The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid.

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.     

Suppression by Citrus Exocortis Viroid Infection of the Naturally Occurring Inhibitor of the Conversion of 1-aminocyclopropane-1-carboxylic Acid to Ethylene by Tomato Microsomes

Citrus exocortis viroid (CEVd) infection of tomato cell cultures suppresses the constitutive inhibitor which blocks the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by tomato microsomes. The inhibitor is associated to microsomal membranes and is also found in the water soluble fraction co-isolated from the cells. The inhibitory effect is concentration dependent, heat stable and could be removed from solution by dialysis. Its possible relationship with regulation of the viroid-induced ethylene production is discussed.     

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.     

Identification of the viroid-induced tomato pathogenesis-related (PR) protein P23 as the thaumatin-like tomato protein NP24 associated with osmotic stress

P23, a 23 kDa pathogenesis-related (PR) protein, was purified from citrus exocortis viroid (CEVd)-infected tomato leaves. Partial amino acid sequencing of this protein including the N-terminal and nine additional tryptic fragments covering about 50% of its primary structure revealed extensive homologies to the members of the family of plant thaumatin-like proteins. Sequence alignment revealed that tomato P23 is the previously described NP24 protein found to be associated to osmotic stress in tomato. In view of this fact the possible role of pathogenesis-related P23 protein as a component of a general mechanism of response of the plant is discussed.     

Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon

The levels of polyamines in leaves of Gynura aurantiaca DC and tomato, Lycopersicon esculentum Mill. cv Rutgers, infected with citrus exocortis viroid (CEVd) or treated with silver nitrate or ethephon (2-chloroethylphosphonic acid) were measured by HPLC in relation to development of symptoms. Previously it had been demonstrated that treatment of G. aurantiaca plants with silver nitrate or ethephon closely mimicked the effects of viroid infection in the plants. In the studies reported here, a marked decrease in putrescine level was observed in plants infected by CEVd or treated with silver ions or ethephon. There was no significant change in either spermidine or spermine levels. Treatment of G. aurantiaca plants with specific inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, Co²⁺) or ethylene action (norbornadiene) prevented the decrease of putrescine associated with silver nitrate treatment and had no effect on spermidine or spermine levels. The development of viroid-like symptoms, the production of associated pathogenesis-related proteins, and the rise in protease activity induced by silver nitrate, were all suppressed by exogenous application of putrescine. The decreased level of putrescine as an ethylene-mediated step in the transduction of the viroid and silver or ethephon signaling is discussed.     

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P_L and P_R, of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P_L is the pathogenicity-modulating domain. The role of the P_R domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.     

Biological and molecular analysis of the pathogenic variant C3 of potato spindle tuber viroid (PSTVd) evolved during adaptation to chamomile (Matricaria chamomilla)

Abstract Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.     

Microarray analysis of Etrog citron (Citrus medica L.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection

Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.     

Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.     

Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity.

Full-length double-stranded cDNA was prepared from purified circular RNA of two new Australian field isolates of citrus exocortis viroid (CEV) using two synthetic oligodeoxynucleotide primers. The cDNA was then cloned into the phage vector M13mp9 for sequence analysis. Sequencing of nine cDNA clones of isolate CEV-DE30 and eleven cDNA clones of isolate CEV-J indicated that both isolates consisted of a mixture of viroid species and led to the discovery of eleven new sequence variants of CEV. These new variants, together with the six reported previously, form two classes of sequence which differ by a minimum of 26 nucleotides in a total of 370 to 375 residues. These two classes correlate with two biologically distinct groups when propagated on tomato plants where one produces severe symptoms and the other gives rise to mild symptoms. Two regions of the native structure of CEV, comprising 18% of the total residues, differ between the sequence variants of mild and severe isolates. Whether or not both of these regions are essential for the variation in pathogenicity has yet to be determined.     

Single nucleotide polymorphisms associated with aggressive periodontitis and severe chronic periodontitis in Japanese

Periodontitis is a common inflammatory disease causing destruction of periodontal tissues. It is a multifactor disease involving genetic factors and oral environmental factors. To determine genetic risk factors associated with aggressive periodontitis or severe chronic periodontitis, single nucleotide polymorphisms (SNPs) in multiple candidate genes were investigated in Japanese. We studied 134 patients with aggressive periodontitis, 117 patients with severe chronic periodontitis, and 125 healthy volunteers without periodontitis, under case-control setting, and 310 SNPs in 125 candidate genes were genotyped. Association evaluation by Fisher's exact test (p < 0.01) revealed statistically significant SNPs in multiple genes, not only in inflammatory mediators (IL6ST and PTGDS, associated with aggressive periodontitis; and CTSD, associated with severe chronic periodontitis), but also in structural factors of periodontal tissues (COL4A1, COL1A1, and KRT23, associated with aggressive periodontitis; and HSPG2, COL17A1, and EGF, associated with severe chronic periodontitis). These appear to be good candidates as genetic factors for future study.     

