Metabolomic analysis reveals the potential metabolites and pathogenesis involved in mulberry yellow dwarf disease

To analyse the molecular mechanisms of phytoplasma pathogenicity, the comprehensive metabolomic changes of mulberry leaf and phloem sap in response to phytoplasma infection were examined using gas chromatography‐mass spectrometry. The metabolic profiles obtained revealed that the metabolite compositions of leaf and phloem sap were different, and phytoplasma infection has a greater impact on the metabolome of phloem sap than of leaf. Phytoplasma infection brought about the content changes in various metabolites, such as carbohydrates, amino acids, organic acids, etc. Meanwhile, the results of biochemical analysis showed that the degradation of starch was repressed, and the starch content was increased in the infected leaves. In addition, we found that phytoplasma infection changed the levels of abscisic acid and cytokinin and break phytohormone balance. Interestingly, our data showed that the contents of H₂O₂ and superoxide were increased in the infected leaves, but not in the phloem saps. Based on the results, the expression levels of the genes involved in the metabolism of some changed metabolites were examined, and the potential molecular mechanisms of these changes were discussed. It can be concluded that both the leaf and phloem saps have a complicated metabolic response to phytoplasma infection, but their response mechanisms were different.     

Effect of phytoplasma infection on metabolite content and antioxidant enzyme activity in lime (<Emphasis Type="Italic">Citrus aurantifolia</Emphasis>)

The objective of the present work was to study biochemical alterations in lime plants infected by the Candidatus Phytoplasma aurantifoliae. Changes in antioxidant activities, content of chlorophylls (Chl), carotenoids (Car), soluble proteins, sugars and auxin (IAA) in infected plant were investigated. The activities of polyphenol oxidase (PPO), peroxidase (POX) and superoxide dismutase (SOD) were observed to be greater in infected leaves than the healthy control. Also according to non-denaturing PAGE, in infected leaves all the antioxidative enzymes isoforms were stronger than that of the healthy control. These results suggest that antioxidant enzymes can be activated in response to infection by phytoplasma. The decrease in content of proteins, total soluble and reducing sugars in infected plants point out changes in host metabolism due to the phytoplasma infection. The reduction in chlorophylls and auxin content shows that the phytoplasma can interfere in photosynthesis and induces senescence in the leaf. In conclusion, this study provides new insights into the lime response to phytoplasma infection.     

A role of mulberry leaves in improving resistance to virus‐mediated disease in Allomyrina dichotoma

The purpose of this study was to develop a safe and effective method for preventing Allomyrina dichotoma nudivirus (AdNV) infection in the Korean horned beetle, Allomyrina dichotoma, found on the farms in the Republic of Korea. Mulberry leaf powder was added to fermented oak sawdust to minimize mortality in AdNV‐infected A. dichotoma. Mulberry leaves were found to contain 1‐deoxynojirimycin, which has anti‐inflammatory, antiviral, and anti‐tumor effects. Based on the proposed antiviral effects of mulberry leaves, a feed of fermented sawdust combined with 1% or 5% mulberry leaf powder was fed to AdNV‐infected second or third stage A. dichotoma larvae. The larval mortality rate was recorded over 10 weeks. The second and third instar larvae that were fed with the sawdust mixture with 5% mulberry leaf powder had mortality rates of 60% and 30%, respectively. In contrast, the control group that was fed with the sawdust without mulberry leaf powder had a mortality rate of 100%. Also, we confirmed that AdNV was not detected in the experimental group that was subjected to an outdoor application test for 8 months with mulberry leaf powder treatment. A reduced mortality rate after treatment with 1% mulberry leaf powder was observed in the field application. In addition, a comparison of the control colony and mulberry leaf treated group showed a statistical difference in growth of larvae at various states, and demonstrated the efficiency of mulberry leaf powder combined with fermented sawdust for treatment of AdNV‐ infected A. dichotoma.     

SWATH‐based quantitative proteomics reveals the mechanism of enhanced Bombyx mori nucleopolyhedrovirus‐resistance in silkworm reared on UV‐B treated mulberry leaves

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most acute infectious diseases in silkworm, which has led to great economic loss in sericulture. Previous study showed that the content of secondary metabolites in mulberry leaves, particularly for moracin N, was increased after UV‐B irradiation. In this study, the BmNPV resistance of silkworms reared on UV‐B treated and moracin N spread mulberry leaves was improved. To uncover the mechanism of enhanced BmNPV resistance, silkworm midguts from UV‐B treated mulberry leaves (BUM) and moracin N (BNM) groups were analyzed by SWATH‐based proteomic technique. Of note, the abundance of ribosomal proteins in BUM and BNM groups was significantly changed to maintain the synthesis of total protein levels and cell survival. While, cytochrome c oxidase subunit II, calcium ATPase and programmed cell death 4 involved in apoptotic process were up‐regulated in BNM group. Expressions of lipase‐1, serine protease precursor, Rab1 protein, and histone genes were increased significantly in BNM group. These results suggest that moracin N might be the main active component in UV‐B treated mulberry leaves which could improve the BmNPV‐resistance of silkworm through promoting apoptotic cell death, enhancing the organism immunity, and regulating the intercellular environment of cells in silkworm. It also presents an innovative process to reduce the mortality rate of silkworms infected with BmNPV.     

Shotgun proteomic analysis of mulberry dwarf phytoplasma

Background  

Mulberry dwarf (MD), which is caused by phytoplasma, is one of the most serious infectious diseases of mulberry. Phytoplasmas have been associated with diseases in several hundred plant species. The inability to culture phytoplasmas in vitro has hindered their characterization at the molecular level. Though the complete genomes of two phytoplasmas have been published, little information has been obtained about the proteome of phytoplasma. Therefore, the proteomic information of phytoplasmas would be useful to elucidate the functional mechanisms of phytoplasma in many biological processes.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

Differential expression of LEA proteins in two genotypes of mulberry under salinity

The relative water content (RWC), cell membrane integrity, protein pattern and the expression of late embryogenesis abundant proteins (LEA; group 1, 2, 3 and 4) under different levels of salt stress (0, 1.0, 1.5 and 2.0 % NaCl) were investigated in mulberry (Morus alba L.) cultivars (S1 and ATP) with contrasting salt tolerance. RWC and membrane integrity decreased with increase in NaCl concentration more in cv. ATP than in cv. S1. SDS-PAGE protein profile of mulberry leaves after the NaCl treatments showed a significant increase in 35, 41, 45 and 70 kDa proteins and significant decrease in 14.3, 18, 23, 28, 30, 42, 47 and 65 kDa proteins. Exposure of plants to NaCl resulted in higher accumulation of LEA proteins in S1 than ATP. The maximum content of LEA (group 3 and 4) was detected in S1 at 2.0 % NaCl, which correlates with its salt tolerance.     

Flavescence dorée phytoplasma deregulates stomatal control of photosynthesis in Vitis vinifera

Flavescence dorée (FD) is among the major grapevine diseases causing high management costs; curative methods against FD are unavailable. In FD‐infected plants, decrease in photosynthesis is usually recorded, but deregulation in stomatal control of leaf gas exchange during FD infection and recovery is unknown. We measured the seasonal time course of gas exchange rates in two cultivars (‘Barbera’ and ‘Nebbiolo’) during the term of 1 year when grapevines experienced a water stress and another with no drought, with difference in gas exchange rates in response to FD infection and recovery as assessed by symptom observation and phytoplasma detection through PCR analysis. Chlorophyll fluorescence was also evaluated at the time of maximum symptom severity in ‘Barbera’, the cultivar showing the most severe stress response to FD infection, causing the highest damage in vineyards of north‐western Italy. In FD‐infected plants, net photosynthesis and transpiration gradually decreased during the season, more during the no drought year than during drought. During recovery, healthy (PCR negative) plants infected 2 years before, but not those infected an year before, regained the gas exchange performances to the level as measured before infection. The relationships between stomatal conductance and the residual leaf intercellular CO₂ concentration (c_i) discriminated healthy versus FD‐infected and recovered plants; at the same c_i, FD‐infected leaves had higher non‐photochemical quenching than healthy ones. We conclude that metabolic, not stomatal, leaf gas exchange limitation in FD‐infected and recovered grapevines is the basis of plant response to FD disease. In addition, we also suggest that such response is dependent upon water stress, by showing that water stress superimposes on FD infection in terms of stomatal and metabolic non‐stomatal limitations to carbon assimilation.     

Effects of Phytoplasma Infection on Pigments, Chlorophyll-Protein Complex and Photosynthetic Activities in Field Grown Apple Leaves

Changes in contents of pigments, chlorophyll-protein complex, and photosynthetic activities were investigated in field grown apple (Malus pumila Mill.) leaves infected by Apple Proliferation phytoplasma. The contents of chlorophyll a+b (Chl) and carotenoids (Car) markedly decreased in infected leaves. Similar results were also observed for content of total soluble proteins and ribulose-1,5-bisphosphate carboxylase activity. When various photosynthetic activities were followed in isolated thylakoids, phytoplasma infection caused a marked inhibition of whole chain and photosystem 2 (PS2) activity. Smaller inhibition of photosystem 1 (PS1) activity was observed even in severely infected leaves. The artificial exogenous electron donors, MnCl₂ diphenyl carbazide, and NH₂OH, did not restore the loss of PS2 activity in both mildly and severely infected leaves. Similar results were obtained by Chl fluorescence measurements. The marked loss of PS2 activity in infected leaves was due to the reduction of contents of chlorophyll and light-harvesting chlorophyll-protein 2 complexes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Phytoplasma Infection on Growth and Photosynthesis in Leaves of Field Grown apple (Malus Pumila Mill. cv. Golden Delicious)

The contents of chlorophyll (Chl), leaf biomass, and soluble proteins were markedly decreased in phytoplasma infected apple leaves. Similar results were also observed for ribulose-1,5-bisphosphate carboxylase, ¹⁴CO₂ fixation, and nitrate reductase activity. In contrast, the contents of sugars, starch, amino acids, and total saccharides were significantly increased in phytoplasma infected leaves. In isolated chloroplasts, phytoplasma infection caused marked inhibition of whole photosynthetic electron chain and photosystem 2 (PS2) activity. The artificial exogenous electron donor, diphenyl carbazide, significantly restored the loss of PS2 activity in infected leaves. Similar results were obtained when F_v/F_m was evaluated by in vivo Chl a fluorescence kinetic measurements.     

Proteomic Analysis of Differentially Expressed Proteins in Resistant Soybean Leaves after Phakopsora pachyrhizi Infection

Soybean rust caused by Phakopsora pachyrhizi is a destructive foliar disease in nearly all soybean‐producing countries. Understanding the host responses at the molecular level is certainly essential for effective control of the disease. To identify proteins involved in the resistance to soybean rust, differential proteomic analysis was conducted in soybean leaves of a resistant genotype after P. pachyrhizi infection. A total of 41 protein spots exhibiting a fold change >1.5 between the non‐inoculated and P. pachyrhizi‐inoculated soybean leaves at 12 and 24 h postinoculation (hpi) were unambiguously identified and functionally grouped into seven categories. Twenty proteins were up‐regulated and four proteins were down‐regulated at 12 hpi, whereas 18 proteins were up‐regulated and eight proteins were down‐regulated at 24 hpi. Generally, proteins involved in photosynthesis were down‐regulated, whereas proteins associated with disease and defense response, protein folding and assembly, carbohydrate metabolism and energy production were up‐regulated. Results are discussed in terms of the functional implications of the proteins identified, with special emphasis on their putative roles in defense. Abundance changes of these proteins, together with their putative functions reveal a comprehensive picture of the host response in rust‐resistant soybean leaves and provide a useful platform for better understanding of the molecular basis of soybean rust resistance.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

Global quantitative proteomic analysis profiles host protein expression in response to Sendai virus infection

Sendai virus (SeV) is an enveloped nonsegmented negative‐strand RNA virus that belongs to the genus Respirovirus of the Paramyxoviridae family. As a model pathogen, SeV has been extensively studied to define the basic biochemical and molecular biologic properties of the paramyxoviruses. In addition, SeV‐infected host cells were widely employed to uncover the mechanism of innate immune response. To identify proteins involved in the SeV infection process or the SeV‐induced innate immune response process, system‐wide evaluations of SeV–host interactions have been performed. cDNA microarray, siRNA screening and phosphoproteomic analysis suggested that multiple signaling pathways are involved in SeV infection process. Here, to study SeV–host interaction, a global quantitative proteomic analysis was performed on SeV‐infected HEK 293T cells. A total of 4699 host proteins were quantified, with 742 proteins being differentially regulated. Bioinformatics analysis indicated that regulated proteins were mainly involved in “interferon type I (IFN‐I) signaling pathway” and “defense response to virus,” suggesting that these processes play roles in SeV infection. Further RNAi‐based functional studies indicated that the regulated proteins, tripartite motif (TRIM24) and TRIM27, affect SeV‐induced IFN‐I production. Our data provided a comprehensive view of host cell response to SeV and identified host proteins involved in the SeV infection process or the SeV‐induced innate immune response process.     

Auxin influences symptom expression and phytoplasma colonisation in periwinkle infected with periwinkle leaf yellowing phytoplasma

Auxin imbalance was suggested as a key factor in phytoplasma symptom development. Furthermore, remission of the symptoms of phytoplasma‐infected shoots can be promoted by culturing them in vitro in high‐auxin‐containing media. Therefore, effect of spraying 1‐naphthaleneacetic acid (NAA) on infected periwinkle (Catharanthus roseus) with periwinkle leaf yellowing (PLY) phytoplasma was examined. 1‐Naphthaleneacetic acid stimulated symptom development in phytoplasma‐inoculated shoots. Accelerated symptom development was associated with early accumulation of phytoplasmas. Two PATHOGENESIS‐RELATED (PR) genes, CrPR1a and CrPR1b, were induced by PLY phytoplasma infection, and the induction was suppressed by NAA. Therefore, the accelerated symptom development may be due to the suppression effect of NAA on PR‐related defence. However, while NAA promoted symptom development on shoots inoculated with phytoplasma, more non‐symptomatic shoots containing no phytoplasma were observed, suggesting that NAA prevents phytoplasma colonisation in non‐symptomatic shoots. The expression of two genes encoding jasmonic acid (JA) biosynthesis key enzymes, lipoxygenase and allene oxide cyclase, was downregulated in non‐symptomatic shoots of infected plants, and remained downregulated after auxin treatment. Therefore, the auxin‐promoted resistance should be JA independent. Because auxin may promote symptom development of PLY phytoplasma‐infected periwinkles, it may not link to plant resistance to phytoplasma infection.     

Detection and Identification of Aster Yellows (16SrI) Phytoplasma in Peach Trees in Jordan by RFLP Analysis of PCR-Amplified Products (16S rDNAs)

Aster yellows phytoplasma were detected, for the first time, in peach trees in Al‐Jubiha and Homret Al‐Sahen area. Leaves of infected trees showed yellow or reddish, irregular water‐soaked blotches. Discoloured areas become dry and brittle and the dead tissues dropped out. Under severe infections, leaves fall down and fruits dropped prematurely. Phytoplasmas were detected from all symptomatic peach trees by polymerase chain reaction (PCR) using universal phytoplasmas primers P1/P7 followed by R16F2/R2. No amplification products were obtained from templates of asymptomatic peaches. PCR products (1.2 kb) used for restriction fragment length polymorphism analysis (RFLP) after digestion with endonuclease AluI, HpaII, KpnI and RsaI produced the same restriction profiles for all samples, and they were identical with those of American aster yellows (16SrI) phytoplasma strain. This paper is the first report on aster yellows phytoplasma affecting peach trees in Jordan.     

Laser microdissection of grapevine leaf phloem infected by stolbur reveals site‐specific gene responses associated to sucrose transport and metabolism

Bois Noir is an emergent disease of grapevine that has been associated to a phytoplasma belonging to the XII‐A stolbur group. In plants, phytoplasmas have been found mainly in phloem sieve elements, from where they spread moving through the pores of plates, accumulating especially in source leaves. To examine the expression of grapevine genes involved in sucrose transport and metabolism, phloem tissue, including sieve element/companion cell complexes and some parenchyma cells, was isolated from healthy and infected leaves by means of laser microdissection pressure catapulting (LMPC). Site‐specific expression analysis dramatically increased sensitivity, allowing us to identify specific process components almost completely masked in whole‐leaf analysis. Our findings showed decreased phloem loading through inhibition of sucrose transport and increased sucrose cleavage activity, which are metabolic changes strongly suggesting the establishment of a phytoplasma‐induced switch from carbohydrate source to sink. The analysis focused at the infection site also showed a differential regulation and specificity of two pathogenesis‐related thaumatin‐like genes (TL4 and TL5) of the PR‐5 family.     

Candidatus Phytoplasma trifolii (16SrVI) infection modifies the polyphenols concentration in pepper (Capsicum annuum) plant tissues

Of late, the presence of Candidatus Phytoplasma trifolii was reported as a serious threat to the pepper crop in Zacatecas, México; therefore, asymptomatic and symptomatic pepper plants were collected from a commercial field among three samplings after the fruit set stage was reached. Total DNA was extracted using the CTAB‐based method and tested for phytoplasma using a nested PCR assay, followed by a BLAST, and restriction fragment length polymorphism (RFLP) analysis of 16S rDNA sequences, which confirmed the presence of phytoplasma group 16SrVI, “Candidatus Phytoplasma trifolii” in the symptomatic plants. As the metabolic pathways of pathogen‐infected plants tend to change, resulting in a biochemical differentiation with the noninfected plants, the polyphenolic compound concentrations were quantified from the vegetative tissues (root, stem, leaves and developed fruit/big bud) and were analysed based on a principal component analysis (PCA). Results revealed that, in general, plants tend to a progressive increase in total phenols, flavonoids, condensed tannins and anthocyanins related to the plants exposure to “Ca. P. trifolii” infection, and PCA demonstrated that almost 90% of the observed variance was explained by the first two components. Hence, the phenolic content of the plants increases as a response of the defence mechanism, which reflects its condition and resistance.     

Infection of ‘Candidatus Phytoplasma ulmi’ reduces the protein content and alters the activity of detoxifying enzymes in Amplicephalus curtulus

It has been reported that insecticide‐detoxifying enzymes such as glutathione S‐transferases (GST) and esterases are affected by microbial infections in hemipteran insect vectors. The total protein content, and GST and α‐ and β‐esterase activities were quantified in ‘Candidatus Phytoplasma ulmi’‐infected and uninfected adults of Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) at 25, 35, and 45 days after the acquisition access period (AAP) in the head‐thorax and abdomen sections. The total protein content was lower in phytoplasma‐infected leafhoppers 25, 35, and 45 days after the AAP. Thirty‐five days after the AAP, the GST and β‐esterase activities had increased (26 and 69%, respectively) compared to the control. However, 45 days after the AAP, the phytoplasma‐infected leafhoppers displayed lower GST (87%) and β‐esterase (253%) activities than the uninfected individuals. On the other hand, the α‐esterase activity proved to be unaffected by the phytoplasma infection. Forty‐five days after the AAP, females had a higher phytoplasma titer (46%) in their head‐thorax than in their abdomen sections, whereas males showed a higher titer in their abdomens (75%). In addition, the GST and β‐esterase activities in the abdomen were affected negatively by 96–98% as a result of the increasing ‘Ca. Phytoplasma ulmi’ titer. These results indicate that an infection of ‘Ca. Phytoplasma ulmi’ alters the metabolic activities of A. curtulus.