Integrated Analysis of lncRNAs and mRNAs Reveals Complex Gene Network Mediated by lncRNAs and Regulatory Function of MuLRR-RLK-AS in Response to Phytoplasma Infection in Mulberry.

Phytoplasma disease is one of the most serious infectious diseases that affects the growth and development of mulberry. Long non-coding RNAs (lncRNAs) play an important role in plants' defense systems; however, the contribution of lncRNAs in the response to phytoplasma infection in mulberry is still largely unknown. Herein, strand-specific RNA sequencing was performed to profile the mRNAs and lncRNAs involved in the response to phytoplasma infection in mulberry, and a total of 4169 genes were found to be differentially expressed (DE) between healthy and phytoplasma-infected leaves. Moreover, 1794 lncRNAs were identified, of which 742 lncRNAs were DE between healthy and infected leaves. Target prediction showed that there were 68 and 44 DE lncRNAs which may function as cis and trans-regulators, targeting 54 and 44 DE genes, respectively. These DE target genes are associated with biological processes such as metabolism, signaling, development, transcriptional regulation, etc. In addition, it was found that the expression of the antisense lncRNA (MuLRR-RLK-AS) of the leucine-rich repeat receptor-like protein kinase gene (MuLRR-RLK) was decreased in the phytoplasma-infected leaves. Interestingly, it was found that overexpression of MuLRR-RLK-AS can inhibit the expression of MuLRR-RLK. Moreover, it was found that the expression levels of PTI-related and MAPK genes in the transgenic MuLRR-RLK Arabidopsis plants were significantly higher than those in the wild-type plants when inoculated with pathogens, and the transgenic plants were conferred with strong disease resistance. Our results demonstrate that MuLRR-RLK-AS, as a trans-regulatory factor, can inhibit the expression of the MuLRR-RLK gene and is a negative regulatory factor for mulberry resistance. The information provided is particularly useful for understanding the functions and mechanisms of lncRNAs in the response to phytoplasma infection in mulberry.

Flavescence Dorée Strain-Specific Impact on Phenolic Metabolism Dynamics in Grapevine (Vitis vinifera) throughout the Development of Phytoplasma Infection.

Flavescence dorée phytoplasma (FDp) is a phytopathogenic bacterium associated with Grapevine yellowS disease, which causes heavy damage to viticultural production. Epidemiological data revealed that some FDp strains appear to be more widespread and aggressive. However, there is no data on mechanisms underlying the variable pathogenicity among strains. In this research, we employed chromatographic and spectrophotometric techniques to assess how two strains of FDp influence the levels of grapevine phenolic compounds, which are frequently utilized as indicative markers of stress conditions. The results pointed to the upregulation of all branches of phenolic metabolism through the development of infection, correlating with the increase in antioxidative capacity. The more aggressive strain M54 induced stronger downregulation of phenolics' accumulation at the beginning and higher upregulation by the end of the season than the less aggressive M38 strain. These findings reveal potential targets of FDp effectors and provide the first functional demonstration of variable pathogenicity between FDp strains, suggesting the need for future comparative genomic analyses of FDp strains as an important factor in exploring the management possibilities of FDp.

Ceratobasidium sp. is associated with cassava witches' broom disease, a re-emerging threat to cassava cultivation in Southeast Asia.

Cassava witches' broom disease (CWBD) is a devastating disease of cassava in Southeast Asia (SEA), of unknown etiology. Affected plants show reduced internodal length, proliferation of leaves and weakening of stems. This results in poor germination of infected stem cuttings (i.e., planting material) and significant reductions in fresh root yields and starch content, causing economic losses for farmers and processors. Using a metagenomic approach, we identified a fungus belonging to the Ceratobasidium genus, sharing more than 98.3-99.7% nucleotide identity at the Internal Transcribed Spacer (ITS), with Ceratobasidium theobromae a pathogen causing similar symptoms in cacao. Microscopy analysis confirmed the identity of the fungus and specific designed PCR tests readily showed (1) Ceratobasidium sp. of cassava is strongly associated with CWBD symptoms, (2) the fungus is present in diseased samples collected since the first recorded CWBD outbreaks in SEA and (3) the fungus is transmissible by grafting. No phytoplasma sequences were detected in diseased plants. Current disease management efforts include adjustment of quarantine protocols and guarantee the production and distribution of Ceratobasidium-free planting material. Implications of related Ceratobasidium fungi, infecting cassava, and cacao in SEA and in other potential risk areas are discussed.

Genome-Wide Identification and Characterization of the TLP Gene Family in Phyllostachys edulis and Association with Witches' Broom Disease Resistance in Bamboo.

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins with pivotal roles in plant defense mechanisms. In this study, various bioinformatics and RNA-seq methods were used to analyze the biotic and abiotic stress responses of the TLP family in Phyllostachys edulis. Overall, 81 TLP genes were identified in P. edulis; 166 TLPs from four plant species were divided into three groups and ten subclasses, with genetic covariance observed between these species. Subcellular localization in silico studies indicated that TLPs were primarily distributed in the extracellular. Analysis of the upstream sequences of TLPs demonstrated the presence of cis-acting elements related to disease defense, environmental stress, and hormonal responses. Multiple sequence alignment demonstrated that most TLPs possessed five conserved REDDD amino acid sequences with only a few amino acid residue differences. RNA-seq analysis of P. edulis responses to Aciculosporium take, the pathogenic fungus that causes witches' broom disease, showed that P. edulis TLPs (PeTLPs) were expressed in different organs, with the highest expression in buds. PeTLPs responded to both abscisic acid and salicylic acid stress. These PeTLP expression patterns were consistent with their gene and protein structures. Collectively, our findings provide a basis for further comprehensive analyses of the genes related to witches' broom in P. edulis.

Influences of Jujube Witches' Broom (JWB) Phytoplasma Infection and Oxytetracycline Hydrochloride Treatment on the Gene Expression Profiling in Jujube.

Jujube witches' broom disease (JWB), caused by Candidatus Phytoplasma ziziphi, is the most destructive phytoplasma disease threatening the jujube industry. Tetracycline derivatives treatments have been validated to be capable of recovering jujube trees from phytoplasma infection. In this study, we reported that oxytetracycline hydrochloride (OTC-HCl) trunk injection treatment could recover more than 86% of mild JWB-diseased trees. In order to explore the underlying molecular mechanism, comparative transcriptomic analysis of healthy control (C group), JWB-diseased (D group) and OTC-HCl treated JWB-diseased (T group) jujube leaves was performed. In total, 755 differentially expressed genes (DEGs), including 488 in 'C vs. D', 345 in 'D vs. T' and 94 in 'C vs. T', were identified. Gene enrichment analysis revealed that these DEGs were mainly involved in DNA and RNA metabolisms, signaling, photosynthesis, plant hormone metabolism and transduction, primary and secondary metabolisms, their transportations, etc. Notably, most of the DEGs identified in 'C vs. D' displayed adverse change patterns in 'D vs. T', suggesting that the expression of these genes was restored after OTC-HCl treatment. Our study revealed the influences of JWB phytoplasma infection and OTC-HCl treatment on gene expression profiling in jujube and would be helpful for understanding the chemotherapy effects of OTC-HCl on JWB-diseased jujube.

Small RNA Profiling of Aster Yellows Phytoplasma-Infected Catharanthus roseus Plants Showing Different Symptoms.

Micropropagated Catharantus roseus plants infected with 'Candidatus Phytoplasma asteris' showed virescence symptoms, witches' broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.

State of the Art of the Molecular Biology of the Interaction between Cocoa and Witches' Broom Disease: A Systematic Review.

Significant scientific advances to elucidate the Moniliophthora perniciosa pathosystem have been achieved in recent years, but the molecular biology of this pathogen-host interaction is still a field with many unanswered questions. In order to present insights at the molecular level, we present the first systematic review on the theme. All told, 1118 studies were extracted from public databases. Of these, 109 were eligible for the review, based on the inclusion and exclusion criteria. The results indicated that understanding the transition from the biotrophic-necrotrophic phase of the fungus is crucial for control of the disease. Proteins with strong biotechnological potential or that can be targets for pathosystem intervention were identified, but studies regarding possible applications are still limited. The studies identified revealed important genes in the M. perniciosa-host interaction and efficient molecular markers in the search for genetic variability and sources of resistance, with Theobroma cacao being the most common host. An arsenal of effectors already identified and not explored in the pathosystem were highlighted. This systematic review contributes to the understanding of the pathosystem at the molecular level, offering new insights and proposing different paths for the development of new strategies to control witches' broom disease.

Genome-Wide Identification and Expression of the Paulownia fortunei MADS-Box Gene Family in Response to Phytoplasma Infection.

Paulownia witches' broom (PaWB), caused by phytoplasmas, is the most devastating infectious disease of Paulownia. Although a few MADS-box transcription factors have been reported to be involved in the formation of PaWB, there has been little investigation into all of the MADS-box gene family in Paulownia. The objective of this study is to identify the MADS-box gene family in Paulownia fortunei on a genome-wide scale and explore their response to PaWB infection. Bioinformatics software were used for identification, characterization, subcellular localization, phylogenetic analysis, the prediction of conserved motifs, gene structures, cis-elements, and protein-protein interaction network construction. The tissue expression profiling of PfMADS-box genes was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Transcriptome data and the protein interaction network prediction were combined to screen the genes associated with PaWB formation. We identified 89 MADS-box genes in the P. fortunei genome and categorized them into 14 subfamilies. The comprehensive analysis showed that segment duplication events had significant effects on the evolution of the PfMADS-box gene family; the motif distribution of proteins in the same subfamily are similar; development-related, phytohormone-responsive, and stress-related cis-elements were enriched in the promoter regions. The tissue expression pattern of PfMADS-box genes suggested that they underwent subfunctional differentiation. Three genes, PfMADS3, PfMADS57, and PfMADS87, might be related to the occurrence of PaWB. These results will provide a valuable resource to explore the potential functions of PfMADS-box genes and lay a solid foundation for understanding the roles of PfMADS-box genes in paulownia-phytoplasma interactions.

Walnut Witches'-Broom Disease Threatens Butternut Restoration Efforts in Indiana.

Due to the devastating effects of butternut canker disease, efforts to protect the endangered butternut (Juglans cinerea) tree through resistance breeding have been a primary focus of forest restoration efforts. Walnut witches'-broom (WWB) disease poses a serious threat to these restoration efforts. This study sought to confirm the presence of the WWB disease phytoplasma, 'Candidatus Phytoplasma pruni', in butternut research plantings in Indiana using molecular methods and Sanger sequencing and to identify butternut families affected by the disease. We also sought to better understand the incidence of the WWB phytoplasma in asymptomatic trees and asymptomatic branches of symptomatic trees to better direct management decisions. Sanger sequencing confirmed the presence of the WWB phytoplasma in the butternut restoration plantings, the first confirmation in Indiana based on sequencing to our knowledge, in both symptomatic and some asymptomatic trees. In addition, the WWB phytoplasma was detected in asymptomatic branches of symptomatic trees, indicating that phytoplasma infection is not necessarily localized to symptomatic tissues in a tree. Trees with positive molecular confirmation of the WWB phytoplasma consisted of 23 different butternut families and one family of Japanese walnut (J. ailantifolia), which is considered to be one of the most susceptible species to WWB disease. Based on these findings, future studies should prioritize identifying the hybridity and pedigrees of families and their susceptibility to WWB disease to aid in butternut restoration efforts.

The 'Candidatus Phytoplasma mali' effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

'Candidatus Phytoplasma mali', is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11CaPm, which shares similarity to SAP11AYWB from 'Candidatus Phytoplasma asteris'. SAP11AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.

Comparative Biochemical and Transcriptomic Analyses Provide New Insights into Phytoplasma Infection Responses in Cucumber.

Flat stem and witches' broom phytoplasma-like symptoms in the cucumber inbred line C17 were observed in a greenhouse at Yangzhou University, China for three consecutive planting seasons; these symptoms resulted in a decreased yield. To better understand the cause of these symptoms, 16S rRNA PCR, plant hormones, mineral elements, and RNA-seq profiling were performed using symptomatic and normal stem samples. The results showed that the causal agent was classified as the Candidatus phytoplasma asteris strain, a plant pathogenic prokaryote that could not be cultured in vitro. Measurement of plant hormones showed that auxin, salicylic acid, and jasmonic acid contents were significantly increased, whereas that of ethylene's immediate biosynthetic precursor, 1-aminocyclopropane-1-carboxylic acid, was decreased in the phytoplasma-infected stems compared with the healthy stems. Furthermore, measurement of mineral element composition showed that magnesium, calcium, sodium, iron, and zinc concentrations significantly changed in the phytoplasma-infected cucumber stems compared with the uninfected stems. Comparative RNA-seq identified 253 differentially expressed genes, including 179 upregulated and 74 downregulated genes. Further analyses suggested that genes related to phenylpropanoid biosynthesis, phenylalanine metabolism, and plant hormone signal transduction contributed to phytoplasma infection. Taken together, this study presents the first in-depth assessment of disease symptoms and biochemical content of cucumber stems known to be infected with phytoplasma.

Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters.

MAIN CONCLUSION: Loss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport. Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant's response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Combined transcriptome and metabolome analysis of Nerium indicum L. elaborates the key pathways that are activated in response to witches' broom disease.

BACKGROUND: Nerium indicum Mill. is an ornamental plant that is found in parks, riversides, lakesides, and scenic areas in China and other parts of the world. Our recent survey indicated the prevalence of witches' broom disease (WBD) in Guangdong, China. To find out the possible defense strategies against WBD, we performed a MiSeq based ITS sequencing to identify the possible casual organism, then did a de novo transcriptome sequencing and metabolome profiling in the phloem and stem tip of N. indicum plants suffering from WBD compared to healthy ones. RESULTS: The survey showed that Wengyuen county and Zengcheng district had the highest disease incidence rates. The most prevalent microbial species in the diseased tissues was Cophinforma mamane. The transcriptome sequencing resulted in the identification of 191,224 unigenes of which 142,396 could be annotated. There were 19,031 and 13,284 differentially expressed genes (DEGs) between diseased phloem (NOWP) and healthy phloem (NOHP), and diseased stem (NOWS) and healthy stem (NOHS), respectively. The DEGs were enriched in MAPK-signaling (plant), plant-pathogen interaction, plant-hormone signal transduction, phenylpropanoid and flavonoid biosynthesis, linoleic acid and α-linoleic acid metabolism pathways. Particularly, we found that N. indicum plants activated the phytohormone signaling, MAPK-signaling cascade, defense related proteins, and the biosynthesis of phenylpropanoids and flavonoids as defense responses to the pathogenic infection. The metabolome profiling identified 586 metabolites of which 386 and 324 metabolites were differentially accumulated in NOHP vs NOWP and NOHS and NOWS, respectively. The differential accumulation of metabolites related to phytohormone signaling, linoleic acid metabolism, phenylpropanoid and flavonoid biosynthesis, nicotinate and nicotinamide metabolism, and citrate cycle was observed, indicating the role of these pathways in defense responses against the pathogenic infection. CONCLUSION: Our results showed that Guangdong province has a high incidence of WBD in most of the surveyed areas. C. mamane is suspected to be the causing pathogen of WBD in N. indicum. N. indicum initiated the MAPK-signaling cascade and phytohormone signaling, leading to the activation of pathogen-associated molecular patterns and hypersensitive response. Furthermore, N. indicum accumulated high concentrations of phenolic acids, coumarins and lignans, and flavonoids under WBD. These results provide scientific tools for the formulation of control strategies of WBD in N. indicum.

Distinct Taphrina strains from the phyllosphere of birch exhibiting a range of witches' broom disease symptoms.

The phyllosphere is an important microbial habitat and reservoir of organisms that modify plant health. Taphrina betulina is the causal agent of birch witches' broom disease. Taphrina species are dimorphic, infecting hosts in the filamentous form and residing in the host phyllosphere as non-infectious yeast. As such, they are expected to be found as resident yeasts on their hosts, even on healthy tissues; however, there is little experimental data supporting this supposition. With the aim of exploring the local infection ecology of T. betulina, we isolated yeasts from the phyllosphere of birch leaves, using three sample classes; infected leaves inside symptom-bearing branches, healthy leaves from symptom-free branches on symptom-bearing trees and leaves from symptom-free branches on symptom-free trees. Isolations yielded 224 yeast strains, representing 11 taxa, including T. betulina, which was the most common isolate and was found in all sample classes, including symptom-free samples. Genotyping revealed genetic diversity among these T. betulina isolates, with seven distinct genotypes differentiated by the markers used. Twenty-two representative T. betulina strains were selected for further study, revealing further phenotypic differences. These findings support that T. betulina is ubiquitous on birch and that individual trees host a diversity of T. betulina strains.

Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones.

Witches'-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood. We postulated that the WB symptom is a manifestation of the pathologically induced redistribution of sugar and phytohormones. Employing potato purple top phytoplasma and its alternative host tomato (Solanum lycopersicum), sugar metabolism and transportation, and the spatiotemporal distribution of phytohormones were investigated. A transmission electron microscopy (TEM) analysis revealed that starch breakdown was inhibited, resulting in the degradation of damaged chloroplasts, and in turn, premature leaf senescence. In the infected source leaves, two marker genes encoding asparagine synthetase (Sl-ASN) and trehalose-6-phosphate synthase (Sl-TPS) that induce early leaf senescence were significantly up-regulated. However, the key gibberellin biosynthesis gene that encodes ent-kaurene synthase (Sl-KS) was suppressed. The assessment of sugar content in various infected tissues (mature leaves, stems, roots, and leaf axils) indicated that sucrose transportation through phloem was impeded, leading to sucrose reallocation into the leaf axils. Excessive callose deposition and the resulting reduction in sieve pore size revealed by aniline blue staining and TEM provided additional evidence to support impaired sugar transport. In addition, a spatiotemporal distribution study of cytokinin and auxin using reporter lines detected a cytokinin signal in leaf axils where the axillary buds initiated. However, the auxin responsive signal was rarely present in such leaf axils, but at the tips of the newly elongated buds. These results suggested that redistributed sucrose as well as cytokinin in leaf axils triggered the axillary bud initiation, and auxin played a role in the bud elongation. The expression profiles of genes encoding squamosa promoter-binding proteins (Sl-SBP1), and BRANCHED1 (Sl-BRC1a and Sl-BRC1b) that control axillary bud release, as determined by quantitative reverse transcription (qRT)-PCR, indicated their roles in WB induction. However, their interactions with sugars and cytokinins require further study. Our findings provide a comprehensive insight into the mechanisms by which phytoplasmas induce WB along with leaf chlorosis, little leaf, and stunted growth.

Detecção de 'CA. Phytoplasma phoenicium' em plantas de pervinca (Cathranthus roseus) em Uttar Pradesh, Índia

Cathranthus roseus also known as periwinkle, an ornamental plant contains several medicinal values, was found with the symptoms of little leaf and witches' broom at Shahjahanpur location with the incidence of up to 8%. The phytoplasma etiology was confirmed through scanning electron microscopy examination in all the four-leaf samples. Molecular analysis through PCR with universal primer pairs P1/P6 followed by nested PCR with R16F2n/R16r2 primers yielded ~1.2kbp amplicons in all the four symptomatic leaf samples. One amplicon was eluted, purified, sequenced, and used in BLASTn searches, which showed maximum identity of periwinkle isolate with several isolates of 16SrIX group of phytoplasma. Further, phylogenic analysis and in silico RFLP confirmed the association of 16SrIX-C subgroup phytoplasma in little leaf and witches broom plants which is the first report from India.

Conformational Changes in Three-Dimensional Chromatin Structure in Paulownia fortunei After Phytoplasma Infection.

Higher-order chromatin structures play important roles in regulating multiple biological processes such as growth and development as well as biotic and abiotic stress response. However, little is known about three-dimensional chromatin structures in Paulownia or about whole-genome chromatin conformational changes that occur in response to Paulownia witches' broom (PaWB) disease. We used high-throughput chromosome conformation capture (Hi-C) to obtain genome-wide profiles of chromatin conformation in both healthy and phytoplasma-infected Paulownia fortunei genome. The heat map results indicated that the strongest interactions between chromosomes were in the telomeres. We confirmed that the main structural characteristics of A/B compartments, topologically associated domains, and chromatin loops were prominent in the Paulownia genome and were clearly altered in phytoplasma-infected plants. By combining chromatin immunoprecipitation sequencing, Hi-C signals, and RNA sequencing data, we inferred that the chromatin structure changed and the modification levels of three histones (H3K4me3/K9ac/K36me3) increased in phytoplasma-infected P. fortunei, which was associated with changes of transcriptional activity. We concluded that for epigenetic modifications, transcriptional activity might function in combination to shape chromatin packing in healthy and phytoplasm-infected Paulownia. Finally, 11 genes (e.g., RPN6, Sec61 subunit-α) that were commonly located at specific topologically associated domain boundaries, A/B compartment switching and specific loops, and had been associated with histone marks were identified and considered as closely related to PaWB stress. Our results provide new insights into the nexus between gene regulation and chromatin conformational alterations in nonmodel plants upon phytopathogen infection and plant disease resistance.

Ultrastructure of phytoplasma-infected jujube leaves with witches' broom disease.

The subcellular characteristics of phytoplasma-infected jujube (Ziziphus jujuba) leaves were investigated using transmission electron microscopy. Midrib fragments of witches' broom-diseased jujube leaves were collected from abnormally small leaves at an early stage of branch clustering. The diseased jujube leaves showed multivesicular bodies (MVBs) with vesicles and tubules in the phloem parenchyma cells and sieve elements. The MVBs were connected to the plasma membrane appressed to the cell wall. There were increased callose collars at the pore-plasmodesma unit ends of the sieve elements in the diseased leaves than in control leaves. The proliferation of MVBs in the diseased jujube leaves could be associated with endoplasmic reticulum stress-dependent exosome release. The phytoplasma produced pleomorphic cells in sieve elements. Several types of putative extracellular structures were observed on the phytoplasma cells: (i) fimbriae-like threads, (ii) pili-like projections, (iii) flagella-like appendages, and (iv) tube-like structures. This study provides novel insights into intracellular obligate cell wall-less prokaryotes and host phloem structures.

The role of salicylic acid and benzothiadiazole in decreasing phytoplasma titer of sugarcane white leaf disease.

The objective of this research was to study the effect of Benzothiadiazole (BTH) and Salicylic acid (SA) on the systemic acquired resistance (SAR) of sugarcane the phytoplasma associated with the sugarcane white leaf (SCWL) disease. The experiment was conducted on plants of the sugarcane variety Khon Kaen 3 (KK3) infected with SCWL phytoplasma using insect vectors. Biochemical changes related to the SAR such as SA and total phenolic compounds were followed according to 4 different timepoints: 7, 14, 21 and 28 days after inoculation. Together, phytoplasma were quantified by RT-qPCR using the secA gene of phytoplasma. According to our results, the spraying of BTH and SA tended to increase the amounts of SA, total phenolic compounds and a lower presence of phytoplasma in the plants in comparison with the inoculated control. Spraying BTH at a concentration of 2.4 mM and SA at a concentration of 2.4 mM exhibited the best efficiency to reduce the concentration of phytoplasma. According to RT-qPCR results, the inoculated plants sprayed with BTH displayed a significantly lower concentration of phytoplasma compared to the inoculated controls. Overall, our results indicated that the spray of BTH and SA could induce an efficient SAR response to the phytoplasma associated with the SCWL disease. We expect these results will give support to the development of new products for controlling white leaf disease in sugarcane.

Phytoplasma diseases of plants: molecular diagnostics and way forward.

Phytoplasmas are obligate phytopathogenic bacteria associated with devastating diseases in hundreds of crops across the world. They have been responsible for huge economic losses in many crop plants for decades now. Isolation and establishment of axenic culture of phytoplasma in complex media is a recent progress in phytoplasma research. Earlier methods for phytoplasma disease detection included symptom profiling, microscopy, serology and dodder transmission studies. With advancement in the field of molecular biology, phytoplasma diagnostics and characterisation witnessed radical improvement. Starting from PCR amplification which often necessities a nested PCR on account of low titre of phytoplasmas, to the closed tube quantitative PCR assays and then the ddPCR, an array of diagnostics have been developed for phytoplasma. The isothermal diagnostic platforms are the latest addition to this and the Loop Mediated Isothermal Amplification (LAMP) assay has been applied for the detection of phytoplasma from several hosts. The futuristic approach in phytoplasma detection will be very likely provided by an integration of nanotechnology and molecular diagnostics. Phytoplasma disease management majorly relies on early detection, vector control, use of disease free planting materials and cultivation of resistant varieties. Hence understanding the molecular mechanism of phytoplasma-host interaction is as important as timely and accurate detection, in the management of phytoplasma diseases. Further, the changing climatic scenario and global warming may lead to an upsurge in the phytoplasma diseases spread and severity across the world, making disease management even more challenging.