Editing VvDXS1 for the creation of muscat flavour in Vitis vinifera cv. Scarlet Royal.

Muscat flavour represents a group of unique aromatic attributes in some grape varieties. Biochemically, grape berries with muscat flavour produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway, and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single-point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavour in grapes. In this study, the same substitution in the VvDXS1 protein was successfully created through prime editing in the table grape Vitis vinifera cv. 'Scarlet Royal'. The targeted point mutation was detected in most of the transgenic vines, with varying editing efficiencies. No unintended mutations were detected in the edited alleles, either by PCR Sanger sequencing or by amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in their leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes.

Grape cultivars adapted to hotter, drier growing regions exhibit greater photosynthesis under hot conditions despite less drought-resistant leaves.

BACKGROUND AND AIMS: Many agricultural areas are expected to face hotter, drier conditions from climate change. Understanding the mechanisms crops use to mitigate these stresses can guide breeding for more tolerant plant material. We tested relationships between traits, physiological function under hot conditions, and historical climate associations to evaluate these mechanisms for winegrapes. We expected a more negative leaf osmotic potential at full hydration (πo), which reduces leaf turgor loss during drought, and either a metabolically cheaper or more osmoprotectant leaf chemical composition, to allow cultivars associated with hot, dry regions to maintain greater gas exchange under hot growing conditions. METHODS: We measured πo, gas exchange, and leaf chemistry for 7 commercially important winegrape cultivars that vary widely in historical climate associations. Vines were grown under common garden field conditions in a hot wine-growing region (Davis, California) and measured over the hottest period of the growing season (July - September). KEY RESULTS: □o varied significantly between cultivars, and all cultivars significantly reduced □o (osmotically adjusted) over the study period, though osmotic adjustment did not vary across cultivars. πo was correlated with gas exchange and climate associations, but in the opposite directions than expected. Photosynthesis and πo were higher in the cultivars associated with hotter, less humid regions. Leaf chemical composition varied between cultivars, but was not related to climate associations. CONCLUSIONS: These findings suggest that leaf turgor maintenance is not a primary limitation on grapevine adaptation to hot or atmospherically dry growing conditions. Thus, selecting for a more negative πo or greater osmotic adjustment is not a promising strategy to develop more climate-resilient grape varieties, contrary to findings for other crops. Future work is needed to identify the mechanisms increasing photosynthesis in the cultivars associated with hot, dry regions.

Drought Exacerbates Botryosphaeria Dieback Symptoms in Grapevines and Confounds Host-based Molecular Markers of Infection by Neofusicoccum parvum.

Neofusicoccum parvum, causal fungus of the grapevine trunk disease Botryosphaeria dieback, attacks the wood of Vitis vinifera. Because lesions are internal, using putative host-based markers of infection from leaves for diagnosis is a nondestructive option. However, their specificity under drought stress is unknown. Potted 'Cabernet-Sauvignon' were inoculated with N. parvum in the greenhouse after wounding (IW), and with wounded and nonwounded noninoculated controls. At 2 weeks postinoculation (WPI), half of the plants were severely stressed (SS), receiving 30% water volume of the well-watered (WW) plants. Larger lesions at 12 WPI among IW-SS plants, compared with all other treatments, revealed an interactive effect of inoculation and drought on lesion length. Expression of eight putative marker genes was analyzed in leaves by qPCR at the onset of drought stress, and at 8 and 12 WPI. One marker showed consistent over-expression at 8 WPI in IW plants, regardless of water treatment, suggesting specificity to infection. By 12 WPI, higher expression of seven genes in all SS plants (across inoculation treatments) revealed specificity to drought. Cross-reactivity of markers to drought, therefore, limits their utility for disease diagnosis in the field, where drought induced by climate and deficit irrigation is common.

Vitis rupestris B38 Confers Isolate-Specific Quantitative Resistance to Penetration by Erysiphe necator.