In vitro system for studying interactions between Citrus exocortis viroid and Gynura aurantiaca (Blume) DC. metabolism and growing conditions

Citrus exocortis viroid (CEVd) is an economically important plant pathogen, which infects a broad range of hosts. In order to investigate complex interactions among viroid, host-growing conditions, and plant secondary metabolism, we setup an in vitro system for the cultivation of CEVd-infected and viroid-free Gynura aurantiaca (Blume) DC. shoots. Both basal Murashige and Skoog (MS) medium and MS medium supplemented with cytokinines supported the growth of shoots, but viroid-free shoots performed better than infected ones. The addition of cytokinines induced an increase in the concentration of purple-reddish pigment, cyanidin tetra-glucoside (GAA), which did not depend on the presence of viroid. The differences in single strand conformation polymorphism patterns between CEVd sequence variants obtained from plants grown on medium supplemented with cytokinines and those obtained from plants grown on basal MS medium or in the greenhouse, indicated that cytokinines may influence not only the plant metabolism, but also the viroid population structure.     

The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu³¹⁴ decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe³¹⁵ mutants including the AAloop which retained Glu³¹⁴ had modest AK activity (5–84% of the wild type). All the results above suggested that Glu³¹⁴ played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu³¹⁴ mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7 kJ/mol lower than wild type AK. Therefore Phe³¹⁵ in dimeric AK has a different role from His³¹⁵ in monomeric AK. This might contribute to the stabilization of the native conformation, while His³¹⁵ in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Interference between viroids inducing exocortis and cachexia diseases of citrus

Systemic interactions of long duration among the Group I] Citrus Viroids, CVd-IIa and CVd-IIb were investigated by grafting buds from established citron (Citrus medica L.) sources containing single viroids to a common healthy seedling. In healthy tissues, the two viroids became established in approximately equal titres as a mixed infection. By contrast, tissues that grew from the CVd-IIa or CVd-IIb source buds contained only trace amounts of the heterologous invading viroids. This interference between CVd-IIa, a mild exocortis agent, and CVd-IIb, the cachexia agent, was also demonstrated in the presence of CEVd, the severe exocortis agent but not a Group II viroid. When a CVd-IIb bud was propagated on a citron containing CVd-IIa, a dramatic reduction in the titre of CVd-IIb was observed. The interference between CVd-IIa and CVd-IIb indicates that the mild and relatively innocuous isolate, CVd-IIa, can interfere with the replication and/or accumulation of the severe cachexia agent, CVd-IIb, in citrus. This offers a potential practical approach for the control of cachexia in commercial plantings by “viroid interference”.     

The spectrum of mutations in genes associated with resistance to rifampicin,isoniazid, and fluoroquinolones in the clinical strains of <Emphasis Type="Italic">M. tuberculosis</Emphasis> reflects the transmissibility of mutant clones

To study the transmissibility of drug resistant mutant clones, M. tuberculosis samples were isolated from the patients of the clinical department and the polyclinic of the Central TB Research Institute (n = 1455) for 2011–2014. A number of clones were phenotypically resistant to rifampicin (n = 829), isoniazid (n = 968), and fluoroquinolones (n = 220). We have detected 21 resistance-associated variants in eight codons of rpoB, six variants in three codons of katG, three variants in two positions of inhA, four variants in four positions of ahpC, and nine variants in five codons of gyrA, which were represented in the analyzed samples with varied frequencies. Most common mutations were rpoB 531 Ser→Leu (77.93%), katG 315 (Ser→Thr) (94.11%), and gyrA 94 (Asp→Gly) (45.45%). We found that the mutations at position 15 of inhA (C→T) (frequency of 25.72%) are commonly associated with katG 315 (Ser→Thr). This association of two DNA variants may arise due to the double selection by coexposure of M. tuberculosis to isoniazid and ethionamide. The high transmissibility of mutated strains was observed, which may be explained by the minimal influence of the resistance determinants on strain viability. The high transmissibility of resistant variants may also explain the large populational prevalence of drug-resistant TB strains.