Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus

Biological control measures to prevent or reduce Agrobacterium vitis-caused losses in grapevine cultures are a worldwide increasing challenge. In the present study, tumour development in grapevine (Vitis vinifera L.) was induced in the sensitive cv. Kerner by infection with Agrobacterium vitis strain K306, carrying the p35Sgus-int plasmid with the gus gene as marker for transformation by the wild-type T-DNA. Pre-inoculation with the non-tumorigenic A. vitis strain F2/5 prevented tumour induction by K306(p35gus-int). Strain M1154, a Tn5 mutant of F2/5 in the luxR-like aviR gene, partially reduced the biocontrol efficiency compared to the wild-type F2/5. GUS-labelling by K306gus was poor in grapevine in contrast to A. tumefaciens 281(p35gus-int)-induced tumours in Arabidopsis, indicating plant species-dependent variable gus expression. To use the more reliable direct mRNA expression assay by RTPCR, a new experimental plant/A. vitis system was established with Ricinus communis as model plant. Ricinus/A. vitis galls were available within one week after K306gus inoculation, reached diameters up to 5 cm, and contained more abundant GUS staining. An additional transformation marker, mRNA expression of the T-DNA-located iaaM oncogene, coding auxin synthesis, was apparent only in tumours induced by the wild-type A. vitis strain K306 in the absence of the gus construct, which is under the control of the strong 35S CaMV promoter. F2/5 pre-inoculation suppressed GUS staining and gus mRNA expression. DAPI staining revealed the loss of vital fluorescent cell nuclei in F2/5-inoculated grapevine tissue and thus inhibition of any successful T-DNA transfer into host cell nuclei. Differentiation of typical circular vessels in globular vascular bundles in M1154-pretreated galls suggests interference with plant auxin metabolism. In conclusion, together with successfully establishing a new experimental model system, Ricinus/A. vitis, pre-treatment of host tissue with the non-pathogenic strain F2/5 resulted in preventing the integration and expression of the oncogenic T-DNA of A. vitis strains by locally necrotizing host cell nuclei.

Genetic identification, origin and sanitary status of grapevine cultivars (Vitis vinifera L.) grown in Babar, Algeria

This research focused on present grapevine biodiversity of neglected cultivars grown in 'Babar' region, Northeastern Algeria. The obtained results demonstrate the complex, rich, and even surprising inheritance of grapevine biodiversity in such a small region, with currently residual viticulture practiced only for direct consumption. Babar is one of the oldest inhabited areas in Algeria and part of the Atlas Mountains, considered very favorable for wild and cultivated vine growing since protohistoric times. Thirty-seven vines from the traditional growing area were analyzed using nuclear microsatellite (SSR) markers for cultivar identification and RT-qPCR analysis for virus detection and sanitary status evaluation. As a result, thirteen different genotypes were found, most of them showing a very good sanitary status, then constituting a valuable biological source for clonal selection. A close relatedness was evidenced with some Mediterranean varieties, resulting from previous exchanges of grapevine cultivars in the past. Furthermore, the present study highlighted the existence of three new genotypes, highly probably autochthonous of Babar region, with proposed names 'Babari', 'Babar-Algeria', and 'Amesski-Babar'. They could represent unique Algerian varieties, probably preserved over time. The conservation of these endangered genotypes is highly recommended

