The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Molecular insights into MpAGO1 and its regulatory miRNA, miR11707, in the high-temperature acclimation of Marchantia polymorpha.

As a model plant for bryophytes, Marchantia polymorpha offers insights into the role of RNA silencing in aiding early land plants navigate the challenges posed by high-temperature environments. Genomic analysis revealed unique ARGONAUTE1 ortholog gene (MpAGO1) in M. polymorpha that is regulated by two species-specific microRNAs (miRNAs), miR11707.1 and miR11707.2. Comparative studies of small RNA profiles from M. polymorpha cellular and MpAGO1 immunoprecipitation (MpAGO1-IP) profiles at various temperatures, along with analyses of Arabidopsis AGO1 (AtAGO1), revealed that MpAGO1 has a low-selectivity for a diverse range of small RNA species than AtAGO1. Protein structural comparisons revealed no discernible differences in the MID domains of MpAGO1 and AtAGO1, suggesting the complexity of miRNA species specificity and necessitating further exploration. Small RNA profiling and size exclusion chromatography have pinpointed a subset of M. polymorpha miRNAs, notably miR11707, that remain in free form within the cell at 22°C but are loaded into MpAGO1 at 28°C to engage in RNA silencing. Investigations into the mir11707 gene editing (mir11707ge) mutants provided evidence of the regulation of miR11707 in MpAGO1. Notably, while MpAGO1 mRNA expression decreases at 28°C, the stability of the MpAGO1 protein and its associated miRNAs is essential for enhancing the RISC activity, revealing the importance of RNA silencing in enabling M. polymorpha to survive thermal stress. This study advances our understanding of RNA silencing in bryophytes and provides groundbreaking insights into the evolutionary resilience of land plants to climatic adversities.

Dual Regulation of Cytochrome P450 Gene Expression by Two Distinct Small RNAs, a Novel tasiRNA and miRNA, in Marchantia polymorpha.

The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5'- and 3'-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, which can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha.

Shrimp SIRT4 promotes white spot syndrome virus replication.

In WSSV pathogenesis, the molecular mechanisms and the key host factors that regulate the viral replication and morphogenesis remain unclear. However, like most viruses, WSSV is known to induce metabolic reprogramming in several metabolic pathways including the host glutamine metabolism, and several recent reports have suggested that the sirtuins SIRT3, SIRT4, and SIRT5, which belong to a family of NAD+-dependent deacetylases, play an important role in this regulation. Here we focus on characterizing LvSIRT4 from Litopenaeus vannamei and investigate its role in regulating glutamine dehydrogenase (GDH), an important enzyme that promotes glutaminolysis and viral replication. We found that LvSIRT4 silencing led to significant decreases in both WSSV gene expression and the number of viral genome copies. Conversely, overexpression of LvSIRT4 led to significant increases in the expression of WSSV genes and the WSSV genome copy number. Immunostaining in Sf9 insect cells confirmed the presence of LvSIRT4 in the mitochondria and the co-localization of LvSIRT4 and LvGDH in the same cellular locations. In vivo gene silencing of LvSIRT4 significantly reduced the gene expression of LvGDH whereas LvSIRT4 overexpression had no effect. However, neither silencing nor overexpression had any effect on the protein expression levels of LvGDH. Lastly, although GDH activity in uninfected shrimp was unchanged, the GDH enzyme activity in WSSV-infected shrimp was significantly increased after both LvSIRT4 silencing and overexpression. This suggests that although there may be no direct regulation, LvSIRT4 might still be able to indirectly regulate LvGDH via the mediation of one or more WSSV proteins that have yet to be identified.

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

BACKGROUND: To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. METHODS: Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. RESULTS: Our results indicated that the P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. CONCLUSIONS: In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.

Complete genome sequence of Amazon lily mosaic virus isolated from amaryllis (Hippeastrum hybridum Hort.).

In April 2011, a virus was isolated by single-lesion isolation on Chenopodium quinoa leaves from an amaryllis plant with chlorotic ringspots in a private garden in Changhua County, Taiwan. An Illumina MiSeq sequencing system was used to determine the genomic nucleotide (nt) sequence of the virus. A de novo-assembled contig with 9377 nt, containing an open reading frame encoding a putative potyviral polyprotein, was annotated as the potyvirus Amazon lily mosaic virus (ALiMV), sharing 95.5% nt sequence identity with a partial genomic sequence of ALiMV available in the GenBank database. Therefore, the amaryllis virus was designated as ALiMV-TW. Through 5´ and 3´ rapid amplification of cDNA ends (RACE), the complete 9618-nt genome sequence of ALiMV-TW was determined. Sequence comparisons indicated that the genome and polyprotein of ALiMV-TW share 52.3-65.1% nt and 30.1-64.2% aa sequence identity, respectively, with those of other potyviruses. This is the first report of a complete genome sequence of ALiMV.

