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Dual RNA Sequencing of Vitis Vinifera During Lasiodiplodia Theobromae Infection Unveils Host–Pathogen Interactions

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Dual RNA Sequencing of Vitis Vinifera During Lasiodiplodia Theobromae Infection Unveils Host–Pathogen Interactions International Journal of Molecular Sciences Article Dual RNA Sequencing of Vitis vinifera during Lasiodiplodia theobromae Infection Unveils Host–Pathogen Interactions Micael F. M. Gonçalves 1 , Rui B. Nunes 1, Laurentijn Tilleman 2 , Yves Van de Peer 3,4,5 , Dieter Deforce 2, Filip Van Nieuwerburgh 2, Ana C. Esteves 6 and Artur Alves 1,* 1 Department of Biology, CESAM, University of Aveiro, 3810-193 Aveiro, Portugal; [email protected] (M.F.M.G.); [email protected] (R.B.N.) 2 Laboratory of Pharmaceutical Biotechnology, Campus Heymans, Ottergemsesteenweg 460, B-9000 Ghent, Belgium; [email protected] (L.T.); [email protected] (D.D.); [email protected] (F.V.N.) 3 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; [email protected] 4 Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium 5 Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria 0028, South Africa 6 Faculty of Dental Medicine, Center for Interdisciplinary Research in Health (CIIS), Universidade Católica Portuguesa, Estrada da Circunvalação, 3504-505 Viseu, Portugal; [email protected] * Correspondence: [email protected]; Tel.: +351-234-370-766 Received: 28 October 2019; Accepted: 29 November 2019; Published: 3 December 2019 Abstract: Lasiodiplodia theobromae is one of the most aggressive agents of the grapevine trunk disease Botryosphaeria dieback. Through a dual RNA-sequencing approach, this study aimed to give a broader perspective on the infection strategy deployed by L. theobromae, while understanding grapevine response. Approximately 0.05% and 90% of the reads were mapped to the genomes of L. theobromae and Vitis vinifera, respectively. Over 2500 genes were significantly differentially expressed in infected plants after 10 dpi, many of which are involved in the inducible defense mechanisms of grapevines. Gene expression analysis showed changes in the fungal metabolism of phenolic compounds, carbohydrate metabolism, transmembrane transport, and toxin synthesis. These functions are related to the pathogenicity mechanisms involved in plant cell wall degradation and fungal defense against antimicrobial substances produced by the host. Genes encoding for the degradation of plant phenylpropanoid precursors were up-regulated, suggesting that the fungus could evade the host defense response using the phenylpropanoid pathway. The up-regulation of many distinct components of the phenylpropanoid pathway in plants supports this hypothesis. Moreover, genes related to phytoalexin biosynthesis, hormone metabolism, cell wall modification enzymes, and pathogenesis-related proteins seem to be involved in the host responses observed. This study provides additional insights into the molecular mechanisms of L. theobromae and V. vinifera interactions. Keywords: dual RNA-Seq; grapevine; botryosphaeria dieback; plant defense; pathogenesis 1. Introduction Grapevine (Vitis vinifera) is widely cultivated and an economically important fruit crop worldwide [1]. Diseases of fungal origin such as grapevine trunk diseases (GTDs) are a significant factor limiting grapevine productivity and longevity [2,3]. Botryosphaeria dieback caused by the fungi of the family Botryosphaeriaceae that grow primarily in mature wood causes dieback as a consequence of the development of a necrotic wood canker/lesion [3]. Botryosphaeria dieback leads to a loss of Int. J. Mol. Sci. 2019, 20, 6083; doi:10.3390/ijms20236083 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 6083 2 of 19 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 19 productivity,fungi of the reducing family profitBotryosphaeriaceae and longevity that [4 ].grow Some primarily authors in suggest mature that wood environmental causes dieback changes, as a such as droughtconsequence and increase of the development of temperature, of a necrotic may induce wood some canker/lesion fungi to become[3]. Botryosphaeria aggressive dieback pathogens, leads killing theirto hosts a loss through of productivity, the release reducing of cell profit wall and degrading longevity enzymes,[4]. Some authors inhibitory suggest proteins, that environmental and toxins [ 5–8]. changes,Lasiodiplodia such as theobromae drought andis aincrease common of temperature, phytopathogenic may induce fungus some and fungi one to ofbecome the most aggressive aggressive speciespathogens, found inkilling grapevines their hosts [1, 9through–11]. It the is mostlyrelease of found cell wall in tropical degrading and enzymes, subtropical inhibitory regions proteins, [12,13 ] and has anand optimal toxins [5–8]. temperature range of 27 ◦C–33 ◦C[14]. This pathogen has a great adaptation capacity and hasLasiodiplodia been associated theobromae with is numerous a common hostsphytopathogenic and diseases fungus [13 ].and Although one of the it most is more aggressive frequent in grape-growingspecies found regions in grapevines with high [1,9–11]. temperatures It is mostly andfound low in tropical precipitation and subtropical [9–11], it regions has also [12,13] been and reported has an optimal temperature range of 27 °C–33 °C [14]. This pathogen has a great adaptation capacity in temperate climates [15–18]. and has been associated with numerous hosts and diseases [13]. Although it is more frequent in grape-growingThe interaction regions between with high plants temperatures and their and pathogenslow precipitation is a [9–11], dynamic it has andalso been complex reported process. Thesein temperate interactions climates should [15–18]. be analyzed as a duel process, and the plant’s reactions should not be separatedThe from interaction the ones between of the pathogenplants and [ 19their]. When pathog aens pathogen is a dynamic interacts and withcomplex its host, process. it will These trigger a complexinteractions host defense should responsebe analyzed activating as a duel various process, processes, and the plant’s such reactions as penetration should resistance, not be separated recognition by patternfrom the recognition ones of the receptorspathogen [19]. (PRRs), When phytohormone a pathogen interacts signaling with pathways,its host, it will secretory trigger a pathways, complex and secondaryhost defense metabolite response production activating [various20]. RNA processes, sequencing such as (RNA-Seq) penetration is resistance, a powerful recognition technology by that has beenpattern widely recognition implemented receptors to(PRRs), investigate phytohormone host defense signaling mechanisms pathways, duringsecretory infection. pathways, So and far, this approachsecondary has beenmetabolite applied production mainly to[20]. the RNA host sequencing or to the pathogen (RNA-Seq) separately is a powerful [19]. technology Dual RNA that sequencing has been widely implemented to investigate host defense mechanisms during infection. So far, this allows the study of both host and pathogen transcriptomes simultaneously, detecting pathogen-specific approach has been applied mainly to the host or to the pathogen separately [19]. Dual RNA transcriptssequencing in the allows same the sample, study providingof both host a moreand pa completethogen transcriptomes insight into thesimultaneously, pathogen infection detecting biology andpathogen-specific host defense mechanisms transcripts [ 19in, 21the,22 same]. This sample, approach providing has already a more been complete applied insight in a fewinto studiesthe of plant–pathogenpathogen infection interactions biology inand crops host [defense23–26] mechanisms and trees [20 [19,21,22].,27,28]. This approach has already been appliedUsing ain duala few RNA-Seq studies of plant–pathogen approach, we aimedinteractions to identify in crops the[23–26] pathogenicity and trees [20,27,28]. factors produced by L. theobromaeUsingand a dual determine RNA-Seq the approach,V. vinifera we defenseaimed to responsesidentify the to pathogenicity the pathogen. factors Overall, produced we intended by L. to contributetheobromae to unravelingand determine host–pathogen the V. vinifera defense interactions responses and to provide the pathogen. helpful Overall, information we intended for the to future developmentcontribute ofto strategiesunraveling ofhost–pathogen disease control interactions and management. and provide helpful information for the future development of strategies of disease control and management. 2. Results 2. Results 2.1. Macroscopic Analysis 2.1. Macroscopic Analysis Progression of necrosis was observed throughout the duration of the experiment (Figure1). Progression of necrosis was observed throughout the duration of the experiment (Figure 1). No No lesionslesions beyond the the wound wound site site were were observed observed in “mock” in “mock” inoculated inoculated plants in plants all sampling in all sampling points. At points. At 11 day day post-inoculationpost-inoculation (dpi), (dpi), no nolesions lesions related related to fungal to fungal infection infection were observed. were observed. After 3 dpi, After a slight 3 dpi, a slightbrowning browning around around the theinoculation inoculation sites sitesin inoculated in inoculated plants plantswas observed was observed (0.5 ± 0.1 (0.5 cm). At0.1 7 cm).dpi, the At 7 dpi, ± the lesionlesion in infected plants plants progressed progressed (5 (5± 0.20.2 cm), cm), and and at 10 at dpi, 10 dpi, the thelesion lesion length length was intensified, was intensified, ± coveringcovering 9 90.2 ± 0.2 cm cm in in length. length. ± Figure 1. Symptom development in V. vinifera following
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