Vmp1 Genes Research Articles

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red-leaf Disease ofSalvia miltiorrhizain China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma-specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN-1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII-A), respectively. The phylogenetic trees showed that LN-1 phytoplasma clustered with the members of 16SrXII-A group, including Ca. P. solani. Computer-simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.

FIRST REPORT OF ‘CANDIDATUS PHYTOPLASMA SOLANI’ IN PEPPER AND CELERY IN BOSNIA AND HERZEGOVINA

In August 2015, in Bijeljina (Semberija province of Bosnia and Herzegovina) pepper and celery plants were surveyed for phytoplasma infections. During the survey pepper plants (Capsicum annuum ) of cvs Fortesa, Niska Sipka and Amanda showing stunting and leaf yellowing were observed, while celery plants (Apium graveolens L.) variety ‘Giant Prague’, expressed leaf whitening and stunting; the percentages of symptomatic plants ranged from 20 to 30%, respectively. Leaf samples from symptomatic and asymptomatic plants were tested to verify phytoplasma presence using nested-PCR/RFLP analyses. Results of 16S rDNA RFLP analyses with Tru1I and tuf typing using HpaII (Langer and Maixner, 2004) indicated the presence of a ‘Candidatus Phytoplasma solani’-related strain in all the symptomatic samples tested. There was no variability in the tuf gene and only tuf-type b was detected. Four symptomatic pepper and two celery samples were selected for further characterization amplifying vmp1 and stamp genes (Fialova et al., 2009; Fabre et al., 2011). The nucleotide sequences of the obtained amplicons were submitted to GenBank (accession Nos. KU340846-51 and KU295501-06 for vmp1 and stamp sequences, respectively). Homology with ‘Ca. P. solani’ sequences from database was 96- 100% for the vmp1 and 99-100% for the stamp gene. Combined RFLP analyses using Tru1I and Hpy188I on stamp and RsaI on vmp1 genes distinguished four ‘Ca. P. solani’ lineages indicating the presence of genetic variability among the phytoplasmas studied. This is the first report of ‘Ca. P. solani’ presence in symptomatic pepper and celery in Bosnia and Herzegovina.

Identification and Characterization of New 'Candidatus Phytoplasma solani' Strains Associated with Bois Noir Disease in Vitis vinifera L. Cultivars Showing a Range of Symptom Severity in Georgia, the Caucasus Region.

Evidence from a preliminary survey highlighted that 'Candidatus Phytoplasma solani', the etiological agent of bois noir (BN) disease of grapevine, infects grapevine varieties in Georgia, a country of the South Caucasus. In this study, field surveys were carried out to investigate the BN symptom severity in international and Georgian native varieties. 'Ca. P. solani' was detected and identified by polymerase chain reaction-based amplification and restriction fragment length polymorphism analysis of 16S ribosomal DNA, and further characterized by multiple gene typing analysis (vmp1 and stamp genes). Obtained data highlighted that the majority of Georgian grapevine varieties showed moderate and mild symptoms, whereas international cultivars exhibited severe symptoms. Molecular characterization of 'Ca. P. solani' from grapevine revealed the presence of 11 distinct phytoplasma types. Only one type (VmGe12/StGe7) was identical to a strain previously reported in periwinkle from Lebanon; the other 'Ca. P. solani' types are described here for the first time. Phylogenetic analyses of vmp1 and stamp gene concatenated nucleotide sequences showed that 'Ca. P. solani' strains in Georgia are associated mainly with the bindweed-related BN host system. Moreover, the fact that 'Ca. P. solani' strains are distributed in grapevine cultivars showing a range of symptom intensity suggests a different susceptibility of such local cultivars to BN.

