Single berry reconstitution prior to RNA-sequencing reveals novel insights into transcriptomic remodeling by leafroll virus infections in grapevines.

Leafroll viruses are among the most devastating pathogens in viticulture and are responsible for major economic losses in the wine industry. However, the molecular interactions underlying the effects on fruit quality deterioration are not well understood. The few molecular studies conducted on berries from infected vines, associated quality decreases with the repression of key genes in sugar transport and anthocyanin biosynthesis. Sampling protocols in these studies did however not account for berry heterogeneity and potential virus induced phenological shifts, which could have biased the molecular information. In the present study, we adopted an innovative individual berry sampling protocol to produce homogeneous batches for RNA extraction, thereby circumventing berry heterogeneity and compensating for virus induced phenological shifts. This way a characterization of the transcriptomic modulation by viral infections was possible and explain why our results differ significantly from previously reported repression of anthocyanin biosynthesis and sugar metabolism. The present study provides new insights into the berry transcriptome modulation by leafroll infection, highlighting the virus induced upregulation of plant innate immunity as well as an increased responsiveness of the early ripening berry to biotic stressors. The study furthermore emphasizes the importance of sampling protocols in physiological studies on grapevine berry metabolism.

Oregano essential oil vapour prevents Plasmopara viticola infection in grapevine (Vitis Vinifera) and primes plant immunity mechanisms.

The reduction of synthetic fungicides in agriculture is necessary to guarantee a sustainable production that protects the environment and consumers' health. Downy mildew caused by the oomycete Plasmopara viticola is the major pathogen in viticulture worldwide and responsible for up to 60% of pesticide treatments. Alternatives to reduce fungicides are thus utterly needed to ensure sustainable vineyard-ecosystems, consumer health and public acceptance. Essential oils (EOs) are amongst the most promising natural plant protection alternatives and have shown their antibacterial, antiviral and antifungal properties on several agricultural crops. However, the efficiency of EOs highly depends on timing, application method and the molecular interactions between the host, the pathogen and EO. Despite proven EO efficiency, the underlying processes are still not understood and remain a black box. The objectives of the present study were: a) to evaluate whether a continuous fumigation of a particular EO can control downy mildew in order to circumvent the drawbacks of direct application, b) to decipher molecular mechanisms that could be triggered in the host and the pathogen by EO application and c) to try to differentiate whether essential oils directly repress the oomycete or act as plant resistance primers. To achieve this a custom-made climatic chamber was constructed that enabled a continuous fumigation of potted vines with different EOs during long-term experiments. The grapevine (Vitis vinifera) cv Chasselas was chosen in reason of its high susceptibility to Plasmopara viticola. Grapevine cuttings were infected with P. viticola and subsequently exposed to continuous fumigation of different EOs at different concentrations, during 2 application time spans (24 hours and 10 days). Experiments were stopped when infection symptoms were clearly observed on the leaves of the control plants. Plant physiology (photosynthesis and growth rate parameters) were recorded and leaves were sampled at different time points for subsequent RNA extraction and transcriptomics analysis. Strikingly, the Oregano vulgare EO vapour treatment during 24h post-infection proved to be sufficient to reduce downy mildew development by 95%. Total RNA was extracted from leaves of 24h and 10d treatments and used for whole transcriptome shotgun sequencing (RNA-seq). Sequenced reads were then mapped onto the V. vinifera and P. viticola genomes. Less than 1% of reads could be mapped onto the P. viticola genome from treated samples, whereas up to 30% reads from the controls mapped onto the P. viticola genome, thereby confirming the visual observation of P. viticola absence in the treated plants. On average, 80% of reads could be mapped onto the V. vinifera genome for differential expression analysis, which yielded 4800 modulated genes. Transcriptomic data clearly showed that the treatment triggered the plant's innate immune system with genes involved in salicylic, jasmonic acid and ethylene synthesis and signaling, activating Pathogenesis-Related-proteins as well as phytoalexin synthesis. These results elucidate EO-host-pathogen interactions for the first time and indicate that the antifungal efficiency of EO is mainly due to the triggering of resistance pathways inside the host plants. This is of major importance for the production and research on biopesticides, plant stimulation products and for resistance-breeding strategies.

Microsatellite amplification in plants: optimization procedure of major PCR components.

Microsatellites (SSRs) are the most informative and popular class of molecular markers used for diverse purposes, particularly in plants: genetic diversity study, marker assisted selection, breeding, mapping, phylogenetics and phylogeography, systematics, etc. They have become a routine technique practically in each laboratory for studying molecular plant genetics. Despite their wide utilization, however, setup and optimization of various conditions involved in PCR amplification is a prerequisite for reliable inference of results. In this chapter, we describe optimization of SSR-PCR conditions and give ranges of concentrations for different parameters. The protocol provided here is inspired from bench work on the use of microsatellite to study diversity of Vitis vinifera germplasm.