Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP-F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

Complete genome sequence of "Candidatus Phytoplasma cynodontis" GY2015, a plant pathogen associated with Bermuda grass white leaf disease in Taiwan.

The complete genome sequence of "Candidatus Phytoplasma cynodontis" strain GY2015, which consists of one 498,922-bp circular chromosome, is presented in this work. This uncultivated plant-pathogenic bacterium is associated with Bermuda grass white leaf disease in Taoyuan, Taiwan.

Genomic Characterization of the Periwinkle Leaf Yellowing (PLY) Phytoplasmas in Taiwan.

The periwinkle leaf yellowing (PLY) disease was first reported in Taiwan in 2005. This disease was caused by an uncultivated bacterium in the genus "Candidatus phytoplasma." In subsequent years, this bacterium was linked to other plant diseases and caused losses in agriculture. For genomic investigation of this bacterium and its relatives, we conducted whole genome sequencing of a PLY phytoplasma from an infected periwinkle collected in Taoyuan. The de novo genome assembly produced eight contigs with a total length of 824,596 bp. The annotation contains 775 protein-coding genes, 63 pseudogenes, 32 tRNA genes, and two sets of rRNA operons. To characterize the genomic diversity across populations, a second strain that infects green onions in Yilan was collected for re-sequencing analysis. Comparison between these two strains identified 337 sequence polymorphisms and 10 structural variations. The metabolic pathway analysis indicated that the PLY phytoplasma genome contains two regions with highly conserved gene composition for carbohydrate metabolism. Intriguingly, each region contains several pseudogenes and the remaining functional genes in these two regions complement each other, suggesting a case of duplication followed by differential gene losses. Comparative analysis with other available phytoplasma genomes indicated that this PLY phytoplasma belongs to the 16SrI-B subgroup in the genus, with "Candidatus Phytoplasma asteris" that causes the onion yellowing (OY) disease in Japan as the closest known relative. For characterized effectors that these bacteria use to manipulate their plant hosts, the PLY phytoplasma has homologs for SAP11, SAP54/PHYL1, and TENGU. For genome structure comparison, we found that potential mobile unit (PMU) insertions may be the main factor that drives genome rearrangements in these bacteria. A total of 10 PMU-like regions were found in the PLY phytoplasma genome. Two of these PMUs were found to harbor one SAP11 homolog each, with one more similar to the 16SrI-B type and the other more similar to the 16SrI-A type, suggesting possible horizontal transfer. Taken together, this work provided a first look into population genomics of the PLY phytoplasmas in Taiwan, as well as identified several evolutionary processes that contributed to the genetic diversification of these plant-pathogenic bacteria.

Silencing of CrNPR1 and CrNPR3 Alters Plant Susceptibility to Periwinkle Leaf Yellowing Phytoplasma.

Phytoplasmas are prokaryotic plant pathogens that cause considerable loss in many economically important crops, and an increasing number of phytoplasma diseases are being reported on new hosts. Knowledge of plant defense mechanisms against such pathogens should help to improve strategies for controlling these diseases. Salicylic acid (SA)-mediated defense may play an important role in defense against phytoplasmas. Here, we report that SA accumulated in Madagascar periwinkle (Catharanthus roseus) infected with periwinkle leaf yellowing (PLY) phytoplasma. CrPR1a expression was induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. NPR1 plays a central role in SA signaling, and two NPR1 homologs, CrNPR1 and CrNPR3, were identified from a periwinkle transcriptome database. Similar to CrPR1a, CrNPR1 expression was also induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. Silencing of CrNPR1, but not CrNPR3, significantly repressed CrPR1a induction in Tobacco rattle virus-infected periwinkle plants. In addition, symptoms of PLY progressed fastest in CrNPR1-silenced plants and slowest in CrNPR3-silenced plants. Consistently, expression of CrNPR1, but not CrNPR3, was induced by phytoplasma infection as well as SA treatment. This study highlights the importance of NPR1- and SA-mediated defense against phytoplasma in periwinkle and offers insight into plant-phytoplasma interactions to improve disease control strategies.

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.

Characterization and evaluation of Bacillus amyloliquefaciens strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars.

Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.

Peanut witches' broom (PnWB) phytoplasma-mediated leafy flower symptoms and abnormal vascular bundles development.

The peanut witches' broom (PnWB) phytoplasma causes virescence symptoms such as phyllody (leafy flower) in infected peanuts. However, the obligate nature of phytoplasma limits the study of host-pathogen interactions, and the detailed anatomy of PnWB-infected plants has yet to be reported. Here, we demonstrate that 4',6'-diamidino-2-phenylindole (DAPI) staining can be used to track PnWB infection. The DAPI-stained phytoplasma cells were observed in phloem/internal phloem tissues, and changes in vascular bundle morphology, including increasing pith rays and thinner cell walls in the xylem, were found. We also discerned the cell types comprising PnWB in infected sieve tube members. These results suggest that the presence of PnWB in phloem tissue facilitates the transmission of phytoplasma via sap-feeding insect vectors. In addition, PnWB in sieve tube members and changes in vascular bundle morphology might strongly promote the ability of phytoplasmas to assimilate nutrients. These data will help further an understanding of the obligate life cycle and host-pathogen interactions of phytoplasma.

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.

Development of a Mild Viral Expression System for Gain-Of-Function Study of Phytoplasma Effector In Planta.

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches'-broom (AYWB) phytoplasma and Peanut witches'-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T0 lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-ProR182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T0 lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.

Genetic analyses of the FRNK motif function of Turnip mosaic virus uncover multiple and potentially interactive pathways of cross-protection.

Cross-protection triggered by a mild strain of virus acts as a prophylaxis to prevent subsequent infections by related viruses in plants; however, the underling mechanisms are not fully understood. Through mutagenesis, we isolated a mutant strain of Turnip mosaic virus (TuMV), named Tu-GK, that contains an Arg182Lys substitution in helper component-proteinase (HC-Pro(K)) that confers complete cross-protection against infection by a severe strain of TuMV in Nicotiana benthamiana, Arabidopsis thaliana Col-0, and the Arabidopsis dcl2-4/dcl4-1 double mutant defective in DICER-like ribonuclease (DCL)2/DCL4-mediated silencing. Our analyses showed that HC-Pro(K) loses the ability to interfere with microRNA pathways, although it retains a partial capability for RNA silencing suppression triggered by DCL. We further showed that Tu-GK infection triggers strong salicylic acid (SA)-dependent and SA-independent innate immunity responses. Our data suggest that DCL2/4-dependent and -independent RNA silencing pathways are involved, and may crosstalk with basal innate immunity pathways, in host defense and in cross-protection.

High-throughput transcriptome analysis of the leafy flower transition of Catharanthus roseus induced by peanut witches'-broom phytoplasma infection.

Peanut witches'-broom (PnWB) phytoplasma are obligate bacteria that cause leafy flower symptoms in Catharanthus roseus. The PnWB-mediated leafy flower transitions were studied to understand the mechanisms underlying the pathogen-host interaction; however, our understanding is limited because of the lack of information on the C. roseus genome. In this study, the whole-transcriptome profiles from healthy flowers (HFs) and stage 4 (S4) PnWB-infected leafy flowers of C. roseus were investigated using next-generation sequencing (NGS). More than 60,000 contigs were generated using a de novo assembly approach, and 34.2% of the contigs (20,711 genes) were annotated as putative genes through name-calling, open reading frame determination and gene ontology analyses. Furthermore, a customized microarray based on this sequence information was designed and used to analyze samples further at various stages of PnWB infection. In the NGS profile, 87.8% of the genes showed expression levels that were consistent with those in the microarray profiles, suggesting that accurate gene expression levels can be detected using NGS. The data revealed that defense-related and flowering gene expression levels were altered in S4 PnWB-infected leafy flowers, indicating that the immunity and reproductive stages of C. roseus were compromised. The network analysis suggested that the expression levels of >1,000 candidate genes were highly associated with CrSVP1/2 and CrFT expression, which might be crucial in the leafy flower transition. In conclusion, this study provides a new perspective for understanding plant pathology and the mechanisms underlying the leafy flowering transition caused by host-pathogen interactions through analyzing bioinformatics data obtained using a powerful, rapid high-throughput technique.

Comparative analysis of the peanut witches'-broom phytoplasma genome reveals horizontal transfer of potential mobile units and effectors.

Phytoplasmas are a group of bacteria that are associated with hundreds of plant diseases. Due to their economical importance and the difficulties involved in the experimental study of these obligate pathogens, genome sequencing and comparative analysis have been utilized as powerful tools to understand phytoplasma biology. To date four complete phytoplasma genome sequences have been published. However, these four strains represent limited phylogenetic diversity. In this study, we report the shotgun sequencing and evolutionary analysis of a peanut witches'-broom (PnWB) phytoplasma genome. The availability of this genome provides the first representative of the 16SrII group and substantially improves the taxon sampling to investigate genome evolution. The draft genome assembly contains 13 chromosomal contigs with a total size of 562,473 bp, covering ∼90% of the chromosome. Additionally, a complete plasmid sequence is included. Comparisons among the five available phytoplasma genomes reveal the differentiations in gene content and metabolic capacity. Notably, phylogenetic inferences of the potential mobile units (PMUs) in these genomes indicate that horizontal transfer may have occurred between divergent phytoplasma lineages. Because many effectors are associated with PMUs, the horizontal transfer of these transposon-like elements can contribute to the adaptation and diversification of these pathogens. In summary, the findings from this study highlight the importance of improving taxon sampling when investigating genome evolution. Moreover, the currently available sequences are inadequate to fully characterize the pan-genome of phytoplasmas. Future genome sequencing efforts to expand phylogenetic diversity are essential in improving our understanding of phytoplasma evolution.

Improving initial infectivity of the Turnip mosaic virus (TuMV) infectious clone by an mini binary vector via agro-infiltration.

BACKGROUND: The in vivo infectious clone of Turnip mosaic virus (TuMV), p35S-TuMV, was used on plant pathology research for many years. To activate p35S-TuMV, the plasmid was mechanically introduced to the local lesion host Chenopodium quinoa. However, low infectivity occurred when the TuMV from C. quinoa was transferred to the systemic host Nicotiana benthamiana. RESULTS: To increase the efficiency of initial infectivity on N. benthamiana, the expression of the TuMV infectious clone by a binary vector that directly activates viral RNA through agro-infiltration is considered to be a good alternative. The size of the binary vector by agro-infiltration is usually large and its backbone has numerous restriction sites that increase difficulty for construction. In this study, we attempted to construct a mini binary vector (pBD003) with less restriction sites. The full-length cDNA of TuMV genome, with or without green fluorescence protein, was inserted in pBD003 to generate pBD-TuMV constructs, which were then individually introduced to N. benthamiana plants by agro-infiltration. Symptom development and ELISA positivity with TuMV antiserum indicated that the pBD-TuMV constructs are infectious. Moreover, the initial infectivity of a mild strain TuMV-GK, which contains an R182K mutation on HC-Pro, constructed in the pBD003 vector was significantly increased by agro-infiltration. CONCLUSION: Thus, we concluded that the newly constructed mini binary vector provides a more feasible tool for TuMV researches in areas, such as creating a mild strain for cross-protection, or a viral vector for foreign gene expression. In addition, the multiple cloning sites will be further cloned in pBD003 for convenience in constructing other viral infectious clones.

Comparative analysis of gene content evolution in phytoplasmas and mycoplasmas.

Phytoplasmas and mycoplasmas are two groups of important pathogens in the bacterial class Mollicutes. Because of their economical and clinical importance, these obligate pathogens have attracted much research attention. However, difficulties involved in the empirical study of these bacteria, particularly the fact that phytoplasmas have not yet been successfully cultivated outside of their hosts despite decades of attempts, have greatly hampered research progress. With the rapid advancements in genome sequencing, comparative genome analysis provides a new approach to facilitate our understanding of these bacteria. In this study, our main focus is to investigate the evolution of gene content in phytoplasmas, mycoplasmas, and their common ancestor. By using a phylogenetic framework for comparative analysis of 12 complete genome sequences, we characterized the putative gains and losses of genes in these obligate parasites. Our results demonstrated that the degradation of metabolic capacities in these bacteria has occurred predominantly in the common ancestor of Mollicutes, prior to the evolutionary split of phytoplasmas and mycoplasmas. Furthermore, we identified a list of genes that are acquired by the common ancestor of phytoplasmas and are conserved across all strains with complete genome sequences available. These genes include several putative effectors for the interactions with hosts and may be good candidates for future functional characterization.

Phytoplasma-induced floral abnormalities in Catharanthus roseus are associated with phytoplasma accumulation and transcript repression of floral organ identity genes.

Floral symptoms caused by phytoplasma largely resemble floral reversion in other plants. Periwinkle leaf yellowing (PLY) phytoplasma and peanut witches'-broom (PnWB) phytoplasma caused different degrees of floral abnormalities on infected periwinkle plants. The PLY phytoplasma-infected plants exhibited floral discoloration, virescence, small flowers, and only occasionally full floral reversion. In contrast, PnWB phytoplasma frequently induced complete floral reversion and resulted in a witches'-broom symptom from the floral reversion. Although different degrees of floral symptoms were induced by these two phytoplasmas, the morphological disorders were similar to those of other plants carrying SEPALLATA mutations or gene silencing. Here, we compared expression levels of organ-identity-related genes and pigmentation genes during floral symptom development. Accumulation of phytoplasmas in malformed flowers and their closely surrounding leaves was also compared. In infected plants, transcript abundance of all examined organ identity genes and pigmentation genes was suppressed. Indeed, CrSEP3, a SEPALLALA3 ortholog, showed the greatest suppression among genes examined. Of the pigmentation genes, transcript reduction of chalcone synthase was most highly correlated with the loss in floral pigmentation. Floral symptom severities were associated with the accumulation of either phytoplasmas. Interestingly, both phytoplasmas accumulated to higher levels in malformed flowers than in their surrounding leaves. Many plant pathogens manipulate host plant development to their advantage. It is intriguing to see whether phytoplasmas alter floral development to increase their population.

An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom.

A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches' broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches' broom (PNWB) and the two showed homology based on the results of Western blot analysis, Southern hybridization, Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma and PNWB phytoplasma were not found in other phytoplasmas tested.