Deepening inside the homonyms of 'Wildbacher' by means of SSR markers

Sixteen accessions of the grapevine cultivar 'Wildbacher' coming from different European repositories and from Styrian and Italian private vineyards were analysed by genetic fingerprinting with SSR markers. Five different molecular profiles were found, confirming that the name 'Wildbacher' is commonly used as a homonym. Several new genotypes could be linked to the previous list of the 'Wildbacher' family. In particular, in Italian commercial vineyards two main 'Wildbacher' varieties defined by A and B genetic profiles were found. They correspond to the two reference genotypes from Styria, 'Wildbacher Blau' ('Blauer Wildbacher)' and 'Wildbacher Spätblau'. In both countries 'Wildbacher Blau' represents the most spread and better performing type and it is earlier ripening compared to 'Wildbacher Spätblau'. They were morphologically and genetically very similar to each other and they shared at least one allele at each of the 11 SSR loci analyzed for cultivar identification. Three different other 'Wildbacher' genotypes from a German collection were identified as individual cultivars. While 'Rotblättriger' (genotype C) and 'Frühblau' from Germany (genotype D) showed some genetic similarity with A and B genotypes, E 'Wildbacher', coming from Hungary, proved to have a distinct genetic profile. Close relationship to the key variety (for the development of European diversity) 'Heunisch' is indicated by sharing one allele at all loci investigated so far. There exist some indications that the second parent is an extinct wild vine. Evaluation of morphological parameters resulted in some differences among all five genotypes.

Oxidative DNA damage bypass in Arabidopsis thaliana requires DNA polymerase λ and proliferating cell nuclear antigen 2

The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol l catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol l, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol l, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol l in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol l is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes

Unraveling the genetic origin of 'Glera', 'Ribolla Gialla' and other autochthonous grapevine varieties from Friuli Venezia Giulia (northeastern Italy)

'Glera' and 'Ribolla Gialla' are the most economically relevant local grapevine cultivars of Friuli Venezia Giulia region (north-eastern Italy). 'Glera' is used to produce the world-renowned Prosecco wine. 'Ribolla Gialla' cultivation is constantly increasing due to the strong demand for sparkling wine and is the most important variety in Brda (Slovenia). Knowledge of local varieties history in terms of migration and pedigree relationships has scientific and marketing appeal. Following prospections, genotyping and ampelographic characterization of minor germplasm in Friuli Venezia Giulia, a further research was developed to understand the parentage relationships among the grapevine varieties grown in this region. An integrated strategy was followed combining the analysis of nuclear and chloroplast microsatellites with the Vitis 18k SNP chip. Two main recurrent parents were found, which can be regarded as "founders": 'Vulpea', an Austrian variety parent-offspring related with at least ten Friuli Venezia Giulia cultivars, among them 'Glera', and 'Refosco Nostrano', first degree related with other six Friuli Venezia Giulia varieties. 'Ribolla Gialla' was shown to be another member of the impressively long list of offspring derived from the prolific 'Heunisch Weiss'. Combining molecular markers and historical references was a high-performance strategy for retracing and adjusting the history of cultivars

The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA

Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition

Bithiazole Inhibitors of Phosphatidylinositol 4-Kinase (PI4KIIIβ) as Broad-Spectrum Antivirals Blocking the Replication of SARS-CoV-2, Zika Virus, and Human Rhinoviruses

Over half a century since the description of the first antiviral drug, “old” re-emerging viruses and “new” emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated

Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 53 Garnacha accessions coming from Italy, France and Spain. The samples studied include 28 Italian accessions (named Tocai rosso in Vicenza area; Alicante in Sicily and Elba island; Gamay perugino in Perugia province; Cannonau in Sardinia), 19 Spanish accessions of different types (named Garnacha tinta, Garnacha blanca, Garnacha peluda, Garnacha roja, Garnacha erguida, Garnacha roya) and 6 French accessions (named Grenache and Grenache noir). In order to verify the varietal identity of the samples, analyses based on 14 simple sequence repeat (SSR) loci were performed. The presence of an additional allele at ISV3 locus (151 bp) was found in four Tocai rosso accessions and in a Sardinian Cannonau clone, that are, incidentally, chimeras. In addition to microsatellite analysis, intravarietal variability study was performed using AFLP, SAMPL and M-AFLP molecular markers. AFLPs could discriminate among several Garnacha samples; SAMPLs allowed distinguishing few genotypes on the basis of their geographic origin, whereas M-AFLPs revealed plant-specific markers, differentiating all accessions. Italian samples showed the greatest variability among themselves, especially on the basis of their different provenance, while Spanish samples were the most similar, in spite of their morphological diversity

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients’ age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to “kill two birds with one stone” by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure–activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs