László G. Kovács

Powdery Mildew Induces Defense-Oriented Reprogramming of the Transcriptome in a Susceptible But Not in a Resistant Grapevine

Grapevines exhibit a wide spectrum of resistance to the powdery mildew fungus (PM), Erysiphe necator (Schw.) Burr., but little is known about the transcriptional basis of the defense to PM. Our microscopic observations showed that PM produced less hyphal growth and induced more brown-colored epidermal cells on leaves of PM-resistant Vitis aestivalis ‘Norton’ than on leaves of PM-susceptible Vitis vinifera ‘Cabernet sauvignon’. We found that endogenous salicylic acid levels were higher in V. aestivalis than in V. vinifera in the absence of the fungus and that salicylic acid levels increased in V. vinifera at 120 h postinoculation with PM. To test the hypothesis that gene expression differences would be apparent when V. aestivalis and V. vinifera were mounting a response to PM, we conducted a comprehensive Vitis GeneChip analysis. We examined the transcriptome at 0, 4, 8, 12, 24, and 48 h postinoculation with PM. We found only three PM-responsive transcripts in V. aestivalis and 625 in V. vinifera. There was a significant increase in the abundance of transcripts encoding ENHANCED DISEASE SUSCEPTIBILITY1, mitogen-activated protein kinase kinase, WRKY, PATHOGENESIS-RELATED1, PATHOGENESIS-RELATED10, and stilbene synthase in PM-infected V. vinifera, suggesting an induction of the basal defense response. The overall changes in the PM-responsive V. vinifera transcriptome also indicated a possible reprogramming of metabolism toward the increased synthesis of the secondary metabolites. These results suggested that resistance to PM in V. aestivalis was not associated with overall reprogramming of the transcriptome. However, PM induced defense-oriented transcriptional changes in V. vinifera.

Changes in protein abundance during powdery mildew infection of leaf tissues of Cabernet Sauvignon grapevine (Vitis vinifera L.).

A comparative analysis of differentially expressed proteins in a susceptible grapevine (Vitis vinifera ‘Cabernet Sauvignon’) during the infection of Erysiphe necator, the causal pathogen of grapevine powdery mildew (PM), was conducted using iTRAQ. The quantitative labeling analysis revealed 63 proteins that significantly changed in abundance at 24, 36, 48, and 72 h post inoculation with powdery mildew conidiospores. The functional classification of the PM‐responsive proteins showed that they are involved in photosynthesis, metabolism, disease/defense, protein destination, and protein synthesis. A number of the proteins induced in grapevine in response to E. necator are associated with the plant defense response, suggesting that PM‐susceptible Cabernet Sauvignon is able to initiate a basal defense but unable to restrict fungal growth or slow down disease progression.

Up-regulated transcripts in a compatible powdery mildew-grapevine interaction

Powdery mildews (Erysiphales) are obligate biotrophic pathogens that invade susceptible plant cells without triggering cell death. This suggests a highly adept mechanism of parasitism which enables powdery mildews to avoid detection or evade defenses by their host. To better understand this plant-pathogen interaction, we employed suppression subtractive hybridization (SSH), differential hybridization and quantitative real-time (qRT) PCR for the identification of grapevine (Vitis vinifera L.) genes that were specifically up-regulated in response to the grape powdery mildew Erysiphe necator Schwein. We identified 25 grapevine transcripts that increased in abundance upon infection in leaves of the susceptible host V. vinifera Cabernet Sauvignon. Despite the compatible interaction between the pathogen and plant, several of the E. necator-induced transcripts represented typical defense response genes. Among the transcripts identified were those that encoded a leucine-rich repeat serine/threonine kinase-like receptor, an MYB transcription factor, and two ubiquitination-associated proteins, indicating the stimulation of intracellular signal transduction and regulatory functions. A number of genes characteristic of senescence processes, including metallothioneins, a deoxyribonuclease, an aspartyl protease and a subtilase-like serine protease, also were identified. These transcripts expanded the list of previously identified E. necator-responsive grapevine genes and facilitated a more comprehensive view of the molecular events that underlie this economically important plant-pathogen interaction.