Vitis rupestris B38 is a North American grapevine resistant to the major pathogen of cultivated grapevines, Erysiphe necator. Sources of powdery mildew resistance, like V. rotundifolia, are widely used in grape breeding but are already threatened, even before commercialization, by isolates that can reproduce on Run1 and other rotundifolia-derived breeding lines. Thus, complementary sources of resistance are needed to improve resistance durability. The segregation of foliar powdery mildew severity in an F1 family, derived from a cross of V. rupestris B38×V. vinifera 'Chardonnay', was observed in the field over three growing seasons and in potted vines following single-isolate inoculation. A pattern of continuous variation was observed in every instance. Mechanisms of resistance were analyzed on the resistant and susceptible parent by using microscopy to quantify the ability of the pathogen to penetrate and to form a colony on detached leaves. While 'Chardonnay' was susceptible in all tested conditions, V. rupestris B38 resistance was characterized by a reduction in pathogen penetration, with an effect of leaf position and significant differences among powdery mildew isolates. Segregation of the ability of the pathogen to penetrate and form a colony in F1 individuals showed a pattern of quantitative penetration resistance with no delay or restriction on colony formation once penetration has been achieved. Moreover, V. rupestris B38 showed an enhanced penetration resistance to a powdery mildew isolate with the ability to overcome the Run1 gene, making it an interesting resistance source to prolong the durability of this gene.

Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis.

Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

Mixed infections of fungal trunk pathogens and induced systemic phenolic compound production in grapevines.

As grapevines mature in California vineyards they accumulate chronic wood infections by the Ascomycete fungi that cause trunk diseases, including Botryosphaeria dieback (caused by Diplodia seriata and Neofusicoccum parvum) and Esca (caused by Phaeomoniella chlamydospora). It is thought that such mixed infections become localized to separate internal lesions/cankers of the permanent, woody structure of an individual vine, but nonetheless the fungi all colonize the same vascular system. In response to infection by one pathogen, the host may initiate systemic biochemical changes, which in turn may affect the extent of subsequent infections by other pathogens. To test this hypothesis, we measured changes in phenolic compounds in the wood and lesion lengths of the pathogens, during sequential co-inoculations with different or identical pair-wise sequences of infection by D. seriata, N. parvum, or P. chlamydospora. Prior fungal infections only affected the development of subsequent D. seriata infections. Effects of fungal infections on phenolic compounds were variable, yet initial infection by D. seriata was associated with significantly higher concentrations of most phenolic compounds distally, compared to all other initial inoculation treatments. It was hypothesized that pre-existing phenolic levels can slow initial lesion development of fungal trunk pathogens, especially for D. seriata, but over time the pathogens appeared to overcome or neutralize phenolic compounds and grow unimpeded. These results demonstrate that effects of one fungal trunk pathogen infection is generally unable to distally affect another long-term, albeit shifts in host phenolics and other plant defenses do occur.

Phenolic Compound Induction in Plant-Microbe and Plant-Insect Interactions: A Meta-Analysis.

Plants rely on a variety of ways to protect themselves from being fed upon, including de novo production of specific compounds such as those termed as phenolics. Phenolics are often described as important in plant health and numerous studies have concluded they increase as a result of insect feeding, pathogen infection, or beneficial microorganism colonization. However, there are some studies reaching differing conclusions. Therefore, meta-analyses were conducted to observe whether common trends in phenolic induction in plants can be made when they become hosts to insects or microorganisms. Four hypotheses were tested. The first was that total phenolics increase as a generic response, and meta-analyses confirmed that this occurs when plants are infested with insects or colonized by bacterial or fungal microorganisms, but not for oomycetes. The second hypothesis was that phenolic induction is different when a beneficial microorganism colonizes a plant vs. when a plant is infected by a pathogen. Beneficial bacteria, pathogenic bacteria, and beneficial fungi produced increased phenolic levels in plant hosts, but fungal pathogens did not. The third hypothesis was that insect feeding method on plant hosts determines if phenolics are induced. Chewing induced phenolics but piercing-sucking and wood-boring did not. Lastly, we used meta-analyses to determine if annual or perennials rely on phenolic induction in different amounts, and even though annuals had significantly increased phenolic levels but perennials did not, it was observed that phenolic induction was not statistically different when plant type was considered. These results demonstrate that phenolic induction is a common response in plant hosts exposed to feeding or colonization, with specific exceptions such a pathogenic fungi and piercing-sucking insects.