Elucidation of enzymes involved in the biosynthetic pathway of bioactive polyacetylenes in Bidens pilosa using integrated omics approaches.

Polyacetylene compounds from Bidens pilosa are known to have several pharmacological activities. In this study, we identified major genes encoding enzymes involved in the biosynthesis of polyacetylene in B. pilosa. Seven polyacetylene metabolites present in B. pilosa leaves were induced by methyl jasmonate (MeJA) treatment and physical wounding. Transcriptome analysis via high-throughput sequencing revealed 39 202 annotated gene fragment sequences. A DNA microarray established by the 39 202 annotated genes was used to profile gene expression in B. pilosa leaf and root tissues. As no polyacetylene compounds were found in roots, the gene expression pattern in root tissue was used as a negative control. By subtracting MeJA-induced genes in roots, we obtained 1216 genes in leaves showing an approximate three-fold increase in expression post-MeJA treatment. Nine genes encoding enzymes with desaturation function were selected for confirmation of expression by qRT-PCR. Among them, two genes, BPTC030748 and BPTC012564, were predicted to encode Δ12-oleate desaturase (OD) and Δ12-fatty acid acetylenase (FAA), respectively. In B. pilosa leaves, RNAi knock-down concomitantly decreased, while virus-mediated transient overexpression of either gene elevated polyacetylene content. In summary, we demonstrate that two important enzymes, Δ12-oleate desaturase and Δ12-fatty acid acetylenase, involved in desaturation of linear fatty acid precursors play a role in polyacetylene biosynthesis in an important medicinal plant, Bidens pilosa.

Bile acid and bile acid transporters are involved in the pathogenesis of acute hepatopancreatic necrosis disease in white shrimp Litopenaeus vannamei.

Acute hepatopancreas necrosis disease is a recently emerged shrimp disease that is caused by virulent strains of Vibrio parahaemolyticus. Although AHPND poses a serious threat to the shrimp industry, particularly in Asia, its underlying pathogenic mechanisms are not well characterized. Since a previous transcriptomic study showed upregulation of the apical sodium bile acid transporter (LvASBT), our objective here was to explore the role of bile acids and bile acid transporters in AHPND infection. We confirmed that mRNA expression of LvASBT was upregulated in the stomach of AHPND-infected shrimps. Bile acid concentrations were also higher in the stomach of AHPND-infected shrimp and correlated with high expression of pVA plasmid and Pir toxins. In vitro assays showed that bile acids enhanced biofilm formation and increased the release of PirABvp toxins in AHPND-causing V. parahaemolyticus, while in vivo inhibition of LvASBT by GSK2330672 reduced the copy numbers of pVA plasmid, Pir toxin and reduced the amounts of bile acids in AHPND-infected shrimp stomach. Transcriptomics data for AHPND-causing V. parahaemolyticus treated with bile acids showed upregulation of various genes involved in membrane transport, RND efflux pumps and a bacterial secretion system. Taken together, our results show that AHPND-causing V. parahaemolyticus virulence is positively regulated by bile acids and that LvASBT and bile acids in shrimp stomach have important roles in AHPND pathogenesis.

Structural insights into the interaction between phytoplasmal effector causing phyllody 1 and MADS transcription factors.

Phytoplasmas are bacterial plant pathogens which can induce severe symptoms including dwarfism, phyllody and virescence in an infected plant. Because phytoplasmas infect many important crops such as peanut and papaya they have caused serious agricultural losses. The phytoplasmal effector causing phyllody 1 (PHYL1) is an important phytoplasmal pathogenic factor which affects the biological function of MADS transcription factors by interacting with their K (keratin-like) domain, thus resulting in abnormal plant developments such as phyllody. Until now, lack of information on the structure of PHYL1 has prevented a detailed understanding of the binding mechanism between PHYL1 and the MADS transcription factors. Here, we present the crystal structure of PHYL1 from peanut witches'-broom phytoplasma (PHYL1PnWB ). This protein was found to fold into a unique α-helical hairpin with exposed hydrophobic residues on its surface that may play an important role in its biological function. Using proteomics approaches, we propose a binding mode of PHYL1PnWB with the K domain of the MADS transcription factor SEPALLATA3 (SEP3_K) and identify the residues of PHYL1PnWB that are important for this interaction. Furthermore, using surface plasmon resonance we measure the binding strength of PHYL1PnWB proteins to SEP3_K. Lastly, based on confocal images, we found that α-helix 2 of PHYL1PnWB plays an important role in PHYL1-mediated degradation of SEP3. Taken together, these results provide a structural understanding of the specific binding mechanism between PHYL1PnWB and SEP3_K.