Occurrence of different phytoplasma infections in wild herbaceous dicots growing in vineyards affected by bois noir in Tuscany (Italy)

1 , domenico RIZZO 2 Summary. Wild herbaceous dicotyledonous plants (dicots) showing symptoms ascribable to phytoplasma disor- ders were found to be widely distributed in organic vineyards in central Tuscany (Italy) affected by bois noir, a grapevine yellows disease caused by Candidatus Phytoplasma solani. In 2010 symptomatic dicots were tenta- tively identified to species level and the incidence of symptoms estimated in two selected vineyards in the prov- ince of Florence. Incidence ranged from 2 to 77%, and was not related to the relative abundance of hosts since very common species as well as relatively rare ones were consistently found to be symptomatic. PCR indexing and 16S rRNA sequence analyses indicated that two phytoplasmas co-existed in the vineyards: Ca. P. solani, infecting the root systems of 17 taxa, and a phytoplasma closely related to Ca. P. phoenicium, infecting 11 taxa, and occasion- ally co-infecting the same plant. Regardless of the high frequency of both pathogens in the vineyards, only Ca. P. solani could be detected in the grapevines. Population screening by means of tuf sequence analyses revealed the presence of only the tuf-b Ca. P. solani type both in dicot hosts and grapevine. This supports current notions of bois noir epidemiology, indicating that some infected dicots act as sources of Ca. P. solani inoculum whereas oth- ers are dead-end hosts. When the same specimens were screened by sequence analysis of the vmp1 gene, evidence was found that different phytoplasma genotypes may be predominant in grapevines and dicots.

Vmp1 and stamp genes variability of ‘Candidatus phytoplasma solani’ in Bosnian and Herzegovinian grapevine

Bois noir is one of the most important diseases caused by ‘Candidatus Phytoplasma solani’ affecting grapevine in the Euro-Mediterranean area. ‘Ca. P. solani’ characterization plays a significant role in its diagnosis, control and understanding of its epidemiology. In the present study, two genes encoding membrane proteins, namely vmp1 and stamp, were used to assess the genetic variability of 18 ‘Ca. P. solani’ grapevine isolates from the Herzegovina region (B&H), previously characterised as tuf-b type. Nested-PCR/RFLP and sequence analyses of vmp1 gene showed the presence of three profiles V17, V14 and V4 where V17 was highly prevalent. Phylogenetic analysis of stamp gene revealed the presence of four different genotypes, where Rqg50 was the most frequent. Finally, the combined results of tuf, vmp1 and stamp genes, reported three previously identified genotypes from Serbia, Austria and Montenegro, and two newly described ones (DB1 and DB2). Although performed only on ‘Ca. P. solani’ isolates from infected grapevines, this study provides useful molecular information to get better insight on BN epidemiology in B&H.

‘Candidatus phytoplasma solani’ genotypes associated with potato stolbur in Serbia and the role of Hyalesthes obsoletus and Reptalus panzeri (hemiptera, cixiidae) as natural vectors

A progressive spread of stolbur-associated symptoms observed in potato fields in Serbia over the past few years initiated the study on disease epidemiology and transmission pathways performed during 2013 and 2014. Inspection of potato fields on 12 localities in northern Serbia revealed high incidence (60 % of symptomatic plants) and wide dispersal (100 % of inspected localities) of ‘Candidatus Phytoplasma solani’. A qualitative analysis of Auchenorrhyncha fauna in affected potato fields identified 16 species, however only Hyalesthes obsoletus, Reptalus panzeri and R. quinquecostatus tested positive for ‘Ca. P. solani’. Multilocus typing of strains associated with field collected potato plants and insects had been performed to identify the propagation scenario underlying the threatening epidemics. Combined analyses of the tuf, stamp and vmp1 genes detected ten genotypes, seven of which were shared by the potato plants and insects, confirming their interaction, with no clear species-specific association of certain ‘Ca. P. solani’ genotypes with plausible insect vectors. Semi- field experiments with naturally ‘Ca. P. solani’-infected H. obsoletus and R. panzeri confirmed the ability of both species to successfully transmit the pathogen to potato plants and induce symptoms characteristic of stolbur disease. The third putative vector R. quinquecostatus shared genotypes of ‘Ca. P. solani’ with potato plants and other two cixiids, and though not tested in this study should not be ruled out as a potential vector. Our study revealed rather complex epidemiology of potato stolbur in Serbia involving several possible routes of horizontal transmission and provided experimental evidence for two natural planthopper vectors.

Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.

Genetic Variability of Stolbur Phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) and its Main Host Plants in Vineyard Agroecosystems.

Bois noir is an economically important grapevine yellows that is induced by 'Candidatus Phytoplasma solani' and principally vectored by the planthopper Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). This study explores the 'Ca. P. solani' genetic variability associated to the nettle-H. obsoletus and bindweed-H. obsoletus systems in vineyard agroecosystems of the central-eastern Italy. Molecular characterization of 'Ca. P. solani' isolates was carried out using polymerase chain reaction/restriction fragment length polymorphism to investigate the nonribosomal vmp1 gene. Seven phytoplasma vmp-types were detected among the host plants- and insect-associated field-collected samples. The vmp1 gene showed the highest polymorphism in the bindweed-H. obsoletus system, according to restriction fragment length polymorphism analysis, which is in agreement with nucleotide sequence analysis. Five vmp-types were associated with H. obsoletus from bindweed, of which one was solely restricted to planthoppers, with one genotype also in planthoppers from nettle. Type V12 was the most prevalent in both planthoppers and bindweed. H. obsoletus from nettle harbored three vmp-types, of which V3 was predominant. V3 was the only type detected for nettle. Our data demonstrate that planthoppers might have acquired some 'Ca. P. solani' profiles from other plant hosts before landing on nettle or bindweed. Overall, the different vmp1 gene rearrangements observed in these two plant hosts-H. obsoletus systems might represent different adaptations of the pathogen to the two host plants. Molecular information about the complex of vmp-types provides useful data for better understanding of Bois noir epidemiology in vineyard agroecosystem.

IL-4 Up-Regulates MiR-21 and the MiRNAs Hosted in the CLCN5 Gene in Chronic Lymphocytic Leukemia.

Interleukin 4 (IL-4) induces B-cell differentiation and survival of chronic lymphocytic leukemia (CLL) cells. MicroRNAs (miRNAs) regulate mRNA and protein expression, and several miRNAs, deregulated in CLL, might play roles as oncogenes or tumor suppressors. We have studied the miRNA profile of CLL, and its response to IL-4, by oligonucleotide microarrays, resulting in the detection of a set of 129 mature miRNAs consistently expressed in CLL, which included 41 differentially expressed compared to normal B cells (NBC), and 6 significantly underexpressed in ZAP-70 positive patients. IL-4 stimulation brought about up-regulation of the 5p and 3p mature variants of the miR-21 gene, which maps immediately downstream to the VMP1 gene, and of the mature forms generated from the miR-362 (3p and 5p), miR-500a (3p), miR-502 (3p), and miR-532 (3p and 5p) genes, which map within the third intron of the CLCN5 gene. Both genes are in turn regulated by IL-4, suggesting that these miRNAs were regulated by IL-4 as passengers from their carrier genes. Their levels of up-regulation by IL-4 significantly correlated with cytoprotection. MiR-21 has been reported to be leukemogenic, associated to bad prognosis in CLL, and the miRNA more frequently overexpressed in human cancer. Up-regulation by IL-4 of miR-21 and the miRNAs hosted in the CLCN5 locus may contribute to evasion of apoptosis of CLL cells. These findings indicate that the IL-4 pathway and the miRNAs induced by IL-4 are promising targets for the development of novel therapies in CLL.

The molecular epidemiology of bois noir grapevine yellows caused by ‘Candidatus Phytoplasma solani’ in the Republic of Macedonia

Bois noir (BN), which is induced by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), is an important grapevine yellows disease that causes severe damage in viticultural regions throughout the Euro-Mediterranean basin. An epidemiological survey to determine potential insect vectors and the primary reservoir plants of BN phytoplasma in Macedonian vineyards was undertaken between 2012 and 2013 in the southeastern part of the country. A study on the species diversity from the suborder Auchenorrhycha revealed the prevalence of the principal vector of ‘Ca. P. solani’, which is the planthopper Hyalesthes obsoletus. Reptalus panzeri, which is the second-most documented BN vector, was not recorded in Macedonian vineyards. Three leafhopper species, namely Psammotettix alienus, Artianus manderstjernii and Euscelis incisus, were also widespread in the BN-affected vineyards, but only H. obsoletus tested positive for ‘Ca. P. solani’. Molecular characterizations were performed by the sequencing and/or RFLP typing of tuf, vmp1 and stamp genes, and the results were used to gain detailed insight into the molecular diversity of the ‘Ca. P. solani’ strains associated with grapevines, tentative reservoir plants (Urtica dioica and Convolvulus arvensis) and the H. obsoletus associated with these plants. Among the 91 ‘Ca. P. solani’ strains detected in diverse plant and insect hosts, three tuf, five vmp1 and 11 distinct stamp genotypes were identified. Twelve comprehensive genotypes of ‘Ca. P. solani’ were detected according to the tuf/vmp1/stamp genotyping. The highest diversity of genotypes was detected among the strains from H. obsoletus individuals associated with U. dioica, of which the most frequent genotype was tuf-ab/V18/M1 (43 %). The tuf-b/V2-TA/STOL comprehensive genotype was found in 33 % of naturally infected grapevines. Two ‘Ca. P. solani’ genotypes were associated with U. dioica, namely (i) tuf-ab/V18/M1 (60 %) and tuf-a/V3/M4 (40 %), and only one genotype (tuf-b/V2-TA/Rqg50) was associated with C. arvensis.

A case study of FD and BN phytoplasma variability in Croatia: multigene sequence analysis approach

Uncultivable bacteria from the genus ‘Candidatus Phytoplasma’ are associated with grapevine yellows (GY) diseases worldwide. In Euro-Mediterranean viticultural areas, GY are most frequently caused by Bois Noir (BN) and Flavescence Doree (FD) phytoplasmas. Surveys of GY in Croatia have been conducted regularly since 1997. BN phytoplasmas have been found to be widespread, while FD phytoplasmas were recently discovered in restricted areas of the country. The aim of this study was to assess the variability of genotypes involved in GY pathosystems by a multilocus sequence typing (MLST) approach. Grapevine, weed and insect vector samples were collected from three locations. Species-specific stamp and vmp1 genes, together with house-keeping genes tuf and secY, were amplified from BN strains. In FD strains, the genes secY, map and uvrB-degV were analyzed. MLST revealed a diversity of BN genotypes, one of which was prevalent and identified in samples of grapevine and the insect vector Hyalesthes obsoletus, corroborating their affiliation to the same pathosystem. Distinct BN strains found in bindweed and two grapevine samples indicated the presence of different BN pathosystems involving a yet unidentified vector, possibly from the genus Reptalus. Moreover, a co-occurence of BN and FD phytoplasma in the same vineyard was identified. The genotyping of FD strains from both grapevine and Scaphoideus titanus showed the presence of at least two distinct FD genotypes at two different locations, suggesting separate introductions of the disease into the country. In this study, MLST proved to be a useful and informative tool in advancing the understanding of GY epidemiology in Croatia.

In-vineyard population structure of ‘Candidatus Phytoplasma solani’ using multilocus sequence typing analysis

