Bertazzon et al. BMC Genomics (2019) 20:526 https://doi.org/10.1186/s12864-019-5908-6 RESEARCH ARTICLE Open Access Grapevine comparative early transcriptomic profiling suggests that Flavescence dorée phytoplasma represses plant responses induced by vector feeding in susceptible varieties Nadia Bertazzon1* , Paolo Bagnaresi2, Vally Forte1, Elisabetta Mazzucotelli2, Luisa Filippin1, Davide Guerra2, Antonella Zechini2, Luigi Cattivelli2 and Elisa Angelini1 Abstract Background: Flavescence dorée is the most serious grapevine yellows disease in Europe. It is caused by phytoplasmas which are transmitted from grapevine to grapevine by the leafhopper Scaphoideus titanus. Differences in susceptibility among grapevine varieties suggest the existence of specific genetic features associated with resistance to the phytoplasma and/or possibly with its vector. In this work, RNA-Seq was used to compare early transcriptional changes occurring during the three-trophic interaction between the phytoplasma, its vector and the grapevine, represented by two different cultivars, one very susceptible to the disease and the other scarcely susceptible. Results: The comparative analysis of the constitutive transcriptomic profiles suggests the existence of passive defense strategies against the insect and/or the phytoplasma in the scarcely-susceptible cultivar. Moreover, the attack by the infective vector on the scarcely-susceptible variety prompted immediate and substantial transcriptomic changes that led to the rapid erection of further active defenses. On the other hand, in the most susceptible variety the response was delayed and mainly consisted of the induction of phytoalexin synthesis. Surprisingly, the jasmonic acid- and ethylene-mediated defense reactions, activated by the susceptible cultivar following FD-free insect feeding, were not detected in the presence of the phytoplasma-infected vector. Conclusions: The comparison of the transcriptomic response in two grapevine varieties with different levels of susceptibility to Flavescence dorèe highlighted both passive and active defense mechanisms against the vector and/ or the pathogen in the scarcely-susceptible variety, as well as the capacity of the phytoplasmas to repress the defense reaction against the insect in the susceptible variety. Keywords: Flavescence dorèe, Scaphoideus titanus, Vitis vinifera, Grapevine, RNA-Seq, Passive defense, Active defense * Correspondence: [email protected] 1CREA Research Centre for Viticulture and Enology, 31015 Conegliano (TV), Italy Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bertazzon et al. BMC Genomics (2019) 20:526 Page 2 of 27 Background conditions, thus revealing how the disease and the Phytoplasmas are plant pathogenic wall-less gram- symptomatology modify the transcriptomic grapevine positive bacteria associated with numerous diseases in profile. Generally, the recorded differences consisted of wild and cultivated species worldwide [1]. They are the inhibition of genes involved in the photosynthetic phloem-obligate parasites, are very difficult to cultivate chain and in the phenylpropanoid biosynthesis in sus- in an axenic medium [2] and are characterized by a ceptible varieties, and of the induction of defense genes transmission mediated by specific insect vectors or by from the metabolic pathways, leading to the formation grafting. of flavonoids, anthocyanins, antioxidants and a few PR In grapevine, phytoplasmas cause grapevine yellows proteins, which sometimes differ among varieties with (GY) diseases, which lead to very serious damage to the diverse susceptibility [13–17]. The induction of genes re- grape and wine industry, ranging from a lower yield of sponsible for cell wall reinforcement was observed in berries to plant death. One of the most destructive GY cultivars with both high and low susceptibility [13]. In diseases is Flavescence dorée (FD), a quarantine pest in order to take a step forward in understanding the the European Community ([3]; CE Directive 257/2000). defense mechanisms activated in scarcely-susceptible FD, transmitted by the leafhopper Scaphoideus titanus varieties, it is of paramount importance to examine the (St) [4], is highly epidemic. It is caused by several iso- response of the plant during the early phase of infection, lates of phytoplasmas classified in the phylogenetic though this kind of analysis is very difficult in the group 16SrV, subgroups C and D [3]. In Europe, S. species-specific tri-trophic interaction involving the FD titanus is strongly dependent on the vine, on which it phytoplasma, the grapevine and the vector. Indeed, completes one generation per year. The eggs are laid in transmission trials should be established in controlled the late summer and pass the winter; and hatching be- conditions using the leafhopper S. titanus, which it is gins in May. From the first larval stage onwards, the in- unfortunately not yet possible to rear for the whole life sect can acquire the pathogen passively during feeding cycle. Moreover, as FD is transmitted by an insect vector, on infected plants and, after an incubation period of the scarce susceptibility observed in some grapevine var- about one month, can transmit the phytoplasma to ieties could be a combined plant response to both the healthy plants throughout its remaining life [5]. The only phytoplasma and the vector. effective strategies to limit FD epidemics are strictly pre- In the present work a focused experiment aimed at ventive, and are mostly based on insecticide treatments distinguishing the above effects was set up in controlled targeting the insect vector. However, with a view to cre- conditions on two grapevine varieties which display very ate more sustainable agriculture, they should be com- different susceptibility to FD. The total mRNA expres- bined with modern approaches, such as the elicitation of sion was evaluated in two early infection stages in order grapevine defenses and/or the selection of resistant plant to identify the quantitative and qualitative transcrip- material. tomic differences putatively associated with the diverse In Vitis vinifera, intraspecific variability for susceptibil- plant susceptibility to FD and its vector. ity to GY diseases has already been observed both from field experience [6–11] and in controlled inoculation Results conditions [12]. In this context it is worth to note that Transmission trial resistance to phytoplasmas is defined as absence of All S. titanus specimens used in the experiments were symptoms and growth alteration and low titer of the analyzed singularly by real-time PCR for the presence of pathogen, whereas tolerance is absence or mild symp- FD phytoplasma. The analysis confirmed the absence of toms but high titer of the pathogen. Indeed, some var- the pathogen in all the insects used in HSt treatments. ieties, such as Chardonnay and Pinot gris, show very On the other hand, the phytoplasma was found in 16 S. serious damage from GY, while when some others, such titanus specimens fed on FD-infected leaves, which cor- as Tocai friulano or Moscato bianco, are subjected to responds to 28% of the insects collected from FDSt the same disease pressure, only a few plants display the treatments. symptoms, usually in just one or two branches. It is not All the plantlets used in noSt and HSt treatments and known if the observed differences in susceptibility could analyzed by real-time PCR proved to be negative for the be caused also by insect preference or feeding behavior. presence of the FD phytoplasma. The plantlets of the In the last few years, some molecular studies aimed at FDSt treatments where at least one of the two insects identifying the grapevine genetic determinants involved had been found to be infected were analyzed. In total, in the response to GY disease have been carried out on ten micropropagated plantlets were infected (six out of Chardonnay and a few other varieties in field 11 in Chardonnay; four out of 19 in T. friulano; no sta- experiments [13–18]. These analyses have been per- tistically significant differences). The quantification of formed on healthy and infected plants grown under field the FD phytoplasma gave different 16S rRNA copy Bertazzon et al. BMC Genomics (2019) 20:526 Page 3 of 27 numbers, varying from 1.2 × 103 to 3.7 × 104 for μgof percentages summarize to 89.74% (SD 2.513) and 90.4% RNA, without any significant differences between the (SD 1.79), respectively, for T. friulano and Chardonnay. two varieties and the two time points (Additional file 1). Therefore, within the limits of this similarity assessment based on global read mapping percentages, also with this Selection of biological replicates for RNA-Seq analyses approach the two genomes do not appear dramatically Some plantlets used for the transmission trials were dis- different in such a way to substantially hamper RNA-Seq carded due either to absence of FD phytoplasma in the