A Conserved Helix in the C-Terminal Region of Watermelon Silver Mottle Virus Nonstructural Protein S Is Imperative For Protein Stability Affecting Self-Interaction, RNA Silencing Suppression, and Pathogenicity.

In orthotospovirus, the nonstructural protein S (NSs) is the RNA-silencing suppressor (RSS) and pathogenicity determinant. Here, we demonstrate that a putative α-helix, designated H8, spanning amino acids 338 to 369 of the C-terminal region of the NSs protein, is crucial for self-interaction of watermelon silver mottle virus NSs protein and that the H8 affects RSS function. Co-immunoprecipitation, yeast two-hybrid, and bimolecular fluorescence complementation analyses revealed that the triple point mutation (TPM) of H8 amino acids Y338A, H350A, and F353A resulted in NSs protein self-interaction dysfunction. Transient expression of H8-deleted (ΔH8) and TPM NSs proteins in Nicotiana benthamiana plants by agroinfitration indicated that these proteins have weaker RSS activity and are far less stable than wild-type (WT) NSs. However, an electrophoretic mobility assay revealed that small interfering RNA (siRNA) binding ability of TPM NSs protein is not compromised. The pathogenicity assay of WT NSs protein expressed by the attenuated turnip mosaic virus vector restored severe symptoms in recombinant-infected N. benthamiana plants but not for ΔH8 or TPM proteins. Taken together, we conclude that the H8 helix in the C-terminal region of NSs protein is crucial for stabilizing NSs protein through self-interaction to maintain normal functions of RSS and pathogenicity, but not for NSs-siRNA binding activity.

Transcriptome and microbiome of coconut rhinoceros beetle (Oryctes rhinoceros) larvae.

BACKGROUND: The coconut rhinoceros beetle, Oryctes rhinoceros, is a major pest of palm crops in tropical Asia and the Pacific Islands. Little molecular data exists for this pest, impeding our ability to develop effective countermeasures and deal with the species' growing resistance to viral biocontrols. We present the first molecular biology analyses of this species, including a metagenomic assay to understand the microbiome of different sections of its digestive tract, and a transcriptomics assay to complement the microbiome data and to shed light on genes of interest like plant cell wall degrading enzymes and immunity and xenobiotic resistance genes. RESULTS: The gut microbiota of Oryctes rhinoceros larvae is quite similar to that of the termite gut, as both species feed on decaying wood. We found the first evidence for endogenous beta-1,4-endoglucanase in the beetle, plus evidence for microbial cellobiase, suggesting the beetle can degrade cellulose together with its gut microfauna. A number of antimicrobial peptides are expressed, particularly by the fat body but also by the midgut and hindgut. CONCLUSIONS: This transcriptome provides a wealth of data about the species' defense against chemical and biological threats, has uncovered several potentially new species of microbial symbionts, and significantly expands our knowledge about this pest.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp.

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND-associated plasmid pVA1 encoding a virulent toxin (Pirvp ) that damages the shrimp's hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimp's stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND-induced changes in the stomach of AHPND-causing V. parahaemolyticus (5HP)-infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene-to-gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP-infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.

Gene-to-Gene Network Analysis of the Mediation of Plant Innate Immunity by the Eliciting Plant Response-Like 1 (Epl1) Elicitor of Trichoderma formosa.

A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.

MicroRNAs in Marchantia polymorpha.

Contents Summary 409 I. Introduction 409 II. RNA silencing machinery in Marchantia polymorpha 410 III. miRNA prediction by integrating omics approach 410 IV. miRNAs and their targets in Marchantia polymorpha 410 V. Mpo-miR390-mediated MpTAS3 tasiRNA biogenesis and potential tasiARF target MpARF2 414 VI. Artificial miRNA and CRISPR-CAS9 edited MIR gene in Marchantia polymorpha 414 VII. Conclusions 415 Acknowledgements 415 References 415 SUMMARY: The liverwort Marchantia polymorpha occupies an important phylogenetic position for comparative studies of land plant gene regulation. Multiple gene regulatory pathways mediated by small RNAs, including microRNAs (miRNAs), trans-acting short-interfering RNAs, and heterochromatic siRNAs often associated with RNA-dependent DNA methylation, have been characterized in flowering plants. Genes for essential components for all of these small RNA-mediated gene silencing pathways are found in M. polymorpha as well as the moss Phsycomitrella patens, indicating that these pathways existed in the ancestral land plant. However, only seven miRNAs are conserved across land plants, with both ancestral and novel targets identified in M. polymorpha. There is little or no evidence that any of these conserved miRNAs are present in algae. As with other plants investigated, most miRNAs in M. polypmorpha exhibit lineage-specific evolution. Application of artificial miRNA and CRISPR-Cas9 technologies in genetic studies of M. polymorpha provide avenues to further investigate miRNA biology.

Class C ARFs evolved before the origin of land plants and antagonize differentiation and developmental transitions in Marchantia polymorpha.

A plethora of developmental and physiological processes in land plants is influenced by auxin, to a large extent via alterations in gene expression by AUXIN RESPONSE FACTORs (ARFs). The canonical auxin transcriptional response system is a land plant innovation, however, charophycean algae possess orthologues of at least some classes of ARF and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, suggesting that elements of the canonical land plant system existed in an ancestral alga. We reconstructed the phylogenetic relationships between streptophyte ARF and AUX/IAA genes and functionally characterized the solitary class C ARF, MpARF3, in Marchantia polymorpha. Phylogenetic analyses indicate that multiple ARF classes, including class C ARFs, existed in an ancestral alga. Loss- and gain-of-function MpARF3 alleles result in pleiotropic effects in the gametophyte, with MpARF3 inhibiting differentiation and developmental transitions in multiple stages of the life cycle. Although loss-of-function Mparf3 and Mpmir160 alleles respond to exogenous auxin treatments, strong miR-resistant MpARF3 alleles are auxin-insensitive, suggesting that class C ARFs act in a context-dependent fashion. We conclude that two modules independently evolved to regulate a pre-existing ARF transcriptional network. Whereas the auxin-TIR1-AUX/IAA pathway evolved to repress class A/B ARF activity, miR160 evolved to repress class C ARFs in a dynamic fashion.

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

The Naming of Names: Guidelines for Gene Nomenclature in Marchantia.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.

MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector.

Leafy flowers are the major symptoms of peanut witches' broom (PnWB) phytoplasma infection in Catharanthus roseus. The orthologs of the phyllody symptoms1 (PHYL1) effector of PnWB from other species of phytoplasma can trigger the proteasomal degradation of several MADS box transcription factors, resulting in leafy flower formation. In contrast, the flowering negative regulator gene SHORT VEGETATIVE PHASE (SVP) was up-regulated in PnWB-infected C. roseus plants, but most microRNA (miRNA) genes had repressed expression. Coincidentally, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the PHYL1 gene of PnWB (PHYL1 plants), which show leafy flower phenotypes, up-regulate SVP of Arabidopsis (AtSVP) but repress a putative regulatory miRNA of AtSVP, miR396. However, the mechanism by which PHYL1 regulates AtSVP and miR396 is unknown, and the evidence of miR396-mediated AtSVP degradation is lacking. Here, we show that miR396 triggers AtSVP messenger RNA (mRNA) decay using genetic approaches, a reporter assay, and high-throughput degradome profiles. Genetic evidence indicates that PHYL1 plants and atmir396a-1 mutants have higher AtSVP accumulation, whereas the transgenic plants overexpressing MIR396 display lower AtSVP expression. The reporter assay indicated that target-site mutation results in decreasing the miR396-mediated repression efficiency. Moreover, degradome profiles revealed that miR396 triggers AtSVP mRNA decay rather than miRNA-mediated cleavage, implying that AtSVP caused miR396-mediated translation inhibition. We hypothesize that PHYL1 directly or indirectly interferes with miR396-mediated AtSVP mRNA decay and synergizes with other effects (e.g. MADS box transcription factor degradation), resulting in abnormal flower formation. We anticipate our findings to be a starting point for studying the posttranscriptional regulation of PHYL1 effectors in symptom development.

Application of an Integrated Omics Approach for Identifying Host Proteins That Interact With Odontoglossum ringspot virus Capsid Protein.

The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.