Selection for Run1-Ren1 Dihybrid Grapevines Using Microsatellite Markers

A grapevine hybrid progeny was generated to track the inheritance of the Ren1 and the Run1 powdery mildew resistance alleles and the segregation of the powdery mildew resistance phenotype. Genotypic analysis was carried out using flanking microsatellite markers; phenotypic evaluations were done under in vitro and greenhouse conditions. Pairing the phenotypic and genotypic data demonstrated that Ren1 and Run1 acted as single dominant loci and assorted independently without considerable distortion of segregation. Chromosomal recombination events were detected in the Ren1 but not in the Run1 region, corroborating earlier observations that crossover between homologous chromosomes was suppressed around the Run1 locus. Taken together, the results confirmed that microsatellite marker-assisted selection is a reliable and expeditious method to combine multiple alleles that confer resistance to a pathogen.

Expression of a Grapevine NAC Transcription Factor Gene Is Induced in Response to Powdery Mildew Colonization in Salicylic Acid-Independent Manner.

Tissue colonization by grape powdery mildew (PM) pathogen Erysiphe necator (Schw.) Burr triggers a major remodeling of the transcriptome in the susceptible grapevine Vitis vinifera L. While changes in the expression of many genes bear the signature of salicylic acid (SA) mediated regulation, the breadth of PM-induced changes suggests the involvement of additional regulatory networks. To explore PM-associated gene regulation mediated by other SA-independent systems, we designed a microarray experiment to distinguish between transcriptome changes induced by E. necator colonization and those triggered by elevated SA levels. We found that the majority of genes responded to both SA and PM, but certain genes were responsive to PM infection alone. Among them, we identified genes of stilbene synthases, PR-10 proteins, and several transcription factors. The microarray results demonstrated that the regulation of these genes is either independent of SA, or dependent, but SA alone is insufficient to bring about their regulation. We inserted the promoter-reporter fusion of a PM-responsive transcription factor gene into a wild-type and two SA-signaling deficient Arabidopsis lines and challenged the resulting transgenic plants with an Arabidopsis-adapted PM pathogen. Our results provide experimental evidence that this grape gene promoter is activated by the pathogen in a SA-independent manner.

Screening for Suicidal Behaviour and Mental Disorders With Prime-MD Questionnaire in General Practice

Background: The major risk factors for completed suicide are previous suicide attempts and mental disorders. Lifetime prevalence of suicide attempts is 3-4%; and primary care is a major basis for suicide prevention. Aim: To assess the value of a screening method developed to determine the prevalence of suicidal behaviour, and to describe the characteristics of suicide attempters in primary care, including screening for major mental disorders. Methods: A Hungarian urban general practitioners district with 1248 inhabitants was screened for suicidal behaviour and for major mental disorders. The Prime-MD questionnaire (Primary Care Evaluation of Mental Disorders) was used to recognize the most common psychiatric disorders; suicidal behaviour was assessed by six structured questions of MINI-Plus diagnostic interview. Results: Prevalence of patients with suicide attempts in primary care was 2.9%; 9% of the patients had either suicidal thoughts or gestures the month before. Self-destructive thoughts or behaviour often coexisted with depressive disorders, 60% of suicidal patients and 11.5% of the screened population had depressive episode. According to multivariate logistic regression, suicidal patients were more likely to take antidepressants, they also had a tendency to have more previous psychiatric treatments and suicide attempts; they visited their General Practitioners less frequently and were more likely to have current depressive episodes. Conclusion: The Prime-MD questionnaire, complemented with questions on suicidal behaviour, is an effective, easy-to-use method for general practitioners to assess suicide risk and to recognize the most common mental disorders. This method can be of great help and is proposed for use by general practitioners in every-day practice.

