Növényi víruskórókozók szisztemikus mozgásának vizsgálata korszerű védekezési stratégia kialakításához = Investigation of the systemic movement of cucumoviruses in order to develop new defence strategies

A cucumovírus nemzetségbe tartozó uborka mozaik vírus az egyik legszélesebb gazdanövénykörrel rendelkező növényi vírusoknak. Genomja három RNS-en elosztva, összesen öt fehérjét kódol. Ezek közül korábban a vírus hosszútávú mozgásában a köpenyfehérjének tulajdonítottak kulcsszerepet. OTKA pályázatunk keretében bizonyítottuk. hogy a köpenyfehérje bétaB-bétaC hurok régiójának van központ szerepe a vírus hosszútávú mozgásában uborka növényen, valamint nem egy-egy aminosav megléte vagy hiánya a meghatározó, hanem a víruspartikulum felületének töltéseloszlása fontos. Különböző cucumovírus-növény rendszereken sikerült bizonyítanunk, hogy az RNS 2- kódolódó fehérjéknek is szerepe van a vírus szisztemizálódásában. Földimogyoró satnyulás vírus izolátumok vizsgálatával bizonyítottuk a rekombináns vírus nagymértékű elterjedését hazánkban, valamint az RNS 2-n kódolódó fehérjék szerepét a vírus hosszútávú mozgásában. A különböző, babon lokális illetve szisztemikus tüneteket indukáló CMV izolátumok esetén a szisztematikus fertőzésért felelős részt sikerült a 2a fehérje egyetlen aminosavára lokalizálnunk (631Y) amiben nagy valószínűséggel a fehérje foszforilációja játszik szerepet. A CMV 2a fehérje alanin scanning mutánsai segítségével hat mutánst azonosítottunk melyek Nicotiana clevelandii növényen a vad típusú vírustól eltérően viselkedtek. Ezek közül négy a géncsendesítés szupresszálásában nem volt hatékony, míg kettő a vírus növényen belüli terjedésében bizonyult sérültnek. | Cucumber mosaic virus (CMV) belonging to the genus Cucumovirus has an extremely wild host range. Its genome consists of three genomic RNA and codes for five proteins. Previously the coat protein of cucumoviruses was described as a key factor in long-distance movement. In our present project we have proved, that the betaB-betaC loop region of the coat protein has the central role in long distance movement on cucumber. We have showed that not the presence of specific amino acids is required, but the general electrostatic potential pattern of the particle surface is important. We have also proved the importance of the viral proteins coded by the RNA2 in the long distance movement on different virus-host systems. In the case of peanut stunt virus we have demonstrated the dominance of the recombinant strains in Hungary, and the role of the proteins encoded by RNA 2 in viral long distance movement. Using different CMV isolates we have proved the role of a single amino acid (631Y) of the 2a protein in the systemic movement of the virus on bean and the role of protein phosphorylation could have a function in this case. Using alanin scanning mutatnts of the 2b protein, we have identified six mutants with different pathological characteristics. Four of them were defective in the suppression of gene silencing, while two was defective in viral movement

CAUSEL: an epigenome- and genome-editing pipeline for establishing function of noncoding GWAS variants

The vast majority of disease-associated single nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWAS) are located in the non-protein coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here, we describe a general pipeline in which candidate functional SNPs are first evaluated by fine-mapping, epigenomic profiling, and epigenome editing and then interrogated for causal function by using genome editing to create isogenic cell lines. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top scoring SNP. Epigenome editing confirmed that rs339331 possessed regulatory potential. Using transcription activator-like effector nuclease (TALEN)-mediated genome-editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the “T” risk allele increased transcription of the RFX6 gene, increased HOXB13 binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach to establish functional causality for non-coding sequence variants identified by GWAS

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination

Bababarát szemlélet hatása a gyermekágyi időszak alakulására

Dolgozatom célja, hogy bemutassam milyen hatással van a Bababarát szemlélet alkalmazása a gyermekágyi időszak folyamatainak alakulására, és mely tényezők által váltja ki egyes hatásait. Az általam készített internetes kérdőív és EPDS ív használata segítségével megismerhettem a megkérdezett anyák gyermekágyi időszakának jellemzőit és alakító tényezőit.BSc/BAegészségügyi gondozás és prevenció – védőnőmagyarnappal

p19 interaction with sRNAs <i>in vivo</i>.

