Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect ...hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power NAD(P)H generation from malate in phytoplasmas.
A new defect of potato, Solanum tuberosum L., “ zebra chip,” so named for the characteristic symptoms that develop in fried chips from infected potato tubers, has recently been documented in several ...southwestern states of the United States, in Mexico, and in Central America. This defect is causing millions of dollars in losses to both potato producers and processors. Zebra chip plant symptoms resemble those caused by potato purple top and psyllid yellows diseases. Experiments were conducted to elucidate the association between the psyllid Bactericera cockerelli (Sulc) (Homoptera: Psyllidae) and zebra chip by exposing clean potato plants to this insect under greenhouse and field conditions. Potato plants and tubers exhibiting zebra chip symptoms were tested for phytoplasmas by polymerase chain reaction. Potato psyllids collected from infected potato fields also were tested. Results indicated that there was an association between the potato psyllid and zebra chip. Plants exposed to psyllids in the greenhouse and field developed zebra chip. In the greenhouse, 25.8 and 59.2 of tubers exhibited zebra chip symptoms in the raw tubers and fried chips, respectively. In the field, 15 and 57 of tubers showed symptoms in raw tubers and chips, respectively. No zebra chip was observed in tubers from plants that had not been exposed to psyllids, either in the greenhouse or field. No phytoplasmas were detected from potato plants or tubers with zebra chip symptoms, suggesting that these pathogens are not involved in zebra chip. Of the 47 samples of potato psyllids tested, only two tested positive for the Columbia Basin potato purple top phytoplasma.
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.
Association of 16SrVI group phytoplasmas in symptomatic sweet cherry samples was confirmed in our previous study by amplifying the 16 S rRNA gene in nested PCR assays. However, this method is ...time-consuming and expensive. Therefore, we developed a loop-mediated isothermal amplification (LAMP) assay based detection protocol from a previously identified 16SrVI group phytoplasma strain in cherry samples. Three sets of primers based on the leucyl tRNA synthetase (
leuS
) and 16 S rRNA gene sequences were designed and evaluated to establish a rapid and efficient LAMP assay based detection system for 16SrVI-D subgroup associated cherry phytoplasma strains. The sweet cherry phytoplasma DNA was efficiently amplified by employing a set of
leuS
based LAMP detection primers in a reaction condition of 63
o
C for 70 min. The phytoplasma positive reactions were visualised by ladder like bands on 2% agarose gel, colour change from violet to blue with hydroxynaphthol blue and presence of fluorescence with ethidium bromide after an hour of LAMP reaction. Furthermore, the designed primers were tested for cross reactivity with other groups of phytoplasma strains belonging to 16SrI, 16SrII and 16SrV but could not amplify any of them. The lowest detection limit for the LAMP detection assay was 10 pg/µL. The developed LAMP method was found to be more robust, reliable and sensitive than the conventional nested PCR assay method. As a result, it has the potential to be used in phytoplasma indexing of sweet cherry germplasm and seedlings for disease free vegetative propagative materials.
1 Dipartimento di Biologia Applicata alla Difesa delle Piante, University of Udine, 33100 Udine, Italy
2 Molecular Plant Pathology Laboratory, USDA, ARS, Beltsville, MD 20705, USA
3 Dipartimento di ...Scienze e Tecnologie Agroalimentari, University of Bologna, 40127 Bologna, Italy
4 FLREC, University of Florida, Fort Lauderdale, FL, USA
5 Dipartimento di Scienze Farmaceutiche, University of Salerno, I-84084 Fisciano, Italy
6 Department of Crop Science, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod 123, Oman
Correspondence I.-M. Lee leeim{at}ba.ars.usda.gov
Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV ( rpl22 ) and rpsC ( rps3 ), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.
Abbreviations: NJ, neighbour-joining; rp, ribosomal protein
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are given in Fig. 1.
Figures presenting the position of the degenerate primers and the analysis of putative restriction sites in ribosomal protein operon sequences are available as supplementary material with the online version of this paper.
