2 protein.

Chemical and genetic variation in feral Cannabis sativa populations across the Nebraska climate gradient.

Cannabis sativa is a versatile crop that can be cultivated for fiber, seed, or phytochemicals. To take advantage of this versatility and the potential of Cannabis as a feedstock for the bioeconomy, genomics-enabled breeding programs must be strengthened and expanded. This work contributes to the foundation for such by investigating the phytochemistry and genomics of feral Cannabis populations collected from seventeen counties across the climate gradient of Nebraska. Flower tissue from male and female plants (28 total) was studied using (i) gas chromatography-mass spectrometry to assess cannabinoid profiles and (ii) RNA sequencing to determine transcript abundances. Both male and female flower tissues produced cannabinoids, and, though the compounds were more abundant in female flower tissue, the primary cannabinoid in both was usually cannabidiol. The expression of genes that mediate early steps on the cannabinoid biosynthetic pathway were upregulated in female relative to male flowers, suggesting that female versus male flower tissue cannabinoid abundance may be controlled at least in part at the transcriptional level. DNA sequencing was used to place feral Cannabis plants from Nebraska into a previously described genomic context, revealing that all the plants studied here are much more similar to previously characterized hemp-type Cannabis plants than to drug-type Cannabis plants, at least at the genetic level. This work provides foundational phytochemical knowledge and a large set of high-quality single nucleotide polymorphism markers for future studies of feral Nebraska Cannabis.

Genome of Paspalum vaginatum and the role of trehalose mediated autophagy in increasing maize biomass.

A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

Immunity-Associated Programmed Cell Death as a Tool for the Identification of Genes Essential for Plant Innate Immunity.

Plants have evolved a sophisticated innate immune system to contend with potential infection by various pathogens. Understanding and manipulation of key molecular mechanisms that plants use to defend against various pathogens are critical for developing novel strategies in plant disease control. In plants, resistance to attempted pathogen infection is often associated with hypersensitive response (HR), a form of rapid programmed cell death (PCD) at the site of attempted pathogen invasion. In this chapter, we describe a method for rapid identification of genes that are essential for plant innate immunity. It combines virus-induced gene silencing (VIGS), a tool that is suitable for studying gene function in high-throughput, with the utilization of immunity-associated PCD, particularly HR-linked PCD as the readout of changes in plant innate immunity. The chapter covers from the design of gene fragment for VIGS, the agroinfiltration of the Nicotiana benthamian plants, to the use of immunity-associated PCD induced by twelve elicitors as the indicator of activation of plant immunity.

The Tomato U-Box Type E3 Ligase PUB13 Acts With Group III Ubiquitin E2 Enzymes to Modulate FLS2-Mediated Immune Signaling.

In Arabidopsis and rice, the ubiquitin ligase PUB13-mediated protein degradation plays a significant role in plant pattern-triggered immunity (PTI) and flowering time control. The Arabidopsis PUB13 has been shown to attenuate the pattern recognition receptor FLS2-mediated immune signaling by ubiquitinating FLS2 and consequently promoting its degradation by the 26S proteasome. Nevertheless, the cognate ubiquitin-conjugating enzymes (E2) with which PUB13 acts to modulate FLS2-mediated PTI are unknown. To address this question, we investigate here the tomato (Solanum lycopersicum) homolog of PUB13, SlPUB13 by utilizing the recently characterized complete set of tomato E2s. Of the 13 groups of tomato E2s, only members in group III are found to interact and act with SlPUB13. Knocking-down of the group III E2 genes enhances callose deposition and induction of the RbohB gene in the immunity-associated, early oxidative burst after flg22 treatment. The group III E2s are also found to work with SlPUB13 to ubiquitinate FLS2 in vitro and are required for PUB13-mediated degradation of FLS2 in vivo upon flg22 treatment, suggesting an essential role for group III E2s in the modulation of FLS2-mediated immune signaling by PUB13. Additionally, another immunity-associated E3, NtCMPG1 is shown to also work specifically with members of group III E2 in the in vitro ubiquitination assay, which implies the group III E2 enzymes may cooperate with many E3 ligases to regulate different aspects of PTI. Taken together, these data corroborate the notion that group III E2 enzymes play an important role in PTI and build a foundation for further functional and mechanistic characterization of tomato PUB13.

Conventional and unconventional ubiquitination in plant immunity.

Ubiquitination is one of the most abundant types of protein post-translational modification (PTM) in plant cells. The importance of ubiquitination in the regulation of many aspects of plant immunity has been increasingly appreciated in recent years. Most of the studies linking ubiquitination to the plant immune system, however, have been focused on the E3 ubiquitin ligases and the conventional ubiquitination that leads to the degradation of the substrate proteins by the 26S proteasome. By contrast, our knowledge about the role of unconventional ubiquitination that often serves as non-degradative, regulatory signal remains a significant gap. We discuss, in this review, the recent advances in our understanding of ubiquitination in the modulation of plant immunity, with a particular focus on the E3 ubiquitin ligases. We approach the topic from a perspective of two broadly defined types of ubiquitination in an attempt to highlight the importance, yet current scarcity, in our knowledge about the regulation of plant immunity by unconventional ubiquitination.

Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach.

BACKGROUND: Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets. RESULTS: Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana. Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae. Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s. CONCLUSIONS: We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and plant immunity-associated ROS production.

Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein.

Viroids are plant-pathogenic molecules made up of single-stranded circular non-coding RNAs. How replicating viroids interfere with host silencing remains largely unknown. In this study, we investigated the effects of a nuclear-replicating Potato spindle tuber viroid (PSTVd) on interference with plant RNA silencing. Using transient induction of silencing in GFP transgenic Nicotiana benthamiana plants (line 16c), we found that PSTVd replication accelerated GFP silencing and increased Virp1 mRNA, which encodes bromodomain-containing viroid-binding protein 1 and is required for PSTVd replication. DNA methylation was increased in the GFP transgene promoter of PSTVd-replicating plants, indicating involvement of transcriptional gene silencing. Consistently, accelerated GFP silencing and increased DNA methylation in the of GFP transgene promoter were detected in plants transiently expressing Virp1. Virp1 mRNA was also increased upon PSTVd infection in natural host potato plants. Reduced transcript levels of certain endogenous genes were also consistent with increases in DNA methylation in related gene promoters in PSTVd-infected potato plants. Together, our data demonstrate that PSTVd replication interferes with the nuclear silencing pathway in that host plant, and this is at least partially attributable to Virp1. This study provides new insights into the plant-viroid interaction on viroid pathogenicity by subverting the plant cell silencing machinery.

A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity.

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen.