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.

Versatile Roles of the Receptor-Like Kinase Feronia in Plant Growth, Development and Host-Pathogen Interaction.

As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.

Endophytes enhance the production of root alkaloids ajmalicine and serpentine by modulating the terpenoid indole alkaloid pathway in Catharanthus roseus roots.

AIM: The aim of this study was to identify and characterize promising endophytes capable of enhancing the content of root alkaloids ajmalicine and serpentine in low alkaloid yielding genotype of Catharanthus roseus cultivar Prabal and the possible mechanisms involved. METHOD AND RESULT: Of the four strains isolated from alkaloid-rich genotype of C. roseus cultivar Dhawal, endophytic strains CATDLF5 (Curvularia sp.) and CATDLF6 (Choanephora infundibulifera) enhanced serpentine content by 211·7-337·6%, while CATDRF2 (Aspergillus japonicus) and CATDS5 (Pseudomonas sp.) increased the content of ajmalicine by 123·4-203·8% in cultivar Prabal. Upregulated expression of key genes, geraniol 10-hydroxylase, tryptophan decarboxylase and strictosidine synthase involved in terpenoid indole alkaloid (TIA) biosynthetic pathway was observed in endophyte inoculated plants. Upregulated Octadecanoid-derivative Responsive Catharanthus AP2/ERF domain transcription activators like ORCA3 while, and downregulation of transcriptional repressor, ZCTs (Cys2/His2-type zinc finger protein family) enhanced the expression of genes for secondary metabolite production in endophyte-inoculated plants. CONCLUSION: The present work concluded that the selected endophytes of C. roseus can enhance the ajmalicine and serpentine contents by modulating the expression of structural and regulatory genes of TIA biosynthetic pathway in root. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytes can play an important role to enhance in planta content of pharmaceutically important alkaloids in C. roseus and can therefore be useful in reducing the cost of production of important alkaloids.

Microbispora catharanthi sp. nov., a novel endophytic actinomycete isolated from the root of Catharanthus roseus.

Strain CR1-09T, an actinomycete isolated from the root of Catharanthus roseus, was taxonomically studied based upon polyphasic approaches. The isolate formed a pair of ovular to circular, smooth-surfaced spores on short sporophores alternately branched from aerial mycelia. It contained meso-diaminopimelic acid in cell wall peptidoglycans. The major menaquinones were MK-9 (H4) and MK-9 (H2). The predominant cellular fatty acids were iso-C16 : 0 and C16 : 0. The polar lipids profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxyl phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, and unidentified ninhydrin positive phosphoglycolipids. Strain CR1-09T showed the highest 16S rRNA gene sequence similarity with Microbispora tritici DSM 104650T (99.5 %). Based on the polyphasic approach, DNA-DNA relatedness and average nucleotide identity (ANI), the strain is considered to represent a novel species of the genus Microbispora, for which the name Microbispora catharanthi is proposed. The type strain is strain CR1-09T (=JCM 30045T=TISTR 2273T).

Vinblastine production by the endophytic fungus Curvularia verruculosa from the leaves of Catharanthus roseus and its in vitro cytotoxicity against HeLa cell line.

The current study was to isolate endophytic fungi producing high yields of indole alkaloids such as vinblastine analogous to their host Cathranthus roseus. Endophytic fungi were isolated from the leaves of C. roseus, identified as Curvularia verruculosa by molecular techniques, and the sequence was deposited in NCBI (MK995628). Vinblastine producing endophytic fungus was grown in 1L Vinca medium for 21 days. The extract was examined for vinblastine by chromatographic techniques. TLC plates showed purple colour spot co-migrated with authentic vinblastine and Rf was calculated by HPTLC (Vin 1 vinblastine -0.75; authentic vinblastine-0.78), these results confirmed vinblastine presence in the Vin1 extract. Further, the TLC purified fungal extract was examined by LC-MS, which revealed the exact mass of vinblastine ([M + H]+m/z 811.51). The most important of the study is high yield production of vinblastine; hence, the extract analysed by HPLC revealed 182 µg/L vinblastine. The TLC purified fungal vinblastine was analysed for the cytotoxicity effect on HeLa cell line and it depicted a higher activity with IC50-8.5 µg/mL and apoptotic morphological changes were analysed. All the results revealed that the endophytic fungus Curvularia verruculosa produced vinblastine and for the first time in a surplus amount compared to other fungi.

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

MAIN CONCLUSION: Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Diversity and biological activities of endophytic fungi associated with Catharanthus roseus.

BACKGROUND: The present study involves diversity and bioactivity of the endophytic fungal community from Catharanthus roseus inhabiting the coastal region. This study has been conducted hypothesizing that the microbial communities in the coastal regions would tolerate a range of abiotic stress such as salinity, humidity, temperature and soil composition, and it may produce new metabolites, which may possess bioactive property. Therefore in the current study, the cytotoxicity and free radical scavenging potential of the fungal organic extracts have been investigated. Moreover, the apoptotic and the antioxidant potential of the fungus that exhibited the best activity in preliminary screening has also been demonstrated. RESULTS: Twenty endophytic fungal isolates were obtained from different parts of the plant, and identified using internal transcribed spacer region analysis. Based on the colonization frequency, the dominant genera were found to be Colletotrichum, Alternaria and Chaetomium with colonization frequency % of 8.66, 7.00 and 6.33, respectively. It was observed that the species diversity and richness was the highest in bark followed by leaf and stem regions of the plant. On screening the fungal ethyl acetate extracts for cytotoxicity against the HeLa cells, the Chaetomium nigricolor extract exhibited potent cytotoxic activity of 92.20% at 100 µg mL- 1 concentration. Comparison between the different organic extracts (ethyl acetate, chloroform, dichloromethane and hexane) of Chaetomium nigricolor mycelial and culture filtrate, it was observed that the mycelial as well the culture filtrate ethyl acetate extracts and the culture filtrate hexane extract showed significant cytotoxic potential against the HeLa and MCF-7 cells, respectively. The apoptotic- and mitochondrial membrane depolarisation-induction potential of the Chaetomium nigricolor ethyl acetate extract has also been demonstrated in this study. Further the screening of antioxidant potential of the ethyl acetate fungal extracts using DPPH scavenging assay showed that Chaetomium nigricolor extract exhibited potential activity with a significant EC50 value of 22 µg mL- 1. The ethyl acetate extract of Chaetomium nigricolor also exhibited superoxide radical scavenging potential. CONCLUSION: These results indicated that diverse endophytic fungal population inhabits Catharanthus roseus. One of the fungal isolate Chaetomium nigricolor exhibited significant cytotoxic, apoptotic and antioxidant potential.

The genome of 'Candidatus Phytoplasma solani' strain SA-1 is highly dynamic and prone to adopting foreign sequences.

Bacteria of the genus 'Candidatus Phytoplasma' are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as 'Ca. P. solani', have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of 'Ca. P. solani' strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The 'Ca. P. solani' SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.

First report on a novel Nigrospora sphaerica isolated from Catharanthus roseus plant with anticarcinogenic properties.

This paper reports on the vinca alkaloid produced by a novel Nigrospora sphaerica isolated from Catharanthus roseus. Through liquid chromatography-mass spectrometry (LCMS), only the crude mycelia extract of this fungus was positive for determination of vinblastine. This vinca alkaloid was then purified by using high-performance liquid chromatography (HPLC) and tested for cytotoxicity activity using MTT assays. The breast cell line cancer (MDA-MB 231) was treated with a purified vinblastine which was intracellulary produced by N. sphaerica. The purified vinblastine from extracted leaf of C. roseus was used as a standard comparison. A positive result with a value of half maximal inhibitory concentration (IC50 ) of > 32 µg ml-1 was observed compared with standard (IC50 ) of 350 µg ml-1 only. It showed that a vinblastine produced by N. sphaerica has a high cytotoxicity activity even though the concentration of vinblastine produced by this endophytic fungus was only 0.868 µg ml-1 .

Effect of Pseudomonas fluorescens RB4 and Bacillus subtilis 189 on the phytoremediation potential of Catharanthus roseus (L.) in Cu and Pb-contaminated soils.

The remediation of heavy metal-contaminated soils has become a critical issue due to toxic effects of these metals on living organisms. The current research was conducted to study the effect of Pseudomonas fluorescens RB4 and Bacillus subtilis 189 on the growth and phytoremediation potential of Catharanthus roseus in Cu- and Pb-contaminated soils. The bacterial strains exhibited significantly higher level of water-extractable Pb and Cu in Pb, Cu, and Cu+Pb-contaminated. The P. fluorescens RB4 inoculated plants, produced 102%, 48%, and 45% higher fresh weight (FW) in soils contaminated with Cu, Pb, and both elements, respectively, as compared to un-inoculated control plants. Similarly, B. subtilis 189 inoculated plants produced 108%, 43%, and 114% more FW in the presence of Cu, Pb, and both elements. The plants co-cultivated with both bacteria exhibited 121%, 102%, and 177% higher FW, in Cu, Pb, and both elements contaminated soils, as compared to respective un-inoculated control. Co-cultivation of P. fluorescens RB4, B. subtilis 189, and P. fluorescens RB4 + B. subtilis 189 resulted in higher accumulation of Cu and Pb in shoots of the C. roseus grown in contaminated soils as compared to un-inoculated control. Bacterial treatments also improved the translocation and metal bioconcentration factors. The growth and phytoextraction capability of C. roseus was improved by inoculation of P. fluorescens RB4 and B. subtilis 189.

Generous hosts: What makes Madagascar periwinkle (Catharanthus roseus) the perfect experimental host plant for fastidious bacteria?

Although much attention has been paid to the metabolism and biosynthesis of monoterpene alkaloids in Catharanthus roseus, its value as an experimental host for a variety of agriculturally and economically important phytopathogenic bacteria warrants further study. In the present study, we evaluated the chemical composition of the phloem and xylem saps of C. roseus to infer the nutritional requirements of phloem- and xylem-limited phytopathogens. Periwinkle phloem sap consisted of a rich mixture of sugars, organic acids, amino acids, amines, fatty acids, sugar acids and sugar alcohols while xylem contained similar compounds in lesser concentrations. Plant sap analysis may lead to a better understanding of the biology of fastidious Mollicutes and their complex nutritional requirements, and to successful culture of phytoplasmas and other uncultured phloem-restricted bacteria such as Candidatus Liberibacter asiaticus, the causal agent of huanglongbing in citrus.

Fungal vincristine from Eutypella spp - CrP14 isolated from Catharanthus roseus induces apoptosis in human squamous carcinoma cell line -A431.

BACKGROUND: Catharanthus roseus, a medicinal plant, is known to produce secondary metabolites, vincristine and vinblastine, which are terpenoid indole alkaloids. Previously we have reported that Eutypella spp - CrP14 isolated from stem cutting of this plant had shown significant antiproliferative activity when tested in vitro against HeLa cell line. The present study was conducted to identify the anticancer compound responsible for the anti-proliferative activity of the fungal extract and to evaluate its in vitro anticancer and apoptotic effects. METHODS: The anti-proliferative activity of the fungal anticancer compound, vincristine was analyzed by MTT assay against different cancer cell lines. We examined its efficacy of apoptotic induction on A431 cells. The parameters examined included cell cycle distribution, loss of mitochondrial membrane potential (MMP), DNA fragmentation and reactive oxygen species (ROS) generation. RESULTS: The presence of vincristine in fungal culture filtrate was confirmed through chromatographic and spectroscopic analyses, and the amount was estimated to be 53 ± 5.0 µg/l. The partially purified fungal vincristine had strong cytotoxic activity towards human squamous carcinoma cells - A431 in the MTT assay. Furthermore, we showed that the fungal vincristine was capable of inducing apoptosis in A431 cells through generation of reactive oxygen species and activation of the intrinsic pathway leading to loss of MMP. CONCLUSIONS: We have demonstrated for the first time that the vincristine from Eutypella spp - CrP14 is an efficient inducer of apoptosis in A431 cells, meriting its further evaluation in vivo.

Cholinesterase inhibitor (Altenuene) from an endophytic fungus Alternaria alternata: optimization, purification and characterization.

AIMS: The aim of this study was to screen endophytic fungi isolated from Vinca rosea for their potential to produce acetylcholinesterase (AChE) inhibitors. METHOD AND RESULTS: Endophytic fungi isolated from V. rosea (Catharanthus roseus), were screened for AChE inhibitor production using Ellman's method. Maximum inhibition against AChE (78%) was observed in an isolate VS-10, identified to be Alternaria alternata on morphological and molecular basis. The isolate also inhibited butyrylcholinesterase (73%). Significant increase (1·3 fold) was achieved after optimization of process parameters using one variable at time approach. The inhibitor was purified using chromatographic techniques. The structure elucidation of the inhibitor was carried out using spectroscopic techniques and was identified to be 'altenuene'. The purified inhibitor possessed antioxidant potential as revealed by dot blot assay. The insecticidal potential of purified inhibitor was evaluated by feeding Spodoptora litura on diet amended with inhibitor. It evinced significant larval mortality. CONCLUSIONS: Endophytic A. alternata can serve as a source of dual cholinesterase inhibitor 'altenuene' with significant antioxidant and insecticidal activity. This is the first report on acetylcholinestearse inhibitory activity of altenuene. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternaria alternata has the potential to produce a dual ChE inhibitor with antioxidant activity useful in the treatment of neurodegenerative disorders and in agriculture as biocontrol agent.

'Candidatus Phytoplasma hispanicum', a novel taxon associated with Mexican periwinkle virescence disease of Catharanthus roseus.

Mexican periwinkle virescence (MPV) phytoplasma was originally discovered in diseased plants of Madagascar periwinkle (Catharanthus roseus) in Yucatán, Mexico. On the basis of results from RFLP analysis of PCR-amplified 16S rRNA gene sequences, strain MPV was previously classified as the first known member of phytoplasma group 16SrXIII, and a new subgroup (16SrXIII-A) was established to accommodate MPV phytoplasma. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MPV represents a lineage distinct from previously described 'CandidatusPhytoplasma' species. Nucleotide sequence alignments revealed that strain MPV shared less than 97.5 % 16S rRNA gene sequence similarity with all previously described 'Ca.Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of Mexican periwinkle virescence phytoplasma strain MPV as representative of a novel taxon, 'CandidatusPhytoplasma hispanicum'.

Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis.

Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229-403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant.

Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots.

Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death.

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp--CrP14, obtained from stem tissues, and Talaromyces radicus--CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 µg/ml and 20 µg/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus--CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus--CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 µg/l) in modified M2 medium and of vinblastine (70 µg/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

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.

Loop-Mediated Isothermal Amplification (LAMP) for Detection of Phytoplasmas in the Field.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based detection method with many applications. This chapter details its use for detection of phytoplasma diseases, combining a rapid 2-min DNA extraction method with real-time LAMP product detection such that the entire procedure can be undertaken and completed in the field within 40 min. Furthermore, the assays include an anneal curve validation step to guard against false positives and an assay for host plant DNA to guard against false negatives.