<p>(A) Western blots of p19 (upper panel), Northern blot of p19-bound vsiRNAs (middle panel) and miR159 (lower panel) during the CymRSV and Cym19stop virus infection of p19syn and wt <i>N</i>. <i>benthamiana</i> plants as indicated. The ratio of p19-bound miR159 to the input (IP/input) is indicated below the panels. (B) The percent of vsiRNA sequences derived from the positive and negative viral strands in the input and in p19-IP. (C) Global accumulation of different size classes of vsiRNAs in p19syn and wt <i>N</i>. <i>benthamiana</i> plants infected by CymRSV and Cym19stop as indicated. Size classes of vsiRNAs are shown by color codes presented on the right side of the panel. (D) Ratio of full length and truncated miRNA matching reads. The columns show the percent of full-length (fl), 5’-truncated (5’tr) and 3’-truncated (3’tr) miRNAs in the input and p19-IP of p19syn and wild type plants. Values above the columns indicate the normalized read count ratio of p19-IP/input for each miRNA in a log2 scale.</p

p19 protein expressing transgenic <i>Nicotiana benthamiana</i> (p19syn) plants.

<p>(A) Specific developmental phenotype of p19syn plants compared to wild type plants. Three representative independent transgenic lines are shown alongside wild-type <i>N</i>. <i>benthamiana</i> plant. (B) Western blot of p19syn transgenic and wild-type plants from lines shown in (A). The presence of p19 transgene does not impact NbAGO1 or NbAGO2 protein levels. Protein loading is shown below (StainFree). (C) Stem length from cotyledons to the fourth and last leaf insertion in wt and T1 p19syn plants at 8–9 leaf stage (lines:1–29 and 1–57) were used to show the elongated internode trait of p19syn plants. Bars: standard deviation. (D) Wild type and p19syn plants agroinfiltrated with GFP and GFP+p19 constructs as indicated. Pictures were taken at 4 dpi. (E) p19-mediated inhibition of RNA silencing of tobacco magnesium protoporphyrin chelatase subunit I <i>(ChlI)</i> (a key chlorophyll biosynthetic gene) induced by CMV + Y-satRNA infection (14 dpi, left panel), silencing of <i>ChlI</i> causes yellowing on wt plants. Northern blot hybridization of CMV + Y-satRNA infected wt or p19syn plants; nucleolar small RNA U6 was used as an internal control (right panel).</p

Affinity of p19 to perfect or mismatch-containing duplex si/miRNAs <i>in vitro</i>.

<p>Band shift assay of perfect duplex siR171 (A), and mismatched miR171a (B), miR171b (C) and miR171c (D) duplex RNAs’ with p19 protein. The structure of dsRNAs is shown above the gel pictures. Direct measurement of the absolute apparent dissociation constant Kd values (F) were calculated as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005935#ppat.1005935.ref057" target="_blank">57</a>] based on the quantification of band intensities of p19:dsRNA bound fraction as a function of p19 protein concentration (E). Relative dissociation constant (Krel) was calculated by normalization of Kd values to Kd of siR171 (F).</p

Deep sequencing analysis of AGO1- and AGO2-bound sRNAs.

<p><i>N</i>. <i>benthamiana</i> specific reads from mock-inoculated (A), CymRSV- (C) and Cym19stop-infected wild-type plants (E). The vsiRNA reads of the same samples are presented in (B), (D) and (F) respectively. 5’ nucleotides of vsiRNAs are indicated by color code on the right. Size classes (nucleotide) of sRNAs are indicated by numbers. Read counts were normalized to 10⁶ total reads. Note the different scale in panel A.</p

Northern blot analysis of NbAGO1- and NbAGO2-bound vsiRNAs and miRNAs.

<p>(<b>A</b>) Serum- NbAGO1- and NbAGO2-immunoprecipitations from mock-, CymRSV- or Cym19stop-infected wt <i>N</i>. <i>benthamiana</i> plants as shown: upper panels, Western blots for AGO1, AGO2 and p19 are shown (StainFree gel picture shown as loading control); lower panels, Northern blots of AGO1-specific miR159, AGO2-specific miR390 and vsiRNAs (ethidium-bromide stain shown as loading control). (B) Serum- NbAGO1- and NbAGO2-immunoprecipitations from mock- or Cym19stop-infected transgenic p19syn lines as shown: upper panels, Western blots for AGO1, AGO2 and p19 (StainFree gel picture shown as loading control); lower panels, Northern blots of AGO1-specific vsiRNAs and miR159 (ethidium-bromide stain shown as loading control).</p