MicroRNAs (miRNAs) play an important role in responding to biotic and abiotic stresses in plants. Jujube witches'-broom a phytoplasma disease of Ziziphus jujuba is prevalent in China and is a serious ...problem to the industry. However, the molecular mechanism of the disease is poorly understood. In this study, genome-wide identification and analysis of microRNAs in response to witches'-broom was performed. A total of 85 conserved miRNA unique sequences belonging to 32 miRNA families and 24 novel miRNA unique sequences, including their complementary miRNA* strands were identified from small RNA libraries derived from a uninfected and witches'-broom infected Z. jujuba plant. Differentially expressed miRNAs associated with Jujube witches'-broom disease were investigated between the two libraries, and 12 up-regulated miRNAs and 10 down- regulated miRNAs identified with more than 2 fold changes. Additionally, 40 target genes of 85 conserved miRNAs and 49 target genes of 24 novel miRNAs were predicted and their putative functions assigned. Using the modified 5'-RACE method, we confirmed that SPL and MYB were cleaved by miR156 and miR159, respectively. Our results provide insight into the molecular mechanisms of witches'-broom disease in Z. jujuba.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Jujube witches' broom disease (JWB), one of the most serious phytoplasma diseases, usually results in the destruction of Chinese jujube (
Mill.). Although most jujube cultivars are sensitive to JWB, ...we found a few genotypes that are highly resistant to JWB. However, the molecular mechanism of phytoplasma resistance has seldom been studied. Here, we used Chinese jujube "T13," which has strong resistance to JWB, and a typical susceptible cultivar, "Pozao" ("PZ"), as materials to perform comparative transcriptome, hormone, and regulation analyses. After phytoplasma infection, the differential expression genes (DEGs) were detected at all three growth phases (S1, S2, and S3) in "PZ," but DEGs were detected only at the first growth phase in "T13." Meanwhile, no phytoplasma was detected, and the symptoms especially witches' broom caused by JWB were not observed at the last two growth phases (S2 and S3) in "T13." Protein-protein interaction analysis also showed that the key genes were mainly involved in hormone and reactive oxygen species (ROS) signaling. In addition, during the recovered growth phase in "T13" from S1 to S2, the level of hydrogen peroxide (H
O
) was significantly increased and then decreased from S2 to S3. Moreover, jasmonic acid (JA) was significantly accumulated in "PZ" diseased plants, especially at the S2 phase and at the S2 phase in "T13," while the content of salicylic acid (SA) decreased significantly at the S2 phase of "T13" compared to that in "PZ." The changes in H
O
and JA or SA were consistent with the changes in their key synthesis genes in the transcriptome data. Finally, exogenous application of an SA inhibitor 1-aminobenzotriazole (ABT) rescued witches' broom symptoms, while the contents of both JA and MeJA increased after ABT treatment compared to the control, demonstrating that exogenous application of an SA inhibitor rescued the symptoms of jujube after phytoplasma infection by decreasing the contents of SA and increasing the contents of JA and MeJA. Collectively, our study provides a new perspective on the transcriptional changes of Chinese jujube in response to JWB and novel insights that the crosstalk of JA and SA signaling communicated together to contribute to "T13" JWB resistance.
Verify the presence and the molecular identity of phytoplasmas in Northern and Central Italy vineyards where yellows diseases are widespread. Phytoplasma presence and identity were determined by ...PCR/RFLP analyses on 16S ribosomal gene testing 1424 symptomatic samples. The 65% of samples resulted phytoplasma infected; in particular 256 samples were found positive to phytoplasmas belonging to group 16SrV (mainly Flavescence dorée associated), and the remaining 37% was infected by phytoplasmas belonging to ribosomal subgroup 16SrXII-A (Stolbur or Bois Noir associated). 16SrV ribosomal group representative strains were further typed for variability in SecY and rpS3 genes. The results showed the presence of phytoplasmas belonging to 16SrV-C, 16SrV-D and to a lesser extent, 16SrV-A subgroup. Possible relationships between genetic polymorphisms of phytoplasma strains belonging to subgroup 16SrV-C and their geographic distribution and/or epidemic situations were detected. Bois Noir and Flavescence dorée phytoplasmas are present in significant percentages in the areas under investigation. Molecular tools allowed to identify phytoplasma-infected plants and the genes employed as polymorphism markers resulted useful in distinguishing and monitoring the spreading of the diseases associated with diverse phytoplasmas belonging to 16SrV subgroup in vineyards.
Highlights ► Phytoplasmas have dual-host life cycles involving plants and insects. ► Phytoplasmas have reduced genomes rich in mobile genetic elements. ► The majority of phytoplasma effector genes ...lie on mobile genetic elements. ► Phytoplasma effectors alter plant development and aid insect colonization. ► Phytoplasma effectors suppress plant defense to insects.
In the winter of 2019, typical phyllody, flower sterility, and stunting symptoms were observed on daffodil plants (Narcissus tazetta) in the Mersin province of Türkiye. Molecular analyses were ...carried out on 16S rRNA genes of these plants to identify the cause of these symptoms and detect the presence of phytoplasma. DNA extracted from symptomatic plant samples was amplified via nested polymerase chain reaction using the universal primers P1/P7 followed by R16F2n/R16R2. The 16S rRNA gene amplicons from nine samples which were exhibiting severe phyllody symptoms were sequenced, nucleotide BLAST analysis revealed a 99.9% identity match among them and 99.91% with ‘Candidatus Phytoplasma mali’. Further analyses of the 16S rRNA gene-based phylogenetic tree, computer-simulated restriction fragment length polymorphism (virtual RFLP) and conventional RFLP analyses of 16S rRNA gene using MseI, TaqI, RsaI and SspI restriction endonucleases led to assign the phytoplasma to 16Sr group X and subgroup A. For finer characterization of ‘Ca. P. mali’ strain in daffodils, a PCR based multilocus sequence analysis (MLSA) was performed on three distinct genetic loci including nitroreductase and rhodanase-like genes, ribosomal protein genes rpl22 and rps3, and secY gene. The BLAST results showed 99.9% match to ‘Ca. P. mali’ AP-15 subtype (GenBank Accession Number: AJ542541). Electron microscopic examination of the infected daffodil plants revealed the presence of pleomorphic phytoplasma units in the sieve elements of the phloem tissues. Although 16SrX‑A subgroup phytoplasmas have frequently been reported from woody plants, to our knowledge this is the first report of ‘Ca. P. mali’ (16SrX-A) infecting daffodil plants worldwide.
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