Background: Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew. Results: Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles. Conclusions: The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.

Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from the cultivar “Thompson Seedless” using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance. Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred. Infection assays using leaf discs showed that, compared to wild-type plants, the VvPR4b knockout lines had increased susceptibility to P. viticola. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of P. viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

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The HD-Zip family has a diversity of functions during plant development. In this study, we identify 33 HD-Zip transcription factors in grape and detect their expressions in ovules and somatic embryos, as well as in various vegetative organs. A genome-wide survey for HD-Zip transcription factors in Vitis was conducted based on the 12 X grape genome (V. vinifera L.). A total of 33 members were identified and classified into four subfamilies (I-IV) based on phylogeny analysis with Arabidopsis, rice and maize. VvHDZs in the same subfamily have similar protein motifs and intron/exon structures. An evaluation of duplication events suggests several HD-Zip genes arose before the divergence of the grape and Arabidopsis lineages. The 33 members of HD-Zip were differentially expressed in ovules of the stenospermic grape, Thompson Seedless and of the seeded grape, Pinot noir. Most have higher expressions during ovule abortion in Thompson Seedless. In addition, transcripts of the HD-Zip family were also detected in somatic embryogenesis of Thompson Seedless and in different vegetative organs of Thompson Seedless at varying levels. Additionally, VvHDZ28 is located in the nucleus and had transcriptional activity consistent with the typical features of the HD-Zip family. Our results provide a foundation for future grape HD-Zip gene function research. The identification and expression profiles of the HD-Zip transcription factors in grape, reveal their diverse roles during ovule abortion and organ development. Our results lay a foundation for functional analysis of grape HDZ genes.

The downy mildew disease in grapevines is caused by Plasmopara viticola. This disease poses a serious threat wherever viticulture is practiced. Wild Vitis species showing resistance to P. viticola offer a promising pathway to develop new grapevine cultivars resistant to P. viticola which will allow reduced use of environmentally unfriendly fungicides. Here, transmission and scanning microscopy was used to compare the resistance responses to downy mildew of three resistant genotypes of V. davidii var. cyanocarpa, V. piasesezkii and V. pseudoreticulata and the suceptible V. vinifera cultivar ‘Pinot Noir’. Following inoculation with sporangia of P. viticola isolate ‘YL’ on V. vinifera cv. ‘Pinot Noir’, the infection was characterized by a rapid spread of intercellular hyphae, a high frequency of haustorium formation within the host’s mesophyll cells, the production of sporangia and by the absence of host-cell necrosis. In contrast zoospores were collapsed in the resistant V. pseudoreticulata ‘Baihe-35-1’, or secretions appeared arround stomata at the beginning of the infection period in V. davidii var. cyanocarpa ‘Langao-5’ and V. piasezkii ‘Liuba-8’. The main characteristics of the resistance responses were the rapid depositions of callose and the appearance of empty hyphae and the plasmolysis of penetrated tissue. Moreover, collapsed haustoria were observed in V. davidii var. cyanocarpa ‘Langao-5’ at 5 days post inoculation (dpi) and in V. piasezkii ‘Liuba-8’ at 7 dpi. Lastly, necrosis extended beyond the zone of restricted colonization in all three resistant genotypes. Sporangia were absent in V. piasezkii ‘Liuba-8’ and greatly decreased in V. davidii var. cyanocarpa ‘Langao-5’ and in V. pseudoreticulata ‘Baihe-35-1’ compared with in V. vinifera cv. ‘Pinot Noir’. Overall, these results provide insights into the cellular biological basis of the incompatible interactions between the pathogen and the host. They indicate a number of several resistant Chinese wild species that could be used in developing new cultivars having good levels of downy mildew resistance.