Neofusicoccum parvum Colonization of the Grapevine Woody Stem Triggers Asynchronous Host Responses at the Site of Infection and in the Leaves.

Grapevine trunk diseases cause important economic losses in vineyards worldwide. Neofusicoccum parvum, one of the most aggressive causal agents of the trunk disease Botryosphaeria dieback, colonizes cells and tissues of the grapevine wood, leading to the formation of an internal canker. Symptoms then extend to distal shoots, with wilting of leaves and bud mortality. Our aim was to characterize the transcriptional dynamics of grapevine genes in the woody stem and in the leaves during Neofusicoccum parvum colonization. Genome-wide transcriptional profiling at seven distinct time points (0, 3, and 24 hours; 2, 6, 8, and 12 weeks) showed that both stems and leaves undergo extensive transcriptomic reprogramming in response to infection of the stem. While most intense transcriptional responses were detected in the stems at 24 hours, strong responses were not detected in the leaves until the next sampling point at 2 weeks post-inoculation. Network co-expression analysis identified modules of co-expressed genes common to both organs and showed most of these genes were asynchronously modulated. The temporal shift between stem vs. leaf responses affected transcriptional modulation of genes involved in both signal perception and transduction, as well as downstream biological processes, including oxidative stress, cell wall rearrangement and cell death. Promoter analysis of the genes asynchronously modulated in stem and leaves during N. parvum colonization suggests that the temporal shift of transcriptional reprogramming between the two organs might be due to asynchronous co-regulation by common transcriptional regulators. Topology analysis of stem and leaf co-expression networks pointed to specific transcription factor-encoding genes, including WRKY and MYB, which may be associated with the observed transcriptional responses in the two organs.

Genes expressed in grapevine leaves reveal latent wood infection by the fungal pathogen Neofusicoccum parvum.

Some pathogenic species of the Botryosphaeriaceae have a latent phase, colonizing woody tissues while perennial hosts show no apparent symptoms until conditions for disease development become favorable. Detection of these pathogens is often limited to the later pathogenic phase. The latent phase is poorly characterized, despite the need for non-destructive detection tools and effective quarantine strategies, which would benefit from identification of host-based markers in leaves. Neofusicoccum parvum infects the wood of grapevines and other horticultural crops, killing the fruit-bearing shoots. We used light microscopy and high-resolution computed tomography (HRCT) to examine the spatio-temporal relationship between pathogen colonization and anatomical changes in stem sections. To identify differentially-expressed grape genes, leaves from inoculated and non-inoculated plants were examined using RNA-Seq. The latent phase occurred between 0 and 1.5 months post-inoculation (MPI), during which time the pathogen did not spread significantly beyond the inoculation site nor were there differences in lesion lengths between inoculated and non-inoculated plants. The pathogenic phase occurred between 1.5 and 2 MPI, when recovery beyond the inoculation site increased and lesion lengths of inoculated plants tripled. By 2 MPI, inoculated plants also had decreased starch content in xylem fibers and rays, and increased levels of gel-occluded xylem vessels, the latter of which HRCT revealed at a higher frequency than microscopy. RNA-Seq and screening of 21 grape expression datasets identified 20 candidate genes that were transcriptionally-activated by infection during the latent phase, and confirmed that the four best candidates (galactinol synthase, abscisic acid-induced wheat plasma membrane polypeptide-19 ortholog, embryonic cell protein 63, BURP domain-containing protein) were not affected by a range of common foliar and wood pathogens or abiotic stresses. Assuming such host responses are consistent among cultivars, and do not cross react with other trunk/foliar pathogens, these grape genes may serve as host-based markers of the latent phase of N. parvum infection.