‘Candidatus Phytoplasma solani’ is a phytoplasma of the stolbur group (16SrXII subgroup A) that is associated with ‘Bois noir’ and causes heavy damage to the quality and quantity of grapevine yields in several European countries, and particularly in the Mediterranean area. Analysis of ‘Ca. P. solani’ genetic diversity was carried out for strains infecting a cv. ‘Chardonnay’ vineyard, through multilocus sequence typing analysis for the vmp1, stamp and secY genes. Several types per gene were detected: seven out of 20 types for vmp1, six out of 17 for stamp, and four out of 16 for secY. High correlations were seen among the vmp1, stamp and secY typing with the tuf typing. However, no correlations were seen among the tuf and vmp1 types and the Bois noir severity in the surveyed grapevines. Grouping the ‘Ca. P. solani’ sequences on the basis of their origins (i.e., study vineyard, Italian regions, Euro-Mediterranean countries), dN/dS ratio analysis revealed overall positive selection for stamp (3.99, P=0.019) and vmp1 (2.28, P=0.001). For secY, the dN/dS ratio was 1.02 (P=0.841), showing neutral selection across this gene. Using analysis of the nucleotide sequencing by a Bayesian approach, we determined the population structure of ‘Ca. P. solani’, which appears to be structured in 3, 5 and 6 subpopulations, according to the secY, stamp and vmp1 genes, respectively. The high genetic diversity of ‘Ca. P. solani’ from a single vineyard reflects the population structure across wider geographical scales. This information is useful to trace inoculum source and movement of pathogen strains at the local level and over long distances.

Bois noir phytoplasma variability in a Mediterranean vineyard system: new plant host and putative vectors

The presence of Bois noir phytoplasma (BNp, Stolbur, 16SrXII-A) in Sicily, one of the most important Italian grape-producing regions, has been known since 1996, but its epidemiology and distribution in the region have been studied only recently. Grapevines and weeds showing symptoms of phytoplasma infection were collected in an organic Chardonnay vineyard, chosen as model vineyard, and tested to verify BNp presence. Leaf- and plant-hoppers were also captured in the same field and included in the analysis. Plant and insect infections were assessed by nested PCR with universal and group-specific (16SrI and –V) phytoplasma primer pairs. BNp was detected in the weed species Erigeron bonariensis, which was then determined as a new natural host of this phytoplasma, and in several Auchenorrhyncha species besides Hyalesthes obsoletus, which can be regarded as new potential vectors. To further evaluate the genetic variability of BNp among host plants and potential vectors, PCR-RFLP of the Stolbur phytoplasma-specific vmp1 gene was carried out. The V1 profile was the most representative, other profiles (V4, V9, V10 and V11) were also detected.

Multiple gene analyses identify distinct “bois noir” phytoplasma genotypes in the Republic of Macedonia

“Bois noir” (BN) is a grapevine yellows disease, associated with phytoplasma strains related to ‘ Candidatus Phytoplasma solani’, that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA , tuf , vmp1 , and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf ( tuf -type a, tuf -type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies.

Incidence of Bois Noir phytoplasma in different viticulture regions of Spain and Stolbur isolates distribution in plants and vectors

The incidence of Bois Noir (BN) disease in grapevine plots, the population of the vector Hyalesthes obsoletus, and the distribution of stolbur phytoplasma isolates were studied over a 4 year period in five regions of Spain. BN incidence in affected plots ranged from 1 to 75 %. A study of the H. obsoletus population indicated that individuals of this insect vector were identified in most of the sampled plots with low populations, from 0.25–5 individuals per aspiration. The population peaks of H. obsoletus were reached at different dates between June 6th and July 24th, depending on the sampled zone and the year. The percentage of H. obsoletus individuals carrying the phytoplasma showed an average of 55 %. In Aragon, this percentage rose to 76 %. The strain tuf-b of stolbur phytoplasma was the most prevalent type in grapevine plants and H. obsoletus, except in grapevine plants from La Rioja where the strain tuf-a was detected in most plants. A study of the vmp1 gene revealed the presence of four different isolates in grapevine plants and H. obsoletus.

Spatial and temporal stolbur population structure in a cv. Chardonnay vineyard according to vmp1 gene characterization

Bois noir is a grapevine disease caused by the stolbur phytoplasma. It is widespread in all European and Mediterranean viticultural areas, and it can induce severe damage to the quality and quantity of production. The recent disease recrudescence has encouraged studies on the use of molecular markers to assess the genetic diversity of stolbur strains. The aim of this study was to evaluate the presence of Bois noir symptoms and to monitor the spatial genetic structure of the stolbur population according to vmp1 genotypes, through 2011 and 2012 in a cv. Chardonnay vineyard. In both years, there were increased vines with symptoms from July to September. The analysis of dispersal indices showed that the spatial distribution was uniform in the vineyard. However, the two‐dimensional contour maps show that Bois noir severity was higher in plants located on the borders than in the central parts of the vineyard. Stolbur population was composed of two prevalent vmp genotypes (V14, V12) across both years, along with other minor haplotypes (V3, V4, V9, V11, V15, V18, in 2011; V3, V18 in 2012). The data indicate that the vmp1 gene is an efficient marker to study the population structure of stolbur phytoplasma, to track the movement of the pathogen, and to identify the inoculum source, which will all serve in the planning of control strategies.

VMP1 is a novel inducible gene which activation is a switch for autophagy

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which participates in cell homeostasis as well as in response to disease. Autophagy acts in a myriad of cellular events leading to survival or cell death. The aim of this work was to study the effector molecules that regulate the transcription of the inducible gene vmp1, which expression triggers autophagy. Using a combination of luciferase assays, expression analysis and shRNA strategies, we report the identification of the promoter region of the human vmp1 gene and the molecular pathways that lead to its activation. We show that vmp1 promoter responds to autophagy stimuli through mTOR‐dependent and mTOR‐independent pathways. Besides we present the essential sequence required for this activation containing the TATA Box and several consensus binding sites for transcription factors related to the cellular stress such as CREB, E2F1, c‐Jun and c‐Fos. We demonstrate that overexpression of any of this factors induces VMP1 expression. Moreover using shRNA technology, p62 degradation and LC3 cleavage or recruitment we show that these factors induce VMP1‐mediated autophagy. Finally we found that p300 acts and is required as a coactivator for VMP1 activation and autophagy. Our results define VMP1 as a target for cellular stress mediators, which activation is a switch for autophagy. AL:CONICET;MIV:UBA‐ CONICET‐ ANPCyT.

Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines, bindweeds and vegetables

Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf-a type and 68% by tuf-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf-b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences' analysis and virtual digestions. The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid: Zhou JM, Trifa Y, Silva H, Pontier D, Lam E, Shah J, Klessig DF: Molec Plant Microbe Interact 2000, 13:191–202

‘Candidatus Phytoplasma solani’ is a phytoplasma of the stolbur group (16SrXII subgroup A) that is associated with ‘Bois noir’ and causes heavy damage to the quality and quantity of grapevine yields in several European countries, and particularly in the Mediterranean area. Analysis of ‘Ca. P. solani’ genetic diversity was carried out for strains infecting a cv. ‘Chardonnay’ vineyard, through multilocus sequence typing analysis for the vmp1, stamp and secY genes. Several types per gene were detected: seven out of 20 types for vmp1, six out of 17 for stamp, and four out of 16 for secY. High correlations were seen among the vmp1, stamp and secY typing with the tuf typing. However, no correlations were seen among the tuf and vmp1 types and the Bois noir severity in the surveyed grapevines. Grouping the ‘Ca. P. solani’ sequences on the basis of their origins (i.e., study vineyard, Italian regions, Euro-Mediterranean countries), dN/dS ratio analysis revealed overall positive selection for stamp (3.99, P = 0.019) and vmp1 (2.28, P = 0.001). For secY, the dN/dS ratio was 1.02 (P = 0.841), showing neutral selection across this gene. Using analysis of the nucleotide sequencing by a Bayesian approach, we determined the population structure of ‘Ca. P. solani’, which appears to be structured in 3, 5 and 6 subpopulations, according to the secY, stamp and vmp1 genes, respectively. The high genetic diversity of ‘Ca. P. solani’ from a single vineyard reflects the population structure across wider geographical scales. This information is useful to trace inoculum source and movement of pathogen strains at the local level and over long distances.