Population Structure of Vitis rupestris, an Important Resource for Viticulture

The wild North American grapevine Vitis rupestris Scheele is an important genetic resource for viticulture, but its natural populations have been severely depleted. We collected samples from seven V. rupestris populations from the Ozark Plateau in Missouri and the Ouachita Mountains in Oklahoma and genotyped them with 14 microsatellite markers to assess allelic diversity, heterozygosity, and genetic differentiation at various levels of population structure. We found that genetic diversity in V. rupestris was similar to that measured in many V. vinifera L. ssp. sylvestris populations and in other outcrossing angiosperms. We detected significant genetic differentiation among populations (ΦPT = 0.105); there was no significant deviation from Hardy–Weinberg equilibrium in some populations, and there was moderate inbreeding in others. Pronounced differentiation between Missouri and Oklahoma populations was supported by a Bayesian clustering approach and principle coordinate analyses and was apparently a function of geographic distance. Genetic differentiation among Missouri populations was modest. We posit that population differentiation and genetic drift may be inherent characteristics of V. rupestris.

RNA-seq-based genome annotation and identification of long-noncoding RNAs in the grapevine cultivar ‘Riesling’

BackgroundThe technological advances of RNA-seq and de novo transcriptome assembly have enabled genome annotation and transcriptome profiling in highly heterozygous species such as grapevine (Vitis vinifera L.). This work is an attempt to utilize a de novo-assembled transcriptome of the V. vinifera cultivar ‘Riesling’ to improve annotation of the grapevine reference genome sequence.ResultsHere we show that the transcriptome assembly of a single V. vinifera cultivar is insufficient for a complete genome annotation of the grapevine reference genome constructed from V. vinifera PN40024. Further, we provide evidence that the gene models we identified cannot be completely anchored to the previously published V. vinifera PN40024 gene models. In addition to these findings, we present a computational pipeline for the de novo identification of lncRNAs. Our results demonstrate that, in grapevine, lncRNAs are significantly different from protein coding transcripts in such metrics as length, GC-content, minimum free energy, and length-corrected minimum free energy.ConclusionsIn grapevine, high-level heterozygosity necessitates that transcriptome characterization be based on cultivar-specific reference genome sequences. Our results strengthen the hypothesis that lncRNAs have thermodynamically different properties than protein-coding RNAs. The analyses of both coding and non-coding RNAs will be instrumental in uncovering inter-cultivar variation in wild and cultivated grapevine species.

The frequency of genetic rearrangements during carrot (Daucus carota) somati embryogenesis is dependent on 2,4-D levels and diminished in its absence

For quantification of genetic variations occurring in plant tissue cultures, DNA sequence alterations and repliconsize changes were monitored through subsequent phases of the model carrot tissue culture system, from the 2,4-D-induced proembryiogenic cell cultures to regenerated plantlets, by RAPD and flow cytometry techniques. Banding patterns of random amplified DNA fragments and ploidy-level distributions of cultured cells were significantly different in the presence and in the absence of 2,4-D. In addition, there were marked differences between the cells induced with lower (1.0 mg/l) and higher (2.5 mg/l) doses of 2,4-D. Among those samples that were cultured in the absence of 2,4-D, the epicotyl (C2), hypocotyl control (H1) and the morphologically normal regenerants (IV2) showed identical banding patterns overlapping with the true-to-type seedling controls (N1, N2). In contrast, treatment of starting explants (H2) with 1.0 mg/l 2,4-D resulted in a marked 82% increase in the number of amplified fragments associated with an appearance of cell lines possessing unusual haploid-like and aneuploid-like DNA contents. The induction with 2.5 mg/l 2,4-D resulted in even greater increase in the number of DNA fragments (~100%) amplified from proembryiogenic cell cultures, while it had no further effect on ploidiy-level distributions compared to the treatment with 1.0 mg/l 2,-D. After the withdrawal of the synthetic auxin, banding patterns and ploidy-level distributions were gradually shifted back to the levels of controls resulting in true-to-type regenerants. Conclusions are in short, the combined use of RAPD and flow cytometry can make quantification of genetic variations typical of dedifferentiated plant cells possible. Quantification opens the window for comprehensive evaluation of different methods, treatments and